CA2197773A1 - Methods of preparing organs for cryopreservation and subsequent transplantation - Google Patents

Abstract

The invention relates to the field of organ and tissue perfusion. More particularly, the present invention relates to a method for preparing organs, such as the kidney and liver, for cryopreservation through the introduction of vitrifiable concentrations of cryoprotectant into them. To prepare the organ for cryopreservation, the donor human or animal, is treated in the usual manner and may also be treated with iloprost, or other vasodilators, and/or transforming growth factor .beta.1. Alternatively, or additionally, the organ which is to be cryopreserved can be administered iloprost, or other vasodilators, and/or transforming growth factor .beta.1 directly into its artery. The invention also relates to preparing organs for transplantation by a method for the removal of the cryoprotectant therefrom using low (such as raffinose, sucrose, mannitol, etc.), medium (such as agents with intermediate molecular weights of around 600-2,000) and high (such as hydroxyethyl starch) molecular weight agents osmotic buffering agents. The invention is also directed to new post-transplantation treatments such as the use of transforming growth factor .beta.1, N-acetylcysteine and aurothioglucose.

Description

21 9777~
wo 96/05727 Pcrlu595110223 Methods Of Preparing Organs For Cryopreservation And Subsequent Transplantation Pield of ~he Inven~on This invention relates to the field of organ perfusion. More particularly, it relates to a computer controlled apparatus and method for perfusing isolated animal, including human, organs. Still more p~llic~lally, this invention relates to an apparatus and methods for ill~lU iU1illg Vitriflable n,~ of ~,lyu~lu,~,~,livc agents into isolated organs or tissues in preparation for their ~,lyuplc~l,. v. lion and for removing these agents from the organs and tissues after their ~,lyuylc:,c. vd~iOn in plCL/dla~iUII for their IIA~ .n into an animai, including into a human.

21 f~7''77'~' ' ' WO 96/05727 ~ ~ PCT/US951102~3 Background of the InYen~ion GyuL ~ dlion (that is, preservation at very low h~ ldLu~ca) of organs would allow organ banks to be establisbed for use by transplant surgeons in much the same way that blood banks are used by the medical S cu~ ulliLy today. At the present time, ulyUIJlCacl~aliUll can be d,u,uluaullcd by freezing an organ or by vitrifying the organ. If an organ is frozen, ice crystals form within the organ which mPrh~nir~lly disrupt its structure and hence damage its ability to function correctly when it is nalla~JlallLcd into a recipient. Vitrification, by contrast, means cr,li~1ifir~fion, as in a glass, without ice crystal formation.
The main difficulty with wyu~Jlcaell/dLion is that it requires the perfusion of organs with high . u ~ c of u~yuLJluLccLive agents (water soluble organic molecules that minimize or prevent freezing injury during cooling to very low ~ ldLulc >). Islo fully suitable equipment or method(s) has been developed to date for carrying out this perfusion process. This has prevented the establishmen~ of viable organ banks that could potentially save lives.
Devices and methods for perfusing organs with ~ lyu,ulute~,Lalll have been described in thê literature since the early 1970's. See, Pegg, D.E., in Cl~rrent Trends in Cryo~iology (A.U. Smith, editor) Plenum Press, New Yûrk, N.Y., 1970, pp. 153-180, but particularly pages 175-177; and Pegg, D.E., Cryo~iology 9:411-419 (1972).
In the apparatus initially described by Pegg, two perfusion circuits operatcd ~ y~ one with and one without Clyu~Jlu;~,~,Ldlll.
Gyu~luL~LallL was introduced and removed by abruptly switching from the ~yul,~uLc~La,l~-free circuit to the ~.lyu~ut~ Ldln-containing circuit, then backagain. The pressure was controlled by undescribed techniques, and data was fed into a data logger which provided a paper tape output which was processed by a L ,uyl~ hlp desk-top Wang calculator. The c~c~hll~.lLdl results were 2 t ~ 3 WO 96/05727 PCTIUS95/lOZZ3 poor. The equipment and technique described were considered inadequate by Pegg and his colleagues, who later modified them considerably.
In 1973, Sherwood et al. (in Organ PreserYanor, D.E. Pegg, ed., Churchill Livingstone, London (1973), pp. 152-174), described four potential S perfusion systems, none of which are known to have been built. The first system consisted of a family of reservoirs connected directly to the organ via a multiway valve, changes being made in steps simply by switching from one reservoir to another.
The second system created changes in concentration by metering flow from a diluent reservoir and from a ~,Lyu~lut~,LAIlL cu~lce~ ALe reservoir into a mixing chamber and then to the kidney. No separate pump for controlling flow to the kidney was included. Total flow was controlled by the output of the metering pumps used for mixing. A heat exchanger was used before rather than after the filLer (thus limiting heat exchanger clrt~.,LN."Ic~), and there was an absence of most arterial sensing. As will become readily apparent below, the only similarity between this sysoem and the present invention was the use of two ~ .,n Alioll sensors, one in the arterial line and one in the venous line of the kidney. Organ flow rate was forced to vary in order to minimize arteriovenous (A-V) cu"~ .lU Al;l~ll differences. The sensing of cunu~.lLlaliull before and after the kidney in the circuit is analogous to but s~lh~t~miAlly inferior to the use of a l~r~ . r. . and a differential .rflA~ t, in the present invention. The present inventors' experience has shown that the use of a differential IrrlAl ~ . is necessary for its greater sensitivity. The concept of controlling organ A-V gradient by controlling 2~i organ flow is distinctly inferior to the system of the present invention.
The third system described by Sherwood et al. also lacked a kidney perfusion pump, relying on a "b.l~h~,lt~ ; control valve" to recirculate perfusate from the filter in such a way as to maintain the desired perfusion pressure to the kidney. As with the second Sherwood system, the heat exchanger is proximal to the filter and no bubble trap is present. The perfusate reservoir's ~ ..nAlio~ is controlled by metered addition of WO 96/05727 2 1 9 7 7 7 3 PCT/US95/10223 ~

cryuplu;~ulllL or diluent as in the second Sherwood sys~em, and if flow from the organ is not recirculated7 major problems arise in ~ hllAill;llg and control-ling perfusate volume and ~un~ r ~lu~ll;ull None of these features is desirable.The fourth system was noted by Pegg in an appendix to the main paper. In this system, perfusate is drained by gravity directly from the mixing reservoir to the kidney through a heat exchanger, re-entering the reservoir after passing through the kidney. Coll~ nAl;r)~l is sensed also by directly and separately pumping liquid from the reservoir to the lr,flr~l,Lu~ and back.
Morlific~ticnc and additional details were reported by Pegg e~al.
(Cr,vobiology 14:168-178 (1977)). The apparatus used one mixing reservoir and one reservoir for adding glycerol concentrate or glycerol-free perfusate to the mixing reservoir to control ~u~ o~ The volume of the mixing reservoir was held constant during perfusion, n~ c~ccit~ting an exponentially increasing rate of diluent addition during cr~u~,luti~ul"~ washout to maintain a linear rate of c",~ r .o~llsnl change. The constant mixing reservoir volume and the presence of only a single delivery reservoir also made it impossible to abruptly change perfusate ~u ~ ~ ,ui., All, u~ ollr ~l~ of the circuit other than the kidney and a pre-kidney heat exchanger were located on a lab bench at ambient ~"~lu~ ul~, with the reservoir being ~ Ic,~L~ il,d at a constant 30~C. The kidney and the heat exchanger were located in a styrofoam box whose internal te.llLJ~ ulr, was not controlled. Despite this lack of control ofthe air tC.ll~ .dLul~ ~UIIUUIIdillg the kidney, only the arterial t~,l.l~.,.dlUl~ but not the venous ~ . .,nl~r or even the kidney surface te.ll~,.dLul~ was measured. The use of a sLyrofoam box also did not allow for perfusion under sterile conditions. The only possible way of measuring organ flow rate was by switching off the effluent l~ ;,J, pump and manually recording the time required for a given volume of fluid to ' in the effluent reservoir, since there was no perfusion pump which specifically supplied the organ, unlike the present invention. Pressure was controlled, not on the basis of kidney resistance, but on the basis of the combined resistance of the kidney and a manually adjustable bypass valve used to allow rapid circulation of 21 q~7~
W096/05727 r~ l ,."1 L~

perfusate through the heat exchanger and back to the mixing reservoir. The pressure sensor was located at the arterial cannula, creating a fluid dead spacerequiring manual cleaning and potentially h~L~udu~ lg undesired addition of unmixed dead space fluid into the arterial cannula. Pressure control was achieved by means of a specially-fabricated pressure control unit whose electrical circuit was described in an earlier paper (Pegg et al., Cryobiology 10:56-66 (1973)). Anerial cu", . ~ ;r.l. but not venous conrr~n~r~irJn was measured. No computer control or monitoring was used. C~ was controlled by feeding the output of the recording Ir r"..1. ." . ., into a "process controller" for Cu~ l iau.. to the output of a linear voltage ramp generator and )IUIJI' ' adjustment of c . r- l~ '. or diluent flow rate. Glycerol were measured manually at 5 minute intervals at both the mixing reservoir and the arterial sample port: evidently, the 1 r., ~ . was not used to send a ,.,~ ,.I.lt signal to a recording device. TC.~ ILU~ and flow were recorded manually at 5 minute intervals. Arterial pressure and kidney weight were recorded as pen traces on a strip chart recorder. None of these features is desirable.
Further rc;rlu~ La were reported by Jacobsen et al. (Cryobiology 15:18-26 (1978)). A bubble trap was added, the sample port on the kidney bypass was eliminated (ru . ~,.I;rl, was measured at the distal end of the bypass line instead)7 and t~ u~ was recorded as a trace on a strip chart recorder rather than manually every 5 minutes. Additionally, these authors reported that bypass c"", ~ u, u iO~l Iagged reservoir l 0~r ~n.niu~, by 5 min (v.
3 min or less for arterial cll ~IAliul~ in the present invention) and that terminal ~,lyu~ute~ rrnrPntr~irn could not be brought to less than 70 mM
after adding S liters of diluent to the mixing reservoir (v. near-zero terminal co~ ,n~liOI~ in the present invention using less than 3 liters of diluent and using peak ~,lyuuluL~Ld~ u~ uiu~ )Iu~hlldt~'y twice those of Jacobsen et al., supra).
A variation on the system was also reported the same year by l.A.
Jacobsen (Cryobiology 15:302-311 (1978)). Jacobsen measured but did not wo s6/0s727 PcrluS95/10223 ~
-6- _ report air t,lll~ UlC~ aul.uulldillg the kidney during perfusion. He reduced the mixing reservoir volume to 70 ml, which was a small fraction of the 400 ml ~otal volume of the circuit. No electronic-output l~r.~l ~u",~ ~f ' appears tO
have been used to directly sense giycerol ronrrnt~tion and control addition and washout. Instead, the calculated values of rr~nrrntr~f~ or diluent flow ratewere drawn on paper with India ink and read by a Leeds and Northrup Trendtrak P~u~lcu~ which then controlled the u.. . ~lnrll Idiluent pump.
Despite the low circuit volume, the minimum c~", . .III,.li..ll of cryu~Jlu;~
which could be achieved was about 100 mM.
Additional alterations of the same sysLem were reported by Armitage et al. (Cryobiology 18:370-377 (1981)). Essentially, the entire perfusion circuit previously used was placed into a refrigerated cabinet. Instead of a voltage ramp controller, a cam-follower was used. Again, however, it was necessary to calculate the required rates of addition of glycerol or diluent using theoretical equations in order to cut the cam properly, an approach which may introduce errors in the actual a~,h;~ ,llL of the desired concentra-tion-time histories. Finally, a ."o~ lio" was made in which an additional reservoir was added to the circuit. This reservoir was apparently accessed by manual stopcocks (the mode of switching to and from this reservoir was not clearly explained), and use of the new reservoir was at the expense of being able to filter the perfusate or send it through a bubble trap. The new reservoirwas not used to change ~.lyuplutf~Lall~ ~'u~ f ~ rli~ rather, it was used to change the ionic ~Jl,,pn~,lioll of the medium after the cryu~Jlua~Ldll~ had beenadded. The volume of the mixing reservoir was set at 500 ml, allowing a final ulyuplut~L l.lL cnnrPn~r~ n of 40 mM to be achieved.
To the best of the inventors7 knowledge, the devices and methods described above represent the current state of the art of ~.IyU~lULC~L~IlL
perfusion as practiced by others.
An approach to organ ~lca~ilvaLiull at cryogenic t~.l"pe.~.t~.lcs previously described by one of the Applicants involved vitrifying rather than froezing organs during cooling (see, for example, Fahy et al., C~fobiology 219~ 73 WO 96/0~727 PcTruS9S/10223 21:407-426 (1984); and U.S. Patent 4,559,298). "Vitrification" means col jfii f jrAtion without freezing and is a form of c~yu~ e~ tion. Vitrification can be brought about in living systems, such as isolated human or other animal organs, by replacing large fractions of the water in these systems with ~,lyu~luLr~Liv~ agents (also known as cryul~lu~lduLa) whose presence inhibiLs crystallization of water (i.e., ice formation) when the system or organ is cooled. Vitrification typically requires ronrPntr-tir~n~ greater than 6 molar (M) cryu~u~,L~-IL. However, using known rf-rhniflllf-~ it has not been possible to use sufficiently high 1Iyul)luu,~,Lf~llL, to vitrify an organ without killing it. The limiting ~ O~lff ~ on for organ survival was typically just over 4 M.
One type of damage caused by ~,lyu~JIuL~ La is osmotic damage.
Cryub;olo~ i~La learned of the osmotic effects of clyul!lutf~LfA~Ia in the 1950's and of the necessity of controlling these effects so as to prevent Illlll~c~,;.a~lly damage during the addition and removal of l,lyuLJlut~,.,~l~La to isolated cells and tissues. Similar lessons were learned when e~y~ ~ rH~ ' moved on to studies of whole organ perfusion with clyuLJIutl,~,L~.lLa. Attention to the principles of osmosis were essential to induce tolerance to ~,Iyu~Jlutl~
addition to organs. DespiLe efforts to control the deleterious osmotic effects of ~;lyuLJIut_Lf~llLa, limits of tolerance to ~,IyulJlUt~LdllL~ are still observed.
There appear to be genuine, inherent toxic effects of l,lyul~lut~Lf~llLa that are il,fir~ll ,.i, ( of the transient osmotic effects of these chemical agents.
Studies by the present inventors and others have examined methods of controlling the non-osmotic, inherent toxicity of cryuLJlut~Livr agents. The results indicate that several techniques can be effective alone and in ~v l.. .~ These include (a) exposure to the highest c~. ~.ln~liu~ at ~ reduced t~ .,ldlulca, (b) the use of specific c ulllI,hldLiul~s of ulyulJIut~L~ a whose effects cancel out each other's toxicities; (c) exposure to .,lyuLJlut~L~lLa in vehicle solutions that are optimized for those particular ~.lyu~lut~~ lLa, (d) the use of non-pe.le.~dti.,g agents that can substitute for a portion of the F;~.l. jIAIh~g agent otherwise needed, thus sparing the cellular interior from 2 ~ ~7~3 exposure to additional inrrar~ r agent; and (e) ~ ,u~ . of the time spent within the cnnr~n~rltir~n range of rapid time-dependent toxicity. Means by which these principles could be applied to whole organs so as to permit them to be treated with vitrifiable solutions without perishing, however, have not been clear or available.
Some of these techniques are in potential conflict with the need to control osmotic forces. For example, reduced LelllLJ~ldLulr~ also reduce the influx and efflux rate of ~;IyulJlul~ll~s, thereby prolonging and i '~,;"g their osmotic effects. Similarly, " :.,;.,.;,;.,~ exposure time to ~,lyuulut~,~,L~
lû maximizes their potential osmotic effects. Thus, there must be a balance reached between the control of osmotic damage and the control of toxicity.
Adequate means for obtaining this balance have not been described in the literature. In some cases, hl;~"S;r~;,.g the osmotic effects of ~,~yu~ul~,~uL~
by ~ ,.;,;"~ exposure times to these agents can be beneficial and Culllpl~.. l.,.lLdly to the reduced toxicity that results, but safe means for achieving this in whole organs have not been described.
Organ preservation at cryogenic ~t:lllp.,.d~UI~ would permit the reduction of the wastage of valuable human organs and would facilitate better matching of donor and recipient, a factor which continues to be important despite the many recent advances in controlling rejection (see, Takiff et el., T~(~n~rlnn~nh~)n 47:102-105 (1989); Gilks et al., T~an~rln~ n'~n 43:669-674 (1987)). Fu~ ul~ most techniques now being explored for inducing recipient imm.lnrlogir~ tolerance of a specific donor organ would be facilitated by the availability of more time for recipient u.r~
One major limitation in organ Clyuplc~vaLiull studies has been the lack of suitable equipment for controlling perfusion parameters such as ~,lyuplut~L~IlL conrPntr~rir~n-time history, pressure, and tU.Il~C.~iu.~.
Previously described standard perfusion machines are not designed for this application and are unable to meet the leu,~. Ir~lll.,lliS addressed here. Patented techniques heretofore known are described in:

WO96/05727 2 t ~ PCTIIJS95110223 ~ g U.S. Patent No. 3,753,865 to Bel~r e~ al.;
U.S. Patent No. 3,772,153 to De Roissart et al.;
U.S. Patent No. 3,843,455 to Bier, M.
U.S. Patent No. 3,892,628 to Thorne et al.;
U.S. Patent No. 3,914,954 to Doerig, R.K.;
U.S. Patent No. 3,995,444 to Clark et al.;
U.S. Patent No. 4,629,686 to Gruenberg, M.L.; and U.S. Patent No. 4,837,390 to Reneau, R.P.
Equipment described for cryu~ sclv~lion applications in the past has permitted ûnly relatively simple ~p~ llell~l protocols to be carried out, and has often been awkward to use. Only Adem et al. have reported using a computer for organ perfusion with cryu~Jlultut~llL (see, for example, J. Biomed. ~ngineering 3:134-139 (1981)). However, their specific design has several major flaws that limit its utility.
The present invention overcomes substantially all of the ,1~ ri. . :. ~ of known apparatus and methods.

Summary of the Inl~ent~on In one emhorlimPnt the present invention is directed to a computer-controlled apparatus and methods for perfusing a human or other animal organ, such as a kidney, liver, heart, etc., with a perfusate, and may include preparing the organ for such perfusion. The perfusion of the organ may be done for any one of a number of reasons including, but not limited to, for example: to prepare the organ for cryopreservation; to prepare the organ for u ~ n~ iu. after its cl yu~ . vc~iun, to preserve it by cull ~, ' means above 0~C; to keep it alive tclll!~ulalily at high t~ u.cO to study its physiology; to test the organ's viability; to attempt 1~ liu,. of the organ;
and to fix the organ for structural studies. The apparatus and methods may also be used to superfuse an organ or tissue slice. In another . ,,I..~,ii,,~,. l this invention is directed to the treatment of the donor animal and/or the about-2~ ~7773 wo 96/0s727 PcrluS9S/10223 ,~

to-be donated organ with iloprost and/or other drugs to prepare it for perfusion. In another ,~",1"~/1;",. .1l this invention is directed to an apparatus and method whicll is used to prepare the organ for cryu~ulc~clvdtiunl such as by vitrification. In another cnnhn~imrn~, this invention is directed to an apparatus and methods for preparing an organ for transplantation into an a,uiuluiuiiaLc host after its cryuiult~clvaliom In one . ,,ho~l;,,,. ,I, this invention is directed to a method of preparing a biological organ for cryopreservation, comprishIg the steps of:
(a) perfusing the organ with gradually increasing ronrf n~r l-irnc of I,lyuylui,~,Ldlll solution to a first pled~tcln~ un~ lA~;rln while cuucul.cllLly reducing the i~,~ll,u~dLule of the organ;
(b) IIIA;I~ the ", ~ ,Ali"n of the (dyuy~uLeuLd~,l for a sufficient time to permit the d~UplU~'dl..a~ osmotic rquilihrA~ir~n of the organ to occur; and (c) increasing the cr~u,uluLt~,Ldlu ~r~ n~liull of the solution to a highersecond p.cd~,;.,..,li,..dlon~c~,~,dLiu~and~ 6~gtlle wyui~lutl-~Ldll~
ronrrn~r~-ion of the solution at the second conren~r~ion for a time sufficient to permit the a~ -uAi--,...~ osmotic equilibratioll of the organ to occur.
The organ is then remûved frûm the perfusiûn apparatus and is .,lyu~ul~ ,.ved using an appropriate method or is further prepared for ulyuiultacl VdLiOn~
After i,lyu~ul~C,Yd~iOII the organ is warmed in an apparatus which is not the apparatus of this invention.
In,~lc,ualdLiull fortheorgan's IIAI~ ;nn intoarecipient, theorgan is then reattaciIed to the perfusiûn apparatus of this hIvention.
In another ~, I.o~l; . l this invention is directed to a methûd of preparing an organ for n A~ I after its ulyuiulc~c. vaLiu., and subsequent warming, I Ulll~UI;:I;II~ .
(a) warming the ûrgan lo a Lenl~..dLulc which permits reperfusion of the organ, wherein damage to the organ is minimized;

WO 96/05727 2 1 ~ 17 ~ 3 PCT/US95/10223 (b) perfusing the organ with a non-vitrifiable confPntr~tion of elyu~J~utc~LdllL for a time sufficient IO permit the d~J~JIuAillldoe osmotic equilibration of the organ to occur; and (c) perfusing substantially all of the cryu~ out of the organ S while ~:on~,ull~ Lly increasing the te~ .dtll~ of the organ to render the organ suitable for ~ tinll In another ~ o~lh....,l this invention is directed to a method of preparing an organ for IIAI~IIIAIII_I;IJI~ further comprising perfusing the organ with a reduced fO . ,nAIillll of ~,lyulJIut~,~,Ldl~L in ~OI~ " with: a low îO molecular weight (LMW) 'hlf",l.. ~ IAIhlg~ osmotic buffering agent (OBA);
or a high molecular weight (HMW) "~ ;llgr OBA; or a CIJII hjl AI;
of LMW and HMW OBAs which are added and removed in an Ull I ' ' ~
fashion which is d~J~JlU~JI for, and may vary from, organ to organ. In the case of the liver, osmotic buffers (OB) do not have to be used at all. In the case of most other organs, the organ is perfused with the d~ u~Jfi_oe wyuplut~L~... solution containing a first OBA ~un~ InAl;ul~ for a time sufficient to permit dp~JIuAillldoe osmotic eqnilihr~ion of the organ to occur.
SnbstAntiAlly all of the ~,lyu~Jluoe~LdllL is then washed out (to a final ~,lyulJIuL~l,Ld~ u"~ nAliul~ of less than 200 millimolar) while decreasing the flJ IllAlio ~ of the OBA to a second, nonzero level substalntially below the first buffering agent f .. . IAliu~ Ievel and while r~.n.. ~lly increasing the t~ ulp~,ldLII~i of the organ. Finally, the organ is perfused to remove the OBA
~urfi~ tly to render the organ suitable for UA~
F~ plifi. Al j~ include the rabbit kidney, the rat liver, and the human kidney.
The apparatus of the invention comprises a compuoer operated perfusion circuit containing a plurality of fluid reservoirs, a means for raising and lowering ~UIlr' ..n~ljf~ and an organ container. Afirst fluid flow path is defined as a loop from the plurality of reservoirs to necessary sensors and oe~ ,ldLul~ ' ~ means and back to the plurality of reservoirs. The reservoirs are selectively ~JJ....~ . I Ahlc tO the first fluid flow path. Pump means 2t 9~7~
WO 96/05727 PCT/US95/1022~f are interposed in a second fluid flow path for pumping fluid from the first fluid flow path to a second fluid flow path. The organ container is located in this ~cond fluid flow path. Pump means may also be included in the second fluid flow path for pumping fluid from the organ container to one or more of the reservoirs or to waste. One or more sensors are interposed in the fluid flow paths for sensing at least one of the r~nrenrr~ n, concentration differential, ~UI~ dLUlt~, pressure, and pH of the fluid flowing in the first and/or second fluid flow paths. Measuring means are interposed in the hrst and second fluid flow paths for measuring u~ U~fiu~ and ~ Lul~
differences between the upstream and downstream sides, in the fluid flow direction, of the organ container. rne sensor(s) and the measuring means are coMected to a ~ dulnulJle computer for providing a continuous i nformation stream from the sensor(s) to the computer. Finally, the computer is coupled to the ~lection means and the pump means to .~ lh...,~llcly selectively control (a) the f ow of fluid from each of the reservoirs individually to the fluid flowpaths, (b) the f ow of f uid from each of the fluid flow paths individually to each of the reservoirs, and (c) at least one of the . ,,,.~ u,.., L~ul~ Lul~, pressure and pH of the fluid flowing in the first and/or second fluid flow path,in accordance with a ~ulcdtu~ .,d computer program without substantial operator intervention.
Additional features of the apparatus of this invention may include a heat exchanger interposed in the hrst fluid flow path for l~onrf~ nine the Lluy~,.dLul~ of fluid flowing in this fluid flow path. A second heat exchanger may be interposed in the second fluid flow path for con~fi~i~ nine the L~ J.,.d~UI~ of fluid flowing in the second fluid flow path.
In describing the apparatus and methods of this invention, many of the various aspects of the same have been numbered. This numbering has been done to create a conceptual organization and structure for this application.
This numbering should not be interpreted to necessarily mean or imply that the particular steps in this invention must be performed in the sequences in which they are presented.

Features and Advantages of the InYention This invention has a multitude of features and advantages, among the most important of which are the following.
1. It permits control of the rnnrr~ntr~tinn of ~:ly~J~Iot~ dlll or any S other fluid or drug in the perfusate of an organ according to a wide variety of p-~d~,;.,.",il.~,d ~ uu~r .ln~uin~-time histories, more or less i~l, p. 1, .~lly of the flow rate of perfusate through the organ, with provision for ~ u -ly varying the ~ Il,ui~. of other drugs or osmotic agents. Step changes in cnnrPrtr~irn are possible, and it is possible to bring ~ l . U,~liO ~ effectively to ~ro.
2. It provides for in-line sensing of ~:ull1CnlldliOn, pH, perfusate t~lU~ LdlUIC;, and other parameters so as to avoid the need for sensors in the perfusate reservoirs and for manual u~u~;u.~uL~.
3. It permits Illill;llli~illg differences between the .. ., ~ i.u~ of ~uyu~llut~L~Ill monitored and the conrrntr~tir)n of l,lyuplut~,~,Ldlll in the perfusate reservoirs by ~ g the time required for perfusate to travel from the reservoirs to the perfusate sensors and back to the reservoirs.

4. It permits "~ ; differences between the nn of clyu~J~uk~L~IlL monitored and the ~o~ n;ou of ~,ly~ ut~,~.LrlllL actually perfused into the organ by l.l;.li.. ,i~;.. g the time required for the perfusate to travel from the main fluid circuit to the perfused organ (or superfused tissue).5. It permits monitoring of the arterio-venous difference in ~:lyulllut~LrllL col~~ ( .ln~lliull across the organ as an index of the degree of, or ulJ~ullu.l;Ly for, organ eql~ hptinn with cryu~"u..,.~
6. It permits control of the t~,.llp~ ulc of the organ essentially ; li ll......... .....1r . Iy of the flow of perfusate through the organ, and permits varying this ~~ lLul~ at will.
7. It permits control of the perfusion pressure, either keeping it fixed or changing it as desired, and, if desired, minimi7ing pulsation.

2 t q~77~
WO 96/OS727 PCI~/US95/10223 8. It protects against perfusion of unmixed solution, air bubbles, particulates, or pathogens into the organ, and avoids inaccessible fluid dead spaces.
9. It interfaces with a computer to control the perfusions, to provide real-time monitoring, display, processing, and recording of the data, to calibrate the sensors and pumps, and to direct the cleaning, ~ h Irr~ l;u~ and priming of the perfusion circuit and to instruct and alert the operator, when necessary.
10. It is readily capable of perfusing and c~yup~u~,Lil~g organs of widely varying size and perfusion ICU,UilClll~ a~ e.g., anything from a rat heart to a human liver, and is capable of tissue or cell culture au~,fl.~;OIl aswell.

Brief Descriptions of the Figures ~igure 1 (comprising Figures lA and lB). Figure lA shows the overall fluidic circuit diagram of this invention. Figure lB shows the cullall u1Liu-- of the Effluent Distribution Block (EDB) and the means by which the effiuent flow is divided to allow sampling by the Q R.l. pump 126 in Fgure IA.
fiigures 2A-C show side, top and bottom views, respectively of a two-chamber gradient former employed as reservoir Rl in this invention.
Figures 3A-C show side, top and bottom views, Ica~ ,Li~ely, of a three-chamber gradient former used as reservoir R3 in this invention.
Figures 4A-C show left, front, and right side views, ICalJ~ y, of the Heat Exchanger/Bubble Trap/Mixer (HBM) used in this invention; Figure 4D shows the basic mixing unit area of the HBM; and Figure 4E shows a top view of the base of the HBM.
~igure 5 shows a typical protocol for introducing and removing a relativeiy dilute ~;L~ifi~lLiull solution. As used in Figure 5 and in some laterfigures the following abbreviations have the following meanings:

w0 96/05727 2 1 9 7 7 7 ~ Ji~

pH5 means phase 5;
epH6 and 7 mean the end of phases 6 and 7, respectively;
pH5:250 means that the con~pnrr~lion of LMW OBA during phase 5 was 250 millimolar;
S epH6:50 and epH7:0 mean that the co~ .u"~ c of LMW OBA at the end of phases 6 and 7 were 50 and 0 millimolar, respectively;
Veh. means vehicle.;
EC means E~urocollins solution;
CPA means ~.lyu~lu~cu~ L agent;
Numbers I to 7 within circles designate the 7 phases referred to later;
P/10 means pressure divided by 10;
M means the target molar ~o l, ~1 ,n ju~
M means the measured molar ~;u . U,.li~ll and F means flow in ml/min:
Figure 6 shows the part of the protocol for the two-step i~LIodu~Liu~
of fully .,UIl~,, 1 \~iLIir,~lion solution that was carried out inside the standard perfusion machine.
Figures 7A-7D comprise a flow chart of activities for organ ulyu~)luLcuLdlll perfusion.
Figure 8 is a schematic diagram of the details of the two-step cooling technique for hlllullu~ high c~ onc of ClyulJIut~L~
I;igure 9 shows an apparatus to perfuse kidneys with vitrification solutions outside of the standard perfusion machine and at ~ u~ ulc~ in the vicinity of-20 to -30~C.
~igure 10 (.. I,.i~.. l~, Figures lûA to 10D) show a typical rat liver perfusion protocol in which neither HES nor LMW OBAs were used.
F'igures IIA and llB comprise a flow chart of the procedure for non-~,-yu,u.uLc~,L~IlL perfusions.
Figure 12 shows the ability of rabbit kidneys I ,~ d after perfusion with the vitrifiable solution known as V49 to function as measured by their control of serum creatinine.

_ _ . ... . .. .. .

2 ~ 97773 W O 96/05?27 P<~rnUS95/10223 Figure 13 shows the effect of cooling to -30~C on rabbit kidneys previously perfused with 7.5 M or 8 M cylu~luu~Live agents, in co~ ar to the results for the non-cooled kidneys exposed to -3~C.
Iiigure 14 shows the results of exposing rabbit kidney slices to high .u"- I .In~ JII~ of cryu,uluLcuLdllL after rather than prior to cooling to -23~C, ~lPmnn~rrAting that bûth the cooling injury and the toxicity associated with high ~O~ AI;~ are prevented by cooling initially in a low (6.1 M) conrentrA~inn Pigure 15 shows that cooling injury is also successfully avoided in the intact kidney at 6.1 M clulJIut.,~,LlllL (100% survival, excellent final creatinine levels), proving the hypothesis that cooling injury is abolished at low 1 A l 10 l l ~
~igure 16 shows the feasibility of the two-step approach for hllludu~
8 M ClyuL~Iut~,~,L~lllL at -22~C; the survival rate was 7/8 and the creatinine levels after two weeks were excellent and identical to those for kidneys exposed only to 6.1 M c~yu~utC~,L.~uL.
~igure 17 shows the feasibility of using the two-step approach to avoid cooling injury down to -32~C with 8 M .,lyu~JIut.,.,L~llL (survival rate = 100%,final creatinine levels identical to those for kidneys exposed only to 6.1 M
cryu~u~uLt~
Figure 18 shows that kidney slices treated with Y55 and cûoled to ~6~C experience maximum cooling injury, no further injury being apparent when slices were cooled all the way to the glass transition tclU~J~,IalUlC.
Figure 19 shows the po~Lu~ Liv~ serum creatinine levels in an intact kidney that was treated with V55 and cooled to 16~C with subsequent life support funtion (survival rate: 111 kidneys so treated; final creatinine levels:acceptable.) Figure 20 (comprising Figures 20A and 20B) show data fiom the perfusion of a human kidney with the vitrifiable solution known as V55 by the method of this invention. Specifically, Figure 20A shows successful control of clyu~lut~,.,LIl~L nu~ I,.l;nn Fgure 20B shows resistance and flow data.

2 l 97773 wo 9610s727 PcrluS95/10223 The data are all from the same 232 gram human kidney. In Figure 20A, P
means pressure in mm Hg. In Figure 20B, resistance is expressed as weight times pressure divided by fow rate ( m~g xg~
(ml / m~n) Pigure 2I shows cooling data from the same kidney as Figure 20. The kidney was cooled after immersion in a 60% w/v mixture of dimethyl sulfoxide and acetamide. These data gave a continuous recording of organ core L~ d~UII; from 0~C, which was reached in about 15 minutes, to about the glass transition i~Ul~ ld~UI~. The data revealed no evidence of ice formation within the kidney.
Pigure 22 shows loading (ascending portion) and unloading (descending portion) of a human pediatric kidney with V55 using the method of this invention. The solid line was the target VSS concentration while the dotted line was the actual measured V55 . u ~ .,n,.~ir)l~ in the circuit. Since the ~,lyu~lut~LdllL was unloaded from this kidney a cooling curve was not generated.
Figure 23 (comprising Figures 23A-23C) shows viability data for rabbit kidney slices (Figure 23A), human kidney slices (Figure 23B) and culllLJdldLi~ ~hbj~ lm~n data (Figure 23C). The human kidney slices showed identical responses to V49 as the rabbit slices but showed slightly lower recovery after cooling to -30~C.

Definit~ons In order to provide a clear and consistent ~..,rlr.~l-",l;"g of the ;ri~ ~;rn and claims, including the scope to be given such terms, the following definitions are provided. Any terms which are not specifically defmed in this or other sections of this patent application have the o~dinary meaning they would have when used by one of skill in the art to which this invention applies at the time of the invention.

W O 96/05727 2 1 q 7 7 7 3 PC~rrUS95/10223 As used herein, '~clyu~ltaclvdlio~ means the ~ .i.,i.,~ of the viability of excised tissues or organs by storh]g them at very low ~ y~ld~lllcS.Cryopreservation is meant to hlclude freezing and vitrification.
Asusedherein, "vitrifica~ion" meanssolifiifr~tir)nofanorganortissue S without freezing ;t.
As used herein, '~cryuplu~c~Lalll means a chemical which inhibits ice crystal formation in a tissue or organ when the organ is cooled to subzero tc~ alllll,a and results in an increase in viability after warming, in Cuul,u~liaull to the effect of cooling without the clyuplut~Lalll.
As used herein, all t~m~ueldt~cs are in ~C unless otherwise specihed.
As used herein, "non-penetrating" means that the great majority of molecules of the chemical do not penetrate into the cells of the tissue or organbut instead remain in the PYtrrrPlllll~r fluid of the tissue or organ.
As used herein, "osmotic buffering agent (OBA)" means a LMW or HMW ",,.,~ ~~ PYtrr~rP~ lr~r solute which counteracts the osmotic effects of grcater intr~rPitlllrr than Py~r~rr~ r . ul~ F.~ n~ of .,lyu~JIut~,~Lalu during the cryup~ult,LanL effluY process.
As used herein, LMW OBAs have relative molecular masses (M,) ûf 1.000 daltons or less. LMW OBAs include, but are not limited to, maltose, potassium and sodium fructose 1,6-~ .h~r, potassium and sodium l~rtl~bi~ , potassium and sodium ~lyu~ -r . ' '~
stachyose, mannitol, sucrose, glucose, mal~otriose, sodium and potassium gluconate, sodium and potassium glucose 6-phosphate, and raffinose. In a more preferred PmhoriimPnt the LMW OBA is selected from the group consisting of mannitol, sucrose and raffinose.
As used herein, HMW OBAs have Mr of 1,000 to 500,000 daltons.
HMW OBAs include, but are not limited to, hy ilu~,llyl starch (HES) 450,000 daltons and lower Mr hydrolysis fragments thereof, especially 1,000 to 100,000dalton fragments), polyvinylpyrrolidone (PVP), potassium rafhnose IlllriF. ~ Alr ( > 1,000 daltons) and Ficoll (1,000 - 100,000 daltons). In a 2 1 97~73 wo 9610s727 PCT/USg~/10223 most preferred ~ o~lh~ the HMW OBA is HESI 450,000 molecular weight.
As used herein, "a~upu osmotic ~ll.,;lil.,,,li."," means that the difference between the arterial and venous co"~ l;u"c is less than about 50 to 200 mM. (A difference of 200 mM at an arterial cu~ l;o,. of 4 M
means that the venous CO~ I;ul~ is 95% of the arterial ~ io ~ A
153 mM difference is equivalent to a 1% w/v co"~ l;on difference for our preferred clyu~Jlu.~,~,LallL formula described below.) As used herein, "animalr means a mammal including, but not limited to, human beings.

Detailed Descriphon of the Preferred Embodiment and Best Mode 1. Description of the Perfusion Apparatus In a preferred I ~ c!~lhll~ -1, the apparatus hl~,oll!ul~Lh~ the principles and features of this invention is contained in a ~crl i~ di~d cabinet 100 (shownby double dashed lines in Figure lA). The ~~.rl;~ t~ cabinet contains two sides, the l~.,.vuh/sul~..lo;d side and the O~ /l r,~ ~.. t~,. side. The cabinet is faced with double paned transparent doors each containing ~ P~ 1 inch of insulating air (which can be reduced in pressure and/or humidity if necessary) between the panes to avoid ~ u. of moisture on the doors and to minimize heat leak into the cabinet. The organ-side door is split to form a "Dutch door". This allows the upper portion of the organ-side door to be opened and closed to place the organ in the system and to remove the organ without changing the Le,,,~ Lu,c below the upper portion of the door, where the organ container and most other equipment are located. The cabinet may also employ a "Dutch door" on the reservoir side of the cabinet to enable the operator to make any needed adju~".~.l,~ (e.g., W096/05727 2 'i 977 73 PCT/US95/10223 fluid addition to the reservoirs, transfer of upper fluid lines, etc.) without disturbing the cabinet's tC,Il~C.d~ul~ to an uunf cf ~d.y degree.
The primary features of the invention and its mode of operation are shown in the fluidic logic schematic of Figure lA. All fluid available for circulation through the system is drawn into the main circuit by a circuit pump 102 through fluid uptake lines UI, U2, U3, or U4 depending upon the computer-controlled actuation pattern of three-way solenoid valves SI, S2, and S3. Uptake lines UI-U4 connect either to fluid delivery lines DI-D4 leading from reservoirs Rl-R4, It~,c~ ,ly, or to cleaning ports Cl-C4, through standard tubing quick ~ , u~l~U~I~ By clamping DI-D4 and unplugging them from uptake lines Ul-U4, lines U1-U4 can be plugged into cleaning ports CI-C4, as indicated by the curved arrows. While this is presently done manually, it will be d~ ' by those skilled in the relevant arts that d~tJlU~
valves, tubing and controls could be added to handle most of these tasks ~nlrmnS.rir sUly In the ...,i,...1., ..l of the invention as presently constructed, the reservoirs RI-R4 are supported on a thick transparent plastic shelf from which four magnetic stir tables hang which stir the four reservoirs (not shown in Fig. 1). Thorough stirring of RI, R3, and R4 is necessary for prûper generation ûf the desired con~Pnrr~tioll-time histories. The on/off states and stir rates of the stir tables are i...l. ~ ly controlled by hl~LIulll~,.lLdLiu located outside the ll,.flfj~,ldltd cabinet.
Ports CI-C4 lead to sources of sterile (distilled) water, air, and d;~:l.f. ~ e..,l Solenoid valves S0 and S00 are interposed in the délivery linesfor these sources and are arranged to ensure that traces of d ~ llf.~ do not enter the perfusion system by accident. Solenoid S0 controls whether air or fluid will enter the perfusion circuit for cleaning, while solenoid S00 deurmines whether the fluid selected will be water or di~."f.~l~.,l The breakup of the main cleaning line into four h~ nd. l channels outside of the cabinet rather than just before reaching CI-C4 (not so indicated in Fig. I) ensures that each channel is in-lPpPnt~Pnt of the others, i.e., not subject to any W o 96/05727 PC~rrUS95/10223 ,f"l cross-.~".;-.";. -1;.. resulting from diffusion of unpurged solution backwards from the fluid uptake lines Ul-U4 into the cleaning lines leading to cleaning ports Cl-C4.
Distilled water and ~ f~l "l are drawn into the system through a sterilizing filter F4, while air is drawn into the system through an air filter F5.
The (i;~ of choice at present is a clinically accepoed dialysis machine cold sterilant such as Actriln' (Minntech, Mi~ Part-lic Minnesota). The cleaning procedure is to wash the perfusate out of the system with water and then to displace the water with sterilant. Prior to the next perfusion, the sterilant is washed out of the system with water and the water is then washed out of the system with air. The system is then primed by displacing the air with ~ JlUL)li~ perfusate. The air flush is used to avoid the persistence of any lingering traces of sterilant dissolved in the rinse water, and to avoid anypossible dilution of the priming 9uid with water (i.e., to reduce the amount of priming fluid needed for displacing water from the system), to allow a visual check of the c~ oF priming, and to reduce spillage of water in the cabinet when the reservoirs, filters, and organ cassette are placed into the system after cleaning but before priming. The air purge can, however, be omitted if desired. The air filter is used to prevent co~l-l"i, ;.," from pathogens in the air, if necessary.
Solenoid valves S9-S12 normally direct fluid to reservoirs Rl-R4 or to the waste line (LW). Reservoirs Rl-R4 can also be detached from the system by removing Ic~ ,ulali~ll lines RL~-RL8 from reservoirs Rl-R4 and plugging them into waste ports Wl-W4,1c~ 1y (as indicated by curved arrows), alloving reservoirs Rl-R4 to be removed from the system for cleaning, sterilizing, and refilling. When reservoirs Rl-R4 are removed, valves S9-S12 direct fluid to waste ports Wl-W4. The four waste lines c4ll~ ouJil~g to waste ports Wl-W4c4nverge to a single common waste line LW. A t vo-way solenoid valve S16 is located on the common waste line.
When the waste ports are not in use, the common waste drainage line is 2 t ~7773 wo s6/0s7t7 PcrluS9~/10223 blocked by closing valve S16 to prevent any possible backflow of waste or pathogens into the sterile cabinet.
The use of this system of uptake lines U1-U4, which are plugged alternately into reservoir delivery lh1es D1-D4 or cleaning ports Cl-C4, in S c~ ull with recirculation lines IRL5-RL8, whicl1 are plugged alternaoely into the reservoir internal return lines (not shown in the hgure) or into waste ports Wl-W4, allows complete sterilization of the perfusion circuit. The blunt ends of the uptake lines U1-U4, delivery lines D1-D4, cleaning ports Cl-C4 and waste ports Wl-W4 may be sterilized by swabbing with .1;~; "f. ~ 1- m when the tubing is being transferred from one alternative position to the other. The tubing transfer is accomplished while applying digital pressure to the tubing so as to occlude it while making the transfer to prevent fluid leaks and furtherreduce the risk of c~JIl~ ,n;ul~
The fluid withdrawn from reservoirs R1-R4 or from ports C1-C4 is delivered through one of several filters FI, F2, and F3, depending upon the state of actuation of solenoid valves S4 through S7. These actuation patterns will be described in more detail below. Experience has shown, however, that a single filter Fl or two filters F1, Fl' in parallel will be adequate for most studies (rendering valves S4-S7 optional, as indicated by broken lines) since virtual step changes in C'~ n;-JII c~n be imposed even when only one or two filters in parallel are present in the circuit.
It is desirable to minimize the distance between the circuit pump 102 head and the solenoids Sl-S7 to minimize circuit dead space and dead time and to minimize the effects of perfusate viscosity. Short distances and adequate tubing inner diameters are p~ lly critical for S1-S3 to assure adequate fluid withdrawal from R1-R4.
Standard Millipore filters appear (Bedford, MA) compatible with our ~,lyu~JIu~ . The filters are capable of sterilizing the perfusate and are ,,..:~L,~I,lc. All filter holders can be removed from the system for cleaning and ~r~rili7~lion by means of the quick ~iicnonn~tC shown in Figure IA. Vent lines Vl-V3 lead to solenoid valves S13-S15, located outside of the 2~97773 W O 96/05727 PC~r/US95/10223 refrigerated portion of the cabinet 100. These vent lines are opened and closed under computer control during priming and cleaning of the system to permit air to escape and thereby prevent the filters from becoming blocked by air or damaged. A manual bypass (shown only for the S13 bypass) is provided for Vl-V3 for emergency purging of air from the circuit.
Obviously, air purges of the system beyond filters F1-F3 are not possible if filters F1-~3 are present in the circuit; hence filters F1-F3 must be removed before beginning the washout of sterilant if an air purge is to be included in that procedure.
In the presently preferred e~l~bo ihl~ L, a 90 mm diameter filter of 0.22 micron pore si~ is located in each filter holder. This si_e filter is able to pass enough ~;L~ir~Liull solution at -6~C to permit the successful perfusion of a rabbit kidney, with an overlying 1.2 micron filter and a coarse prefilter to prevent clogging. The standard configuration for the operative version employs two identical filters in parallel. This is necessary to ~ l.,r~ the flows required for human organs and provides a safety factor for any air which may be inadvertently introduced into the arterial fluid, as well as 11';1-;116~;1.g pressure build-up proximal to the filter. This continuous filter~f-fili7~1-inn and resterili_ation of the perfusate during the perfusion can serve as a back up for pre-sterili~d solutions in case of ~o~ i.. for any reason during the perfusion. (The incidence of renal infections has been 0%
after literally several hundred perfusions.) Once the fluid from the selected reservoir has passed through the ~ t.,u~,l filter, itgoes through somepl~lhl~hl~lly te~ c c~ u~ ;..g in a heat exchanger 104 and then travels to a position as close to the organ as ~ possible, at which point it encounters a "T" type tubing connector Tl. The bulk of the flow passively takes the path L1 ("r- ru., I.-,,.~ t~ . loopn) that leads to a flow-through process control l~fl~ulul~ 106 that measures the index of refraction of the liquid and hence the ~,.yu~lut~L~ u ~f ..l...lin,. The remainder of the flow is directed through an organ loop L2 by means of an organ pump 108. The organ pump speed is controlled by the computer so as 2~ Q777:~
wo s6Jo5727 1 ~ ~ u~3 to maintain the desired organ perfusion pressure despite wide variations in the organ's vascular resistance. By changing the organ pump head and the diameter of the tubing going through it, a wide range of flows can be generated sufficient to perfuse organs of a wide range of sizes: organs as smallS as rat hearts to organs as large as human kidneys have been successfully perfused.
The flow rate delivered by the circuit pump 102, which supplies both the ~cfi~ ,~. loop Ll and the organ loop L2, must be high enough to both exceed the fiow rate through the organ at all times and to ensure that sufficient flow is avaiiable for the ~ ,,. t. ~ 106 and other in-line sensors, generally designated 110, for measuring te,nl,~,.clu~c, pH, and other desired parameters of the perfusate, to permit accurate measurements. The fow must also be high enough to minimize the "dead time" between changes in reservoir c~ n~uiul~ and changes in the sensed ~ "~.~ln,~ n and other sensed parameters in the .. r".m.".,. t ~ loop as well as to minimize the "dead time~
between the reservoir and the organ. The circuit pump flow is limited by the need to prevent fiuid from being delivered to the filters at a rate in excess ofwhat these filters or the tubing leading to them can pass without failing, as weil as by constraints of heat output and wear and tear on the circuit pump tubing. The speed of the cincuit pump is usualiy not varied during an CA~ l and does not therefore usually require computer control, though computer control is available as an option.
After passing through the organ pump 108, the perfusate passes through a second heat exchanger 112 that finalizes perfusate ~.I~ IUlC
- 25 conriirinning This is done by adjusting the fiow of both cold and warm liquid from cold and warm baths 114, 116, respectively, using computer-controlled pumps (not shown) between heat exchanger 112 and baths 114 and 116.
The computer is able to vary fiow through both the cold path and the warm path so as to adjust perfusate ~UI~ U~C in the anerial line and therefore also in the effluent of the organ. The anerial and effluent Icnl~ c~ provide an indication of the actual organ 1~"~ u~c. By , . . . . . . .

21 9777~
W O 96105727 PC~rrUS95/10223 -25- ~

controlling the flow rate of cold and wann bath fluid, organ t~lup.,.d~ulr~ can be adjusted illflrp~ uA. ~lly of organ fow, provided flow is not close to zero.
_xperience has shown that arhrial and venous t~ ,ldlU~ at least as cold as -6~C and at least as high as 25~C can be achieved with this invention.
Generalized cabinet cooling is not an alternative to the heat exchange system shown for subzero perfusions because cooling of the cabinets to subzero ~Illp.,ldlul~,~ will cause freezing of the more dilute solutions in the tubing lines. Specific jacketing and cooling of the organ container is of particular theoretical value, however, and may optionally be included.
The t~ ,ld~u~-cnnAi~if!nPd perfusate is then debubbled and mixed in a bubble trap/mixer 120 just before entering an organ container 122. Arterial and venous hlll~ ltu-r~ probes, generally designated "T" in Figure lA, penetrate the wall of organ container 122 througll simple holes. Pressure and, optionally, hrll~ .ldlulr~ is sensed in the bubble trap. Although shown separately in the drawing for ease of unAPrst~nAi ng, the bubble trap and mixer 120 are in fact an integral part of the heat exchanger 112, so heat exchange continues to be controlled while debubbling and mixing are z~rcnmrli~hpA
FspPriPnfP has shown that mixing was important due to the tendency for layering of dilute solutions on more f~ o. ~, A, denser solutions. Details as to the specific c~ ll u.lio" of the heat exchanger/bubble trap/mixer (HBM) are described below.
Undernormal1h~ ,thecoolingfuideffluentfromthissecond heat exchanger 112 is used to cool the perfusate passing through the pl~lh~ ly heat exchanger 104. This cooling fluid then travels to a solenoid holding block 118 physically containing solenoids Sl-S12, so as to draw off wash heat from these solenoids before returning to the cold bath.
The holding block 118 currently consists of a large aluminum block (but may be either metal or plastic) perforated with cylindrical holes of suff cient diamehr to closely match the outside diameters of the held solenoids. The solenoids are inserted such that the base, containing the fiuid inlets and outlets, faces the operator and the head, from which the electrical i 2iq77~
WO 96105727 PCT/US95~1OZ23 leads penetrate in~o or througll the holding block. The solenoid hoiding block is equipped with an internal fluid path for drawing off waste heat from the solenoids. Feet are provided to position the holding block, prevent it from moving, and protect the fluid inlet and outlet ports when the holding block is removed from the cabinet. The block is positioned behind and above the reservoirs in the refrigerated cabinet so that the solenoid inlets and outlets and their c~-nn~clion~ to the reservoirs are always readily visible.
The solenoids are preferably 3-7 watt (or less) piston type 3-way solenoids of minimal internal fiuid volume having orifices on the order of 0.156 inches or more and Cv values 2 0.16 (e.g., Modei 648T033 solenoids from Neptune Research, Maplewood, NJ) whiie resisting pressures of up to 500 mm Hg or so. The inventors presently prefer Neptune Research (5upra), 3-watt solenoids fitted with RC dropping circuits to reduce heat generation after activation. Solenoids having 1/16 inch orifices and Cv values of 0.01 to 0.03, e.g., Valcor's Model 20-2-3 (Valcor Scientific, Springfield, NJ) are not fully saLi~rd,Lu.y due to the high viscosity of the solutions used for ~,lyu~lc~ t;on (causing difficulty aspirating viscous fluid through Sl-S3), the high flows desired for controlling dead times and for perfusing larger organs, the possibility of clogging, and the buildup of pressure between the circuit pump and S8-S12. The detailed actuation pattern of the solenoids is described below. The solenoids inside the lcfii6~ .d cabinet that are not held in the solenoid block, SRI, SR31 and SR32, are described in more detail below.
An effluent d; ,Llil,uLion block (EDB) 124 (Figure IA) is connected to the output side of the organ container 122. The EDB is designed so that a small amount of effluent is always present at the bottom of the block. This residual f uid is withdrawn by the two-channel "delta R. 1. pump ~ 126 and sent to the differential r~r.r, u~ ("delta R.l. meter") 130 where its refractive index (a measure of ~o", . .u,.~;u") is compared to that of the perfusate from l~ ., loop L1 (pumped at the same rate as the venous effiuent sample) and a difference signal generated and sent to the computer. Since the fiuid in WO 96/05727 r~ 22.s the l~fl~utuu~,tf. loop L1 will ~ uAhuatf the concentration of the fluid entering the artery of the organ, the delta R.l. output provides an estimate of the arte~io-venous ~ lu gradient across the organ. When this gradient is large (in either the positive or negative direction), the organ is far from equilibrium. When the gradient is zero, the organ is at least largely in osmotic equilibrium with the perfusate. The nonlinear baseline resulting from this ulwllllodux use of the differential l~fl~ u~ tf l is accounted for in the software for running the perfusion program.
All effluent from the organ (together with the arterial fluid sampled by the delta R.l. pump) is ultimately collected by the recirculation pump 128 and sent to solenoid S8, which controls whether the effluent is recirculated to the reservoirs or discarded. Effluent to be returned to a reservoir is combined with the fluid flowing through the rPrl ~ uJ~ l~f ~' ~ loop Ll at a T connection T2.
As noted above, return to the correct reservoir is then controlled by the actuation of solenoids S9 through S12.
The recirculation pump 128, like the circuit pump 102, need not require flow adjustment. It is normally set to a rate sufficient to exceed the maximum flow through the organ pump 108. Since the output of the recirculation pump exceeds that of the organ pump, air is continually introduced into the tubing leading to solenoid S8 and usually to the reservoirs Rl-R4. Provisions to prevent excessive bubbling of the reservoirs as a result of this are described below.
Although the delta R.l. pump speed can be changed, it is usually kept constant throughout an f '1' ;' ~'1 In the presently operative version, it has not been under computer control, but computer control would be a desirable option in some cases. The delta R.l. pump employs very small diameter poly~tllylcllf; tubing to reduce delays in fluid transit time. This small tubingis ~ ti~,ulally important because the flow rate through the delta R.l. circuit is limited by the lowest flow rate through the organ, which may be small, and by the limited size of the fluid paths in culllllu,.~.;ally available differential l~r,~ .".. I~

WO 96/05727 2 1 ~ 7 7 7 ~ PCTIUS95/10223 -~8-The retum of the differential ref,duluu~etc. output to the organ effluent line is proximal to the efnuent recirculation pump. This placement rather than placement distal to the pump ensures a steady flow through the differential ,cr"ml)ll.m~" whereas distal placement may prevent or alter differential Irf,Am(.l~ , flow by virtue of a higher exit pressure.
An important elernent of the fluidic circuit is the gradient pump 132 connected to the circuit by a line Pl (Figure lA). The function of the gradient pump is to allow for gradual changes in ~OI~. r m, ~ / within the d~ u~JlidLe reservoirs within the cabinet. The method by which this is ~rC/~mplichP~1 will be described below. The placement of the line P1 to the gradient pump at T3A, just after the pOillt of joining of the Irfl,.. I~ . Ioop L1 and the organ loop L2, presents one option for ensuring the removal of some of the air introduced by the organ effluent ~cu;luuklLioll pump I28 and therefore helps reduce bubbling of the reservoir fluid.
A better option, however, and the one presently used, is to draw no air into line P1. This is accomplished by connecthlg P1 at point T3B and results in fully controlled connpnrrl~rion-time histories. The bubbling problem is then overcome by ~ ly regulating the speed of tl e ICl..;lCUIdLiUll pump 128 to be just slightly in excess of the combined flows of the organ pump 108 and the delta R.l. pump 126 so as to intrûduce little air. Attaching the IC~ ,UI~iUll output of S8 directly to Pl without regulating the speed of pump 128 results in degraded c~ ..n. I;OII control and is not Ir~O I I- 1r~A
The present operative version of the r~lllOl~ 1 of the invention uses silastic tubing of 1/8 inch diameter throughout the system, which is sufficient to - ~~ ~ ~ ~ ~ ' the needed flows and is preferred. Silastic is compatible withActriln' (Minntech, Minneapolis, Minnesota) cold sterilant, is translucent (important for visualizing flow to detect problems and for observing any signs of microbial growth), is impervious to common ~,~yu~ JLccLive agents such as dimethyl sulfoxide, and is soft enough to be easily . ' ' However, silastic tubing should not be used in circuits coming into contact with siliconecooling fluids, which swell and weaken silastic tubing. In addition, C-Flex0 2 1 q7773 Wo s610s727 PCrJUSs5/10223 tubing (Cole Palmer Instrument Co., Chicago, IL) should be used in the pump heads due to its greater strength (silastic tubh1g undergoes spallation) and greater flexibility when cold.
Reservoir R1 is w~ uutl,d as a gradient former (Figure 2).
Essentially the gradient former consists of two concentric cylinders, an outer cylinder 200 and an inner cylinder 201. A fluid path 205 allows fluid to flow from the outer cylinder 200 to the inner cylinder 201 under the influence of gravity in response to a reduction of volume in the inner cylinder. The concentric orientalion of the fluid UO~ ltl~ t~ is very space efficient. The fluid delivery line 201 cullc~ulld~ to the line Dl of Figure IA. The unit shown is a l~ U( ll of a ~ly available gradient former. The necessary ,..1~.1;1;. -l;o..~ for use with this invention are as follows.
I) The stopcock normally used to control flow from the outer cylinder to the inner cylinder in the cc.ll,ll..".,;al device is replaced by a pinch-type two-way (on/off) solenoid valve 202 (currently, a Bio-Chem Valve Corp.
model IOOP2WNC, East Hanover, NJ) (Figure 2C). A pinch-type valve is preferable for this application to a piston-type valve because of the small pressure difference available to drive fluid flow and the r~nCpri~len~ need for a large working diameter fluid path 202b. It is also preferable for casy removal from its tubing 202b when the reservoir is to be removed from the cabinet for cleaning, leaving the solenoid behind. The base of the gradient former has been modified, at 203, to make room for the solenoid and to support it on a platform. Platform 203 is equipped with a vertical metal post 203b. Solenoid 202 is lashed to this post with a rubber band so as to keep the solenoid oriented correctly. The solenoid is located a sufficient distance from the reservoir to avoid excessive heating of the reservoir fluids.
2) The diameter of the fluid path 205 from the outer cylinder 200 to the inner cylinder 201 has been enlarged to pennit flow at an adequate rate of the viscous solutions required for organ cryopreservation. An inner diameter of 118 to 3/16 inch is adequate.

, . . . ... . . . .

wo 96/0s727 2 1 ~ 7 7 7 ~ PCrNsss/l0223 3) A lid 206 has been provided (Fig. 2B). The lid has an outer overhang 207 that prevents the lid from moving from side to side after it is placed on the cylinder as well as concentric grooves into which the wells of 200 and 201 fit. The lid has built-in outer and h1ller filling funnels 208a and 208b with removable lids, and a It~,h~,ulatiOll port 209.
4) Funnels 208a and 208b extend hlto respective internal fill tubes 210a and 210b. The internal fill tubes are preferably rigid hollow rods located next to the wall of the inner and outer cylinders and are perforated at 1-2 cm intervals with holes 211a and 211b, respectively, which are G~,ulu~ilndLcly 3 mm in diameter. The function of the fill tubes is to reduce the creation of bubbles as ~,h~.ul~Lillg fluid impacts the surface of the iiquidin the reservoir. The purpose of the pe.ruldLiu.,~ is to enable air to escape from the tube through the p~..ru~dliulls so as not to force air to the bottom ofthe reservoir to form bubbles. These functions are tJdlLiuu611y important in perfusates containing protein, which tend to stabilize bubbles.

5) A fill mark has been provided to enable the reservoir to be filled IC~lu iuuilJly to the same, predetermilled volume. The operator can establish his/her own fill mark depending upon the details of the :lpplir~rion The gradient formers may have dL)~ en~fillZltiOnC (horizontal lines on bûth the inner and ûuter cylinders, aligned sû as to permit avoidance of parallax error in reading the liquid level in either cylinder) spaced d~ u~dlll~.~ly 0.5 cm apart for a 2 liter gradient former. These y,~
are also important for Pcr~hl jchine slight, deliberdte micm~ltrhPc in liquid level between inner and outer cyliûders, which are necessary to prevent premature mixing of solutions of widely differing densities, such as u-yuL~-ut~,.Ldll~-free perfusate and villirl-~dliUII solution. They also permit a rough ~IUdllLiLdLi~
check by the operator on the progress of the gradient as l~,u~ ~ on the computer screen.

6) The plastic cnmr.)siri(-n of ~ommPrei~lly available gradient formers may create problems for certain types of cryuL~luLt~ldu~ which could conceivably attack the plastic. It is therefore preferred to use reservoirs made W O 96105727 PC~r~US95/10223 of Lldll:lJdll .1~ material (e.g., glass, plexiglass or the like) that is compatible with the ~,lyu~JIvtc,Ldlll chemicals or use reservoirs whose surfaces have been siliconized or otherwise treated to prevent the attack. In the inventors' rYrrrirnrP, acrylic has been found to be an acceptable material.

7) The reservoir R1 contahls a stir bar 212. The stir bar is housed in a jacket 213 attached to a freely spinning vertical pin 214 extending to the stir bar from the lid of the reservoir to prevent the jacket, and hence the stirbar, from moving laterally. This change is necessary to make sure chattering, and therefore poor mixing, does not occur wi1ile the perfusion machine is nn~t~l~n~ Support from above rather tllan below prevents UUIlC~c~ly perfusate frictional heating and wear and tear to the floor of the reservoir.
Reservoir R3 is also cc~ uctcd as a gradient former. The details of reservoir R3 are shown in Figure 3. Reservoir R3 contains an outer cu~ LIII~,... 315 ~R3,), an im1er ~;ulll~JdlLIll~ 318 (R31), and a third - wllltJdl~ ,llL 316 (R32) I".. ,.-"~ t., ~u~ 2drtlll~ 316 is connected to inner cdul~dl~ lL 318 through a fluid conduit 320 controlled by a solenoid 317 (SR31). Cu~ Jd-ul.~ 316 also connects to outer COIll~,dlllll.,lll315 by a fluid conduit 321 controlled by a solenoid 319 (SR32). The use of an outer WIII~Jdl~ is necessary when co", ~ ;on is being reduced to zero or nearly zero, for reasons noted below in the discussion of the function of the gradient pump and the action of the gradient formers. The use of an outer WllltJdlllll~,.ll is greatly preferred compared to a middle wlll~,~lLu.~
having a larger volume of fluid (and no outer compartment) because simply increasing the volume of fluid in the middle CUIIII)dlLIII. ~It would cause the . profile resulting from a constant gradient pump 132 flow rate to become non-linear. Control of cQnrPntr~ n-time history would then become more ccmp~ More importantly, an excessive amount of fluid in the middle cu-,-~ ,l.l would be required to approach a zero conr~ntrPtiQn in the circuit compared to the amount of fluid required in the outer CululJdlI"..,.-t after virtual emptying of the inner and middle coml dlLI"~.IIt~.

2 1 ~773 WO 96~05727 PCI/US95/10223 Automated use of reservoir R3 poses some problems which are successfully addressed in part by software and in part by the spccific construction of R3. Specifcally actuation of solenoid SR32 allows fluid in the outer compartment (R3,) to fiow first into the middle uùuliJdlUI,.,.lL (E~3~) and from this compartment to the inner cylinder (R3,). This is because the pressure hcad present between R33 and R3~ is large when R3, and R31 are ncarly empty which occurs when SR31 is activated. At this point R3, is still full. This large pressure head causes fluid to flow too rapidly into R31 if R3, is connected directly to R3, rather than using R32 as a buffer between R3, and R31. By adjusting ~he level of R3~ the flow can also be partially controlled.
But even with these two ~ ~uLiOIls, further control of flow is required by using an ~ ulu~)fl~ duty cycle for SR31. The flow to R31 should be slow at first and more and more rapid as the ~ u ~ .n u io" is brought closer and closerto zero whereas passive flow under the influence of gravity will always be fastest at first and slowest at the end unless the flow is metercd by the sort of tailored duty cycle currently being imposed on SR31.
Tbe other modifications to R3 resemble those of RR
Reservoir R4 is a gradient former constructed in the same manner as R1.
The purpose of the gradient pump 132 is to remove some of the It7;hl uLllillg fluid from the circuit. This removal of fluid causes the flow rate of fluid back to the reservoir of origin to be less than the flow rate of fluid from this reservoir to the circuit. This causes the level in the inner cylinder of the reservoir (R1 R3 or R4) to go down. This lowering of inner cylinder 'S fluid level in turn causes the fluid in the outer or middle cu,.,~,~.Lul~ to flow into the inner cylinder to keep the two levels similar. Thus the two dissimilar conrPn~ onc in the two cylinders are mixed in the inner cylinder generating the c~n. u ~dull gradient which is then sent to the rest of the circuit. This is the manner in which the gradient pump effects the desired gradual changes in r~nrPI~tr~tion which reach the organ and the ~ r".mul~ ,, Any necessary adjustments to the gradient pump speed are made by the computer.

21 q777~
Wo 96/05727 PCTlUSg5/10223 The principle involved is that of an ordinary linear gradient former in which the portion of the circuit external to the gradient former can be regarded, to a first atJ~ dtioll, as extra volume in the inner cylinder.
Withdrawal and discard of fluid from the inner cylinder at a constant rate will S result in a linear molar cnnrr-nrrP~ion change with time despite the presence of the rest of the circuit and the recirculation of fluid back to the reservoir.
However, unlike an ordinary gradient former, the concentration of fluid leaving the gradient former at the moment the volume in the gradient former becomes zero will not be equal to the conrf~n~rPtir)n of fluid in the outer (or middle) cylinder of the gradient former. Therefore, in order to approach a conrr-n~rPlir\ll of zero during cr)~ .u~,L~IlL washout using an ordinary two-w~ lLnl~ i gradient former, it is necessary to add additional fluid to the outer cylinder while continuing to discard fluid from the inner cylinder normally. This is why R3 has been modified to have a third ~ lllJfllLll~"lL.
The extra fluid required to continue cryu~.~ut~~L~ washout is added from this third coll~ LIlu,.lL by the computer more accurately than a human operator could ~rCnmrlich this task manually. During introduction of cryut lut~L~IlL~
on the other hand, the desired final ro - ~ "niol~ can always be reached h,y using a cr nrPntrPtion in the outer cu l ~ u ~ which ~ r~ y exceeds the final ~,~IJ,.rf .~n~.l;(lll desired in the circuit at the end of the gradient. Since the current method involves an upward step change in ~ - f..~ ion (see below), it is convenient to fill R1 's outer ~ ~.UL.~ n . ~ with the same fluid used in R2.
The HBM heat exchange system is shown in detail in Figures 4A-E.
Perfusate enters the HBM through an entry port 403, travels through a central channel 400, and leaves the HBM via an outlet port 406. On either side of the central perfusate path are separate chambers for regulating t~ ,U~ tUI~. The two innermost te..l~,.cLulc control chambers 401 (one on each side of the perfusate path) are used for the circulation of coolant, while the outer chambers 40~ are a pathway for the flow of room tcrl,~Jc.~tul~ fluid for offsetting the coolant. (For specialized applications involving, for example, nl:)lllloL;.~.lllic perfusion, these pathways can be reversed.) 2~7~
WO 96/05727 PCT/US95/lOZ23 The direction of cold fiuid flow is opt;onal. Adequate ~eul,uc.a~u control has been found when all fiuids (perfusate, coolant, and warming fluid) flow in the same dircction (uphill) despite the lack of ~;OUllt~ u~ heat exchange. This mode allows the avoidance of air or carbon dioxide accumuiation in the outer chambers.
Perfusate enters the botlom of the HBM unit through inlet 403 and travels upward in a zig zag patterm It emerges into a small upper reservoir which has an air space above: this is the bubble trap area 404. Perfusate then travels beneath the bubble trap and goes througlI a perfusate mixing area 405 before finally traveling onward to the arterial outlet.
The inlets for cold 407 and warm 408 fluid are each split into two channels within the base of the unit. The outlets 410, 411 for warm and cold fluid, respectively, each receive fluid collected from two channels such that each channel of the same kind (i.e., each cold channel or each warm channel) is the same length and nominally ~ ,fl~ the same pressure difference from start to hnish, so that flow rate througll each like channel should be a~ u~hlla;~ly equal.
Ail of the cold and warm fluid pathways include a length of flexible tubing 412 at the redr of the unit. These tubing segments serve twû purpûses~
First, by hlLlul iu~,hlg an air gap between the four channels, heat exchange between them is minimized. This is particularly desirable when all of the cold and warm fluid is f owing in the direction opposite to that of perfusate flow (i.e., in orthograde direction) and has not already undergone heat exchange with the perfusate Second, each tube can be clamped. In this way, if by chance one cold channel or one warm channel should take al! of the cold or warm fluid delivered while the other channel "airlocks", this situation can be corrected by clamping the channel receiving all of the flow and purging the air out of the inactive channel, bringing each channel into full function and equal flow.
Because in the orthograde mode the tcul,u~,.dLul~ cnndi~inning fluid enters the heat exchanging portion of the unit at the top and exits at the wo 96/0s727 2 1 ~ 3 Pcrluss5llo22 bottom, it is necessary upon installation to run the cold and hot pumps in retrograde direction in order to purge all air out of the cold and warm channels. This is best accomplished if the cold and warm tubing leading to and from the bath is no more than about 1/8 inch in internal diameter, since at this diameter fluid flow will displace air from the tubing rather than allowing it to flow uphill in a direction opposite to the direction of fiuid flow or otherwise to remain unpurged in various parts of the tubing. Thus, when the pump direction is reversed again from retrograde to orthogradel no air will be present in the tubing and none will be trapped in Ihe heat exchange chambers of the unit.
In addition to serving a heat exchange function, the zig zag pattern is also designed to force mixing of previousiy perfused dense perfusate or, when perfusate density is rising rather thall falling, to purge the less dense perfusate from the perfusate path.
As the perfusate emerges from the zig zag heat exchange area, it enters the bubble trap 404 at trap entry area 418. Perfusate exits the bubble trap through exit region 419. The zig zag pattern, in fact, is also designed to allowany air bubbles to exit the heat exchange area and to emerge into the bubble trap area. The bubble trap area is designed to have the foliowing features.
l. Its volume is ~ufri~,lF,,.. ly large to reduce the pulsatile action of the perfusion pump to a minimum by d,~LIiiJU~ g the shock of each stroke over a relatively large air volume. This simplifies pressure control and mF~ and may be Iess damaging to the organ.
2. Its volume is sufficiently low to minimize the liquid volume present in the trap and thereby to minimize the dead time and dead volume between the organ pump and the organ itself. A minimal voiume is also desirable to minimize layering of more dilute perfusate over more dense perfusate.
3. A pressure sensing port 413 is provided. Port 413 has no fluid connection to the perfusate, thus eliminating a "blind alley" in which fluid cannot be mixed properly or in whicll l1icinfFr~nt might fail to penetrate or might be trapped. Bo~h an electronic pressure transducer (to provide a signal to the computer) and a sphyg.l.t/~ l...ll.. .S, gauge (For calibration and visual checking) are used.
4. The lid 414 of the trap is removable for cleaning.
5. A vent port 416is provided whicl- is used to adjust fluid level in the trap so as to make it the minimum required to serve the bubble trap function and to maximize pressure wave damping. The tubing from this vent leads to the outside of the cabinet, permitting adjustments to be made without opening the cabinet door.
6. A third port 417 is provided througl1 the bubble trap lid to permit the injection of drugs, vascular labeling materials, fixative, etc.
7. The walls of the bubble trap are angled near the trap entry and exit points 418, 419, respectively, to produce a certain amount of mixing of the perfusate both as it enters and as it leaves the trap, and to break up and minimize the volume of layers of dilute perfusate overlying more dense perfusate.

8. The option exists of introducing probes, such as a tc~llt~ ul~
probe via one of the ports in the trap lid to make ~ ulclll~,nta in the perfusate without permanent embedding of the sensor: the port consists of flexible tubing attached to a plastic threaded htting. A probe can be freely admitted or withdrawn and the tubing clamped with hemostats or an equivalent clamp to effect a pressure-tight seal. This simplifies removal and reir~ ion of the HBM when it must be cleaned and allows flexibility in probe selection and the UtJ~JUI~ y of using the probe for other III~UIUIll~ elsewhere.
After leaving the bubble trap, the perfusate descends to a mixing area 405 (see Figure 41~). The basic unit of the 3-unit mixing path is a narrow horizontal entry area HE emerging into a "wide" basal area BA which rjses to an area of flow restriction FR and ends in a descent D to the next horizontal entry area. Fluid entering HE is forced through an opening too small to support much layering of low density fluid on top of high density fluid, especially considering the right angle turn required just before HE.

~ W O 96/05727 2 1 9 7 7 7 3 P~rnUS95/10223 Fluid flowing into BA may, if less dense, rise immediately upward toward FR. If more dense, it may be driven into the wall and rise upward along this wall. Upon rlll U~ FR, however, the denser liquid will be :~rrelPr~trd toward the less dense liquid rising directly from HE, creating turbulence and mixing. If BA fills with dense perfusate, again the speed of the fluid flowing directly upwards at FR should cause the dense liquid to mix with any low density fluid layered above FR. Fullh~ uul~, the narrow descending path D
should draw layered liquid down the angle along with denser liquid, again preventing stagnant layers from persisting. In practice, three such mixing units aligned in series as shown in Figure 4B are sufficient to mix initially very poorly mixed perfusate, whicll is cnuuu.~ d frequently in the course of abruptly raising or lowering cr~u~lu~ a~lll rrnrrntr~tion One final function of the mixing units is to serve as a trap for any small bubbles which for any reason are not removed in the bubble trap area. (Bubbles in the mixing area are, however, easily purged by the operator prior to initiation of organ perfusion.) After leaving the mixing region, the perfusate descends to an outlet port 406 leading directly to the organ. The path from the final mixing unit to port 406 is deliberately created at an angle to the horizontal in order to provide one last chance of stopping any bubbles from reaching the organ, since in order to reach the organ a bubble in this pathway would have to flow downhill, contrary to its tendency to flow uphill.
The mixing area and subsequent areas are purged of air by occluding the outlet tubing affixed to port 406 with the vent open until ~Iy~)lu~d~ t~,ly 1/2 inch of fluid has ~ tf d in the bubble trap. The vent is then closed until the pressure has reached about 60-120 mmHg. Finally, fluid is once again allowed to flow freely through port 406. The jet of fluid through the mixing area and out port 406 sweeps all air out of the fluid path from the bubble trap to port 406. If some air persists, it can be removed by repeating the process.
After air has been purged, the vent is opened to aliow ulluc~ ly fluid in the bubble trap to exit the trap under the infiuence of gravity, reaching a final WO 96/05727 2 1 q 7 7 7 3 PCl'lUS95/10223 depth of about 1/8 inch. A final depth of 1/8 inch cannot be set before purging the line of air because insufficient volume exists to avoid refilling the mixingarea with air from the bubble trap during the purging process, The HBM is designed to require removal for cleaning only hlr~c~lu~.lLly. Disinfection and removal of dishlfectant from the bubble trap area is effected ~ntom~ti~ y but presently requires some operator attention afterwards to ensure that all uppermost exposed surfaces are disinfected and later washed free of riicinfPct~nt without con~min~ting the outlet tubes. The option exists of arranging the outlet tubes at 413, 416, and 417 in such a way that, with specific solenoids attached to them, they could be individually purged with water, disinfectant, and air under automated control, thus relieving the operator of the need for diligence in cleaning the bubble trap.
After the per~usate exits the HBM unit throug]1 port 406, it enters the organ in the organ container 122 (Fig. 1). In the preferred embodiment, the organ container comprises a rectangular box with a hinged lid, lid stop, lid handle, sloped floor, specially sloped feet, arterial and venous tL~l~,louuu,uleinlets, perfusate inlet, and effluent outlet in the foot opposite the inlet. Theslope of the floor is downward in both the right to left and the back to front directions to ensure that all fluid runs to the foot outlet with very little fluid ~ ,,, anywhere in the container. One needle probe is inserted directly through the wall of the arterial line. A second probe is placed directly in the stream of fluid emerging from the organ. In typical results, the arterial and venous tc.n~rd~ule~ differ by only tenths of a degree, but both are useful for quality control. The organ container may employ a soft mesh support for the organ similar to that used in the MOX-100 DCMTU organ cassette (Waters In~lulll~,.lL~ Inc., Rochester, Minnesota) or the organ can be placed directly on the floor of the organ container or on a specially designed i"~ lr ~1 and removable support. The latter option is preferred and is presently in use.
The organ container 122 and the organ pump 108 are placed in maximum proximity to reduce dead times and dead volumes between the two, _, ~I Wo 96/05727 2 1 9 f~ PCT/US95/10223 and the tubing leading from the organ pump to the organ container is chosen to be as small in inner diameter as possible for the same reason.
Most perfusate does not go through the organ loop 12 as described above but travels instead from the filters to the in-line analog refldLlu,n~
106. The presently preferred ~mhorlim~nf of the invention uses a modified commercially available refractometer from Anacon Inc. (Burlington, Mass.).
In particular, small diameter tubing inlet and outlets are used rather than the very large standard Anacon pipe fittings.
The modification of the refractometer sensing head appropriate for the final invention could also contain the following additional changes from the ordinarily available Anacon unit.
1. The internal volume of the fluid path could be further minimi7P~I
2. Presently, it is necessary to purge the air space of the unit with a slow flow of dry nitrogen gas to prevent ~ on.lf ~noll of moisture due to the low tel..~e.~ , and high humidities prevailing in the cabinet. In a modified version, the electronics area of the sensing device could be h~rm~lir:llly sealed with some desiccant inside to eliminate the need for a nitrogen purge.
3. The present unit must be oriented with the fluid fiow direction being vertical and upwards. However, the unit is not ~uilt to be used in this nrj~nt:~tifm and body changes could be made to adapt the unit's shape to this ()ri~ntotin~n The invention allows the operator to access reservoirs in any sequence and to otherwise custom-design the process which may be of interest. The operator is even free to switch solenoid positions depending on what he may ~ want to do. Neif.Li,eh,~, the following nominal application illustrates the actuation patterns required to deliver fuid from and to each individual reservoir and filter. It also illustrates the "standard protocols" for organ ~,-.yu~ ,~"t perfusion and for cleaning of the system which the system was designed primarily to carry out.

W O 96/05727 2 ~ q 7 7 ~ 3 PC~r~S95/10223 ~0-Solenoid S1 admits fluid from Rl whelI off, or from R2 when activated. Solenoid S2 is open to R3 whelI not energi~d, or to R4 when energized. The output of Sl and S2 is to S3, which accepts fluid from Sl (that is, from Rl or R2) when in the resting state and which accepts fuid from S2 (i.e., from R3 or R4) when activated. The common outlet for S3 (always open) leads to the circuit pump 102, which then withdraws f uid from the solenoid-selected reservoir.
If differential filters are to be included, then the output of the circuit pump 102 is to S4's common port (always open). When S4 is not energized, its output is directed to filter Fl. The return from filter Fl returns to the normally open port of S5 and exits through the S5 common outlet to the ". S . Ioop Ll and the organ loop i~2. If, on the other hand, S4 is energized, then its output is directed to the common inlet port of S6. When S6 is in the resting state, its output is directed to filter F2, and the return from filter F2 enters S7 through its normally open port. The output from S7 tQvels to the normally closed port of S5, whiclI must be energized to accept this output. Once f uid has entered S5, it f ows out the S5 common outlet to the Irr~A, l(",.. loop and the organ loop. Finally, if S4 is energized and S6 is also energized, fuid will be directed tl~rough both of these valves and will reach filter F3. The return from filter F3 occurs via the energized S7 and the energized S5 solenoids and goes to the two loops Ll and L2 as above. As noted earlier, the use of filters F2 and F3 and therefore of solenoids S4, S5, S6, and S7 is optional and will be useful primarily when very abrupt changes from one solution to another are required, or when particularly heavy particulate CU";~'""'Al~' must be removed.
Effluent from the organ eventually returns to S8. If S8 is activated, the f uid is discarded. If S8 is not activated, the fluid is directed from S8 tocombine with fiuid from the ~f~ u,nct~, loop and is returned to a desired reservoir.
Fluid traveling through the ,rrlA. I.. lllrl~l loop travels successively to soienoids S9, S10, Sll, and S12 and thell to the waste Ihle if none of these ~I WO 96/05727 2 t 9 ~ 7 7 3 PCT/US95110223 ~1_ solenoids are energi~ed. Energi~ g S9 diverts flow to the Rl recirculation line. SlO's activation (hl the absence of activation of S9) diverts flow to R2.
Similarly, selective activation of Sll or S12 will, respectively, recirculate fluid to R3 or R4.
There are two basic processes of solenoid-actuated fluid control, one for actual perfusions and one for system cleaning and priming. The perfusion process typically proceeds from Rl through R4 whereas priming must occur in the reverse order to load the fluid uptake and fluid recirculation lines for reservoirs R2-R4, particularly if filters F2 and F3 and their associated lines are used, leaving the circuit primed with fluid from (typically) Rl (or Cl) at the end of the priming (or cleaning) process. The typical sequence of solenoid activations required to prime the complete system (or to clean it) is listed in tabular form below.

Solenoid Control Seqllence For Slandardized Rinsing/Priming The conditions of the solenoid control processes are set forth in Tables I and 2. The uses of these control processes are to: replace perfusate with filter-sterilized H~O at the end of the process; replace cleaning HlO with chemical sterilant between perfusions; remove disi"rtutd"t using filter-sterili~ed distilied HlO; remove water using air; remove air using reservoir fluid, i.e. prime the system.
When only F1 (not F2 or F3) is present, priming (and cleaning) may proceed in any order of reservoirs, provided, in the case of priming, that the final reservoir l.;Ull~:~pUlld~ to the first reservoir used for the subsequent perfusion. Applicants now use a procedure involving Illolln,.lldly aspiration from R2, then R3, then R4, then Rl, taking just enough time to prime U2, U3, U4, and Ul, respectively, followed by computer/user interactive activation of S12, Sll, S10, and S9 to allow manual filling of RL~, RL7, RL6, and RL5 by syringe with retrograde exhaust via Pl, because this procedure saYes large quamities Of perfusate and is fast.

w096/05727 2 l ~7~ Pcrlusssllo223 The standard process of solenoid aCIuatioll for withdrawing fluid from Rl-R4 and for creating gradients for a normaf perfusion is as follows (assuming (I) use of oplional filters F2 and F~, (2) straiglltforward or typicaluse of the gradient-controlling solenoids, and (3) the existence of a gradient former as R2). The staged completion of a closed circuit upon going from one reservoir to another is to avoid l~uhl,ul~Lillg solu~ion of undesired CUIll,uu~;LiOll to the new reservoir before its contents have displaced the previous solution from the circuit. If there is no problem wi~h recirculating the previous solution, the precaution of delayed recirculation can be dropped.

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o ~ o , v, Table 2 Sorcnold Conhol S~qn~ntc For S~ndnrd P~sbn Su~Tnsk AccompbslYd Solenoid ~ (+ = i~trf~ktd) S 00~0~ 1 2 3 ~ S 6 7 ~ 9 10 11 12 1 Ini~bl rccircublion lo Rl - - - -- - - - +
2 Rl gndkm S~mc es l, bm aCliV~lC SRI
3 From R2 jusl lo Fl, no rccircublion-- + - - - - - - +
~1 dcih~cJR2 fNs~ soh~lon Ihrough F2, + - + + - - +
nù rtcirrul-tion S Rccircula~c R2 solulion c~rspt + - - + + - - + - +
from orgAn ~0 6 Rccircub~c ~11 R2 solulion + - - + + - - - - + - - ~~
7 Run ~ 8ndkm from rcscrvoir R2~S-mc ~s 6, bul ncllnlc SR2 8 Fcrfusc from R3 3usl lo S6/F2-~ - - + + + - - + - - - - I ~~

9 Pcrfusc from R3 lo F3 circuh - - + + + + + + +
opcn 10 Rcc;rcubsc ~o R3 Umou-h F3 circuil - - + + + + + + +
p~nblly opcn Il. Rccirculllc sll R3 ûurd + + + + + +
12 RunrusZpnnofR33r-dkm Samelsll,bul-c~iv-lcSR31 13 Runsccondp~rlR3gndknl--- S~me~s lI,plusSR31 ~ndSR32 14 Opcn circuL pcrfuse frorn R4 - + + + + + + +
~hrough F3 15 Rccircublc lo R4 c~ccp~ from - + + + + + + + - +
org~n 16 Rccircublc from bmh loop- to R~ + + + + + + +

11 Run R4 ~ndicnl S~mc Is 16, bm ~c~ivcrc SRd ' '-' '"~'~ '' .
C
' ~ b -, ~ -' ' ~

' ' ' Y
. ~

~ ~777~

~6-The number of reservoirs could be less than or greater than the number specified here, with cullu~onding changes in soienoid number. Fu~ u~o~e, the number of layers of R1-R4 need not conform to the descriptions given above. The limits would be one reservoir at the least and perhaps eight S reservoirs at the maximum, in which any reservoir could have from one to four ~olul~alLlll~ The upper limits are based partly on volume and crowding constraints and partly on the improbability of any procedure complex enough to require more than 8 reservoirs for its control.
Another variation would be to employ different capacity reservoirs at different positions (e.g., instead of the herein preferred embodiment, one might have a 2-liter reservoir followed by a one-liter reservoir followed by a 3-liter reservoir followed by a one-liter reservoir, and so on).
In principle, the use of individual reservoirs could be abandoned in favor of one mnl~ lllpA 1 Ull~ reservoir consisting of perhaps four to twenty concentric cylinders each activated by solenoids or even by manual levers external to the ltu~ ..ltul~-controlled area, all stirred by a single central stir table. Abrupt or step changes in fO~.. . ,In,.~ l could still be ~ ."""o.l ~- d if the stepped change is not delivered via the stirred central area. The relative positions of the reservoirs could also change.
Finally, a fiuid metering system could be employed rather than a gradient former. In this system, a pump would deliver ~,,.. u,.r, d Wyu~ ,t~lll or diluent to a mixing reservoir rather than relying on gravity.
This pump would be computer operated to adjust for departures from the pl~ UlllllC;i cnnrrn-r~ion The gradient pump, however, would be retained in order to control overall circuit volume.
The arterial conro.ntr~ion sensor could be located proximal to, rather than distal to, the origin of the organ loop in the circuit, but should not be located proximal to the filters.
A pressure sensor to sense pressure developing on the circuit pump side of the filters could be illUOlU'~ ' ~ as a warning device.

WO 96/05727 PCr/USsS110223 More generally, the device could be separated into two devices, the frst for preparing organs for clyu~urc~clvlltion and the second for preparing previously cryu~ulc~cl~v organs for transplantation. The first device would omit R3 and R4 (and assûciated solenoids) while the second would omit Rl and R2 (and associated solenûids) while otherwise being sllhst~nti:~lly the sameas the unifed device. Given that cryopreservation and the recovery from l~yu~ v~:Lion may occur at different locations and under the direction of different individuals, this variation is likely to be of use under practical conditions. Essentially, these two devices would be identical except for the use of different software and the use of different reservoirs for adding and forremoving ~,lyu~lu~tcv~ . Another usage could involve the unorthodox use of only two reservoirs to :~nrornrlicll both loading and unloading; for example, loading could be done using Rl and R3 if only the ilmer cullll altlllc.lt of R3 were used (R3 standing in for R2), and unlûading could be done using Rl and R3 if R1 substituted for R4.

II. Descriphon of the Mefhods A. Preparing an Organ lior G~ c~c~ ~;vn and c~))seqrrPnt Tnnn~rl~ntnt~~n Into an Animal The complete cryulJ-c~t- V~tiU~I method using the above-described apparatus comprises several parts. One part consists of the plc~c~ of the donor animal and/or the organ prior to its remûval from the animal to prepare the organ for its Clyu~JIc~cl V~tiUII. Another part consists of the choice of the cryu~ulu~cc~ivc agents. Another part is the actual protocol for perfusing the ~,lyu~J~u~vlut into the organ prior to its cryopreservation. Another part is thec~yu~u-c~c~ v~tion~ storage and warming of the organ using ~J,ulu~ lte techniques none of which are part of this invention. Another part of this invention is the protocol for removing the clyu~ulute~llt(s) from the organ wo 96/05727 2 1 9 7 7 7 ~ PCI~/US9~110223 after its warming in preparation for trana~JldnLdLivl, into a recipient. Anotherpart is treatment of the organ and the recipient upon organ nr~ ion ut,.cnt of the Donor and fhe Donated Organ tn v~vo The donor, in addition to other standard treatments, received an infusion of iloprost (Berlex i_aboratories, Inc., Cedar Knolls, NJ) which is a relatively long-lived anaiog of prostacyclin (PGI~), or a similar agent, starting 10 to 20 min. before organ plu~u-~ul"nL. Applicants have found that iloprost was effective in reducing the apparent toxicity of subsequently-administered ulyu~luttl,L~ after either its intravenous infusion to the systemic circulation or its ~ on directly into the renal artery. The best mode dose of iloprost was about 25 ~lglkg given by either route, althougll di rect intra-arterial infusion is presently preferred to maximize organ exposure to the agent while l..h.;l..;,;.,g iloprost-mediated systemic hypotellsion. Fifteen ~ug/kg was aisoeffective, but was less effective than 25 ~g/kg. Acceptable limits of iloprost c~n~Pnrr~l inn for this appl ication are 5-75 ~lg/kg, dependi ng on species, organ, infusion rate, duration of infusion, etc. Iloprost was typically infused over the course of 20 min; acceptable infusion duration limits are 1-60 min for cadaveric organ donors. When hypotcnsioll is a limiting factor, iloprost may be infused at relatively low rcmrPn~r~tinn over a relatively long time (20-60 min). While not wishing to be bound by any particular theory, iloprost's protective action may not be a direct ~;ylvplu~l,LiVc effect. The h.~rre~ ."ci,aof iloprost in protecting kidney slices from cryu~,lv;~L~ -induced injury suggests that iloprost may simply act as a powerful vasodilator that facilitatesuniform ~,lyu~JIu.~,~,L~lu~ distribution. Therefore, other ~cav iil~Lv~a such asacetylcholine, ui ~IVlJl u~a; it~, nitric oxide, hypertonic and/or hy~, uncu~ic flush solutions etc., may be substituted for it at doses whicl- produce sufficient vasodilation in the organ of interest.
An important option for optimizing results was organ pl~l tcLI"cl,l with Ll.luarul~h~g growth factor beta I (TGF,BI), which prevented (iP~ hmPn~ of 2 ~ 97773 wo 9610s727 PcrluS9S/10223 cultured endothelial cells from their substratum in vitro during ~upe.ru~;ol.
with 52% w/v ~,lyu~lut~Lallt, when added to the culture medium at a conrentrRtion of 10 ng/ml about 24 hours prior to superfusion. The best mode use is to administer a bolus injection of TGF,BI of 0.1 ,ug to 10 ~g per kg, 2 to 4 hours before organ donation with or without additional injections at earlier times. The inventors found that giving 0.5 ~g/kg of human TGF,BI at 3, 16, and 20 hours before organ donation protected rabbit kidneys from a 40-50 min exposure to u M .,lyul~lute~Lallt, thus preventing the otherwise-expected hc,uo"l,,,~ that results from such exposure and allowing one animal (exposed for 50 min) to survive until sacrifced on day 15 postoperatively.
After pre-treatment in vivo, the organ of interest was flushed in situ with cold Euro Collins solution, modifled UW solution or a cu",~ l,ly effective solution in such a manner as to avoid conflicts in multiple organ plul,uielll~ The compositions of these solutions are contained in Table 3.
(Should normothermic preservation techniques supersede hypothermic lLiUII for hearts, the heart can be flushed with warm rather than cold solution.) The flushing solution(s) should initially contain iloprost (I ~g/ml in the best mode, acceptable iloprost Or",~ limits being 0-10 ~g/ml), Rn~ir~l~glllRnt~ (e.g., heparin, 10,000 units/liter in the present emhollim~nt acceptable heparin con~ntrR~ion variations being 500-20,000 units/liter), vasodilators (e.g., papaverine, 40-90 mg/liter in the best mode, 0-90 mg/liter as acceptable limits) and other desired agents. A second flushing solution should be used to wash out all of these agents as cooling and blood washout is completed. The excised organ (except for organs such as the heart that may be best maintained by ~u~u~olh~.ulic perfusion) should be transferred to an iced bath of flush solution and transported to a perfusion machine capable of hlLIudu~,;llg and removing cryu~luLa~Là~ in the fashion to be described.

2 1 ~;~77~
WO 96/0~727 PCT/US9~/10223 Table 3 Composihons of Perfusion Soluhons Euro-Collins*
Compound mM g/l Dextrose 194 34.96 KH2PO4 15 2.06 K2HPO4 42 7.40 KCI 15 1.12 NaHCO3 10 0.84 * pH = 7.4 * milliosmolality = 350-365 milli~m Compound mM g/l Dextrose 180 32.43 K2HPO~ 7.2 1.25 v~l.0 ,. ....
1~,1' O. ' ' . 1 1 NaHCO3 10 0.84 Giutatllione 5 1.53 Adenine HCI I 0.17 CaC12 1 0.111 MgCI2 2 0.407 (Note: RPS-2= solution is RPS-2 without CaC12, and also without MgCI2) 2 1 97~73 wo 9610s727 Pcr/uSss/l0223 7:eble 3 (conl.J

Modified UW Solution #I Modified UW Solution ,Y2 Compound mM g/l Compound mM g/l NaH2PO~ H2o 25 3.45 NaH2PO4 H2O 25 3.45 K gluconate 100 23.42 K gluconate 100 23.42 Mg gluconate 1 0.21 Mg gluconate 1 0.21 glucose 5 0.90 glucose 15 2.70 glutathione 3 0.92 glutatllione 3 0.92 adenosine 5 1.34 adenosine 5 1.34 HEPES 10 2.38 HEPES 10 2.38 adenine 1 0.17 adenine 1 0.17 (hydrochloride) (hydrocllloride) ribose 1 0.15 ribose 1 0.15 CaCI2 0.05 0.0056 CaCI2 0.05 0.0056 HES(g) -- 50 -----(Note: Modified UW Solution #2 does not contain HES but contains more glucose than modified UW Solution #I) 2. C~yoprofective Agents: Formulae of the Vitrifcahon Solllhons V49, VS2, VSS, V49B and VSSB
All perfusion ~ h~ a were carried out using solutions designated here by V49, V52 and V55 (V49 has sometimes been referred to as VS4.
V55 has been referred to as VS41 A.) . At low cooling rates (5- 10~C/min) V49 ~ was found to vitrify at 1,000 atm of applied llydrostatic pressure but not at ordinary ambient pressures. V52 was inferred to vitrify at 500 a~lllOa~ c~
(atm) of applied pressure. V55 was found to vitrify at I atm.
V49 was composed of dimetbyl sulfoxide (D), formamide (F), and 1,2-propanediol (P) such that the mole ratio of D to F was 1: 1, the total mass 2 1 q7773 ~0 96/0~727 PCI'IIJS9~110223 of D+F+P per liter was 490 grams, and the total mass of P per liter was IS0 grams. Thus, per liter, D + F = 340 grams, F = 124.33 grams, and D = 215.67 grams. This mixture of ~,~yOIulu~,L~llLa was preferred based on the results described below. Acceptable variations for the p~UIJ()ltiUlls of D, S F, and P are: D:F weight ratio can be as low as 1.4 and as high as 3.5; for the former, the proportion of P:(D + F) should be elevated to 18:34 and/or the total concentration raised to S0-SI % w/v (grams/deciliter) by the addition of extra P.
The formula for VS2 was obtahled by multiplying the ~,-yu~J-u.~ rlt content of V49 by 52/49, keeping the vehicle solution the same as for V49.
The formula for VSS was obLained by multiplying the cryul -utc~l~rll content of V49 by SS/49, keeping the vehicle solution the same as for V49. Thus, the total ~ ;OII of solutes in VSS was SS0 grams/liter vs. the 490 grams/liter of V49. V49B was a variation of V49 in which the 1,2-lS propanediol content was replaced gram for gram by 2,3-butanediol )1. y form or racemic mixture with less thall 5% w/v meso form present), and VSSB was, similarly, a variation of VS5 in which 2,3-butanediol replaced the 1,2-propanediol gram for gram. The total ~yul~uL~ln molarities of V49, VS2 and VSS were 7.49, 7.95 and 8.41M, Ita~J~Li~
The molarities of V49B and VSSB were slightly lower than those of V49 and VS5 due to the greater molecular weight of butanediol vs. I.lu~Jdll~,diol.
While not wishing to be bound by any theory, V49 and VSS appear to be particularly beneficial due to the exceptional ability of formamide to penetrate kidney tissue, the ability of dimethyl sulfoxide to block the toxicityof formamide, the beneficial balance between the three ingredients (IllAl;llli~;llg vitrification tendency while 1";~ g both toxicity and total solute .r"....alAIir,l.), the lack of a coiloid (typical colloid rl~,....lnAl;ulls of about 4-7% w/v elevate Yiscosity), the extraordinarily slow rate of d~,~iLiirl~Liun of these solutions at appropriate pressures (1,000 atm and I
atm, respectively), and the good stability of VS5 at -135~C during at least 6 months of storage.

, . . . . . .. . . .. . ... . . . . ... .. . .

~ WO 96/05727 2 1 9 7 7 7 3 PCrNS95/10223 The cryoprotectanLs used for organ perfusion were adjusted between the limits represented by V49 and V55, dependillg upon the pressure to which the organ was to be subjected. Balancing an organ's tolerance to high pressures and its tolerance to high wyupl-Jt~,.,Lalll ronrenrt~tinnc allowed u~JLh~ aLion of the tradeoff between pressure and corr~n~r~tinn required to maintain vitrifiability. For example, an organ that cannot tolerate l,000 atm but that can tolerate 500 atm may be perfused witll V52. Conrent~ir,nc in excess of 550 grams/liter, to a maximum of about 600 grams/liter, may be used when ~,t~,lu~,.,ncoua nucleation on cooling is a significant probleml since the nucleation process and the growth of any nuclcated ice crystals will be suppressed at these higher cOlluc.lildLiOlls. One example of a situation in which this problem will arise is the ~h-irll aiiOII of very large organs such asthe human liver that will cool palLiuulally slowly. At elevated pressures, similar proponional increases in solute concentration will be required as the cooling rate is lowered.
E~p.,ril~ .La (see results below) with kidney slices indicated that V49B
provided viability identical to the viability obtained with V49. V49B may have greater stability than V49. Variations between V49B and V55B are to be used as per the d.,~ Lions above for V49 and V55.
All Clyu~JIutC.,61lL solutions must contain, in addition to the clyu~nlut~llLathemseives~slowly-penetrdtingsolutescomprisingthe "carrier~
or Uvehicle" solution for the cryuuruLecLdllLs. Typical examples would be modified UW solutions, Euro Collins solution, or Renal Plcacl ~aLiull Solution 2 (RPS-2) (see Table 3). The best mode method used Euro Collins as the vehicie solution of choice for kidneys, modified UW solution (âS per Table 3) as the vehicle solution of choice for the liver, and cu~ ll.,lc;dl UW solution (Viaspan~ (E.l. DuPont and Nemours) as the vehicle solution of choice for hearts.

w096/05727 ~Iq 7~73 ~ J/~

3. Protocolfor Perfus~ng tlze Olgan witl~ Cryoprotectant Typical protocois forcryul"u;~cLd~ll introduction and removal thatwere shown to yield reliable, higlI-quality survival of rabbit kidneys after ~Iyu,ulu;~,~,LdllL washout, trAn~rl~r,r~ion, and long-term functional and hi~r~ ginnl follow-up, are shown h~ Figures S and 6 and are additionally described in the flow charts of Figures 7A-E. As designated in Figure 5, the protocols were divided into at least 7 discrete phases. Phase I was an oq~ ihr~ n period during which the organ established stable baseline ~u~,,,, r~ prior to the introduction of cryuL"utc,,La.,i. Phase 2 was a gradual increase in ciyùlJlutc~,Ldl~ un~c~lLI dlioll that ended in a f u~ ~ . r,n " l ;. ."
plateau known as phase 3. After spending a certain amount of time in phase 3, during which time the A-V cryu~lotc~,Ld"L ~onr~nrr~tiorl gradient usually became ~ 'y zero, the ~nnf ntr: tinn was stepped to a new plateau, this. new plateau phase being phase 4. As described in more detail below, phase 4 need not be the highest .. ,., ~ 1n attained. In Figure 6, for example, the phase 4 concentration is 6.7 M, but the final uu,l. c.~l~dIiun in the ~-~pf~rimrnr of Figure 6 was actually 8.4 M. Whatever the final ~ ~n~ ~ .,n ,.li~""
the first washout step is indicated in Figure 5 as phase 5, another ~J~ l n al iull plateau. Phase 6 is the C~yupluIt~ washûut phase and phase 7 is a pOSt~ yu~lut~ll~ c~illilihr:ltil~n phase.
a) Perfusion pressure: The organ was perfused at pressures sufficient to overcome the organ's critical closing pressure but otherwise low enough to avoid needless damage to the vascular tree. For example, the best mode perfusion pressure for the rabbit kidney was 40 mm Hg without significant pulsation. A desirable range of acceptable pressures has been found to be 20-70 mm Hg for different organs and species, including man, except for the liver. The liver normally receives most of its fiow through a vein at a pressure typically below 10 mm Hg. Rat livers perfused at S mm Hg were able to achieve d,Upll ' ' osmotic e~uilii,,dtiul, after perfusion with V49 for 20 min when no colloid was present, and half of these livers supported life after transplantation. L~cn~l~fillf ntly, the pressure limits for .. _ . _ _ _ ~ .. . ..

21 q7773 ~, WO 96105727 Pcrlusssllo223 _5~

livers are 540 mm Hg througll the porLal vein, and 5-70 mm Hg through the hepatic artery.
b) Ini~ial pe~usion (phase 1): In the best mode protocol, perfusion was first carried out for 15 min to establish baseline values for vascular resistance and f~lihr~tionc (for pressure and refractive index); to ensure complete blood washout; and to thermally equilibrate the organ, here the rabbit kidney or the rat liver. Clinically, the initial perfusion time is arbitrary, and can be adjusted (from zero minutes to 1-2 days or more) to meet the l~u,uhc.ll~,llLs of the organ l)luuul~llu,.lL and Lldlla,uolLdLion process. In Applicants' laboratory, the perfusate in this period was Euro Collins solution or RPS-2 for kidneys and a modified UW solution for livers. However, this initial perfusate could also be another stabilizing solution in a clinical setting depending upon the needs of the hospital or ~"uuu~"~n~ team.
cJ Initial tc,,.~, ,..tu~c: The initial perfusiûn t~,UI,U.,ldLul~
required for the ,ulu~,ul~lll.,.lL and ndlla~)u~LaLioll of an ûrgan~ such as, fûr example, the kidney, need not be identical to the perfusion t~ ,ufldiule established during phase 1. For example, while most organs may be shipped while surrounded by crushed ice at 0~C, other organs may be shipped while being perfused at nulllluLll~,llllid (37~C). When organs are ready for ~,lyu~luL~,LdllL a~h";,~ ~UAl;UI1~ however, a preselected, aLa.lddd. ii~i perfusion t~,ul~ldLult is established. In the best mode, the initial perfusiûn t~,lU~ u~
was 3.54~C, and the acceptable limits were 0-15~C. The inventûrs consider that ûrgans requiring ~u~wLl.~llllic perfusiûn for best long-term Ill_hlt~
can n~ ,Ll.cle~ be cooled to within this same t~,lul.~,.dLul~ range and can be treated in a manner similar to that of hy~,uLl.. ,.,l,icdlly-preserved organs withûut damage within the relatively short times required for this method.
d) Phase 2: Following the initial baseline perfusion, ~lyul~lut~L~ conrPrtr~tinn was elevated at a constant rate until a first plateauof co ~ ;n" was established. When using a V49-type mixture of ~,lyuuluLtl,LdllL~, the ~ul~ulLioll~ of different ~,lyu~olut~,LIlL~ in the mixture were held constant while the total ~;uuuellLldLiull was allowed to change. The WO 96/05727 2 I q 7 ~ ~ 3 PCT/US95110223 rate of increase in total ron~ rnttQtirlI for V49-type solutions was set to about 5I mM/min (nominally 3 M/hr) in the best mode for the kidney, acceptable variations being 31-150 mM/min. These rates were considerably in excess of the 30 mM/min rates used by known techniques for glycerol and propylene S glycol which were considered to be unnecessarily and undesirably slow for most a,lplirQ~ionc of the method. Linear elevation of concentration promoted eq~'ilihrQtion without creating unnecessarily large osmotic stresses.
e) Temperature reduction during plz~zse 2: The tC.~ d~ulc was lowered during phase 2 to protect the kidney from the chemical toxicity of the cryululutc~r~ In the best mode, the temperature reduction began as the arterial Wyu~JIut~ dll~ concentration reached 1.3 M; acceptable limits are 0.5 M to 3.5 M. Temperature descent was termhlated as phase 3 was reached. The cu~ ldLioll change during coolil1g was about 2.5 M in the best mode but may vary from about I M to 4.4 M.
As noted above, the initial perfusion teu.~,~,.d~ul~ should be between 0~C and 15~C. The le.lll./~,.~l~UI~ after cooling shouid fall within the range of -13~C to +5~C and the total temperature drop during cooling should be between 2~C and 25~C. Cooling should not continue to below the freezing point of the organ. In the best mode, the final arterial telll~ d~UlC WdS -3~C, IC~)IC .~ iilg a fall of 6.5 ~C from the initial oemperature and a cooling r_te of about 0.33~C/min. The overall cooling rate sllould not exceed 3~C/min in order to provide adequate opportunity for cryul ~ut~,.ldul diffusion and in order to avoid possible thermal shock to the organ.
,~ Phase 3: The phase 3 plateau was set in the best mode for the kidney at 25 % w/v total ~,lyulJIultcldlll (250 grams/liter, or about 3.8 M)when 4û49% w/v cryu~"utt.,Ld"l was to follow, or 3û~o wlv, (4.6 M) when higher concentrations (e.g., V55) were to follow, acceptable variations being 2û40% w/v or w/w. The phase 3 plateau was set to a level that was close to half of the phase 4 C~'rf .,n,,lil." Lower phase 3 levels will increase osmotic 3û stress upon moving to phase 4, whereas substantially higher phase 3 levels will produce increased toxicity due to longer exposure times to ~u~e.,l,.t~.~

2 ~ 97773 =
wo 96/n5727 PcT/usssllo223 ~,~yutJlu;ectdnL The duration of phase 3 was set lo about 10 min in the best mode procedure, acceptable variations being 5-30 min, dependi ng on perfusion pressure (and lhus organ flow rate), vascular resistance, organ permcability to ~lyu~Jlute~Ld~ and the rapidity of toxic responses. The duration was long enough to allow the organ to at least dl.,.,.u,.i,.,a~ely osmotically equilibrate with the arterial perfusate, as indicated by an arteriovenous ronrenrr~liorl difference no greater than 50-200 M7 so as to minimize uullf.,~dly osmotic stress during the subsequent jump to higher CrJ~/rf ~mnI jO~
g) Perfusion wilh vitrification solution by a one-step, two-step or Illree-step method: A step change in conrPnrrAîioll from phase 3 to phase 4 was necessary to control the exposure time to higllly conr~ - ' ~,lyulJIulc.,Ld-l~. The phase 4 ~ u~ .,nA~ n may be sufrcient for vitrification (a one-step introduction method) or it may be hlsuffcient for ~d~ir~,d~iu~
(requiring one or two additional steps to achieve vitrifiability).

The concept behind the two- (and the three-) step approach is illustrated ~rhPnnA~irAIIy in Figure 8. In the "one-step" approach, all of the c.yùlJIuL~l,L~llL was added in one continuous process (Cl), and cooling to cryogenic t~ tJ~,IdLu~ then occurred in one step (Tl) as well. In the "two-step" approach, part of the cryu~.u;~Ld..L was added in the first step (Cl), andthe rest of the ~,lyu~lut~L~Illt was added in a second step (C2) carried out at tC.Il~!.,.d~ulu;~ near the freezing point of the solution used in the first step. In this approach. cooling also took place in two steps, the first step (Tl) having been used to prepare for the second cu~lr ..n..liu~ increment (C2), and the second step (T2) being used to cool the organ to cryogenic t~,.n~ Lu~c~. In practice, the hrst cooling step was preferably to tcn~ ,.dLulc~ somewhat above the nominal freczing points to guarantee the avoidance of crystallization prior to i-ltluducill~ higher conrPnrrAtion~ of clyulJIuLcl,~nt.

In the best mode, the phase 4 conrpn~rAlion was set to 40% (6.1 M) to 44% (6.7 M) w/v V49 solutes, a concentration that was not sufficient for WO 96/05727 PCT/US9~/102Z3 v;llirl~Liu.~ gure6). Acceptablevariationsforsub-vitrifiable~u,..r"l"u;.~,.c are 307O w/v to 48% w/v V49 solutes or their equivalent. For the one-step ill~lUdUl,~iUII, the phase 4 ~ ..nAIioll may range from 480-600 grams/liter (about 7.4-9.2 M) for V49- or V49B-type solutions (for example, see Figure 5). For non-V49/V49B type solutions, the method limits for phase 4 are 35 %-60% w/v .,~yu,u~uLe0Ld~
Phase 4 conrf~ntr~ was held steady for 20 min in the best mode, acceptable variations being 10-60 min. The con~Pnlr~ticln should be held steady long enough for the organ to closely approach osmotic equilibrium with the perfusate according to the above-described criterion.
For the two step approach, the organ was removed from the perfusion machine after the completion of phase 4, and was cooled by being placed into precooled vi~lirl,dliun solution for 5-30 min (5 min in the preferred mode for rabbit kidneys, longer for more massive organs) prior to being perfused with the ~;L-iru,d~ioll solution. In the best mode, the organs were cooled toward and ~ ly perfused at a te.~ dLul~ of -22:~2~C (if previously perfusedwith6.1 McryuL~u~euLc.~l)or-25~~2~C(ifpreviouslyperfusedwith 6.7 M cryu~lu~ The L~,.--,u..dLulc chosen at this step will be referred to as the "low t~ul~,.dLul~ perfusion Lel..p~,.dtl..e.'l More generally, the low~Ill,u.,ldLul~; perfusion 1l .,.p. .,UIllr may range from -5~C to -35~C.
One ~.,, ,l ,o~ of the apparatus used for perfusi ng organs at the low-telll,u~,.dLul~; perfusion tulll,u.ldLul~ (to accomplish step C2 in Figure 8) isillustrated in Figure 9. In another 1 ..lholl;~ , the cooling and low-t~ll,u~dLul~ perfusion are carried out inside the primary perfusion machine without substantial operator hlv .i.llliùll.
The inventors have perfused rabbit kidneys with V52 at -22~C or with V55 at -25~C at a perfusion pressure fluctuating between 20 and 40 mm Hg but usually not exceeding 30 mm Hg, having obtained excellent results after subsequent 1l,.l.~l.1 l~ul) ~ Acceptable method limits for perfusion pressure range from 50% to 150% of the previous pressure in the perfusion apparatus 2 ~ 3 wo 96105727 PCr/USss/10223 for organs other than the liver, or from 50% to 400% of the previous perfusion pressure in the case of the liver.
The time required for equi libration with vitrifiable concentrations at the low-lc..~ .dLulc perfusion tclupFIALu~c was determined in the case of the kidney by collecting "urine" produced during the low t~ dLUIc perfusion and dc~ its osmolality after sui6ble dilution. The kidney was deemed to have been PqllilihrA~P~I when the osmolality of the urine dp~,-ua~,l,ed the osmolality of the arterial perfusate. For other organs, the extent of eqllilihrA~inn is determined as usual by the dlh~.iU~IIUUs ro", F..u,.l;.J"
difference. Accep6ble equilibration times were ~iFtrrminrd to range from about 20 to about 60 minutes.
Another embudi~ L that will apply to organs which cannot tolerate exposure to fully vitrifiable solutions at the low t~ IàLulc perfusion te.-lpF-dtulr, is the three-step hlLIu~lu~,liull method. These organs may be successfully Llyupl~.s~,.ved by perfusing a less-than-fully-vitrifiable wllLF.ILldLioll at the low-tcul~dLulc perfusion iulll~h,ldLulF; (step two), which concentration, being higher than the LU~ ,nA~ n used prior to cooling to the IU.. t~ultJCIdtu~ci perfusion tclup~,.dLulc, will depress the freezing point of the organ to snhst~nti-AIly (i.e., 3 to 20~C) below the lu.. t~,ultJFI~Lu~c perfusion t~ ,-dLulr The organ can then be perfused with fully vitrifiable conrFntr~til~n~ near the new organ freezing point telllp.,.d~ulc (step three), at which ~r~ ,.aLulc the fully vitrifiable . V.~ tio..~ will be ~u-rlLi~,lllly non-toxic as to be tolerated. This l -I,o~ r- l will apply also to organs that require it for avoiding cooling injury.
h) Ratlonale for the two-step best mode method:
While not wishing to be bound by any theory, the main rationale for the best mode two-step method was the avoidance of cooling injury.
IIlLludu~,h~g LlyuplotccLdll~ at the low-LF;Ill~J.,-dLulc perfusion t~..Up~..dLulc was hy~)uLll~,~;~d to reduce wyuplutc~,6~1l toxicity as well.
The inventors discovered that kidneys perfused at -3~C with V49 survived 100% of the time (14 survivors out of 14 perfusions) but when they . .

wo 96/05727 2 1 ~ 7 7 ~ 3 PCT/US95/10223 ~

were cooled to -30~C, wanned and washed out using the best techniques known at the time, the survival rate fell cllh~-~nti-Ally (see Figure 13). Kidneys perfused with V52 at -3~C using the optimal techniques of the time survived 75% of the time, but when these kidneys were cooled to -30~C, the survival rate upon warming and washout was 0%. Thus, cooling caused injury at 49%
w/v ~,lyu~lutecLdnt and caused complete loss of viability at 52% w/v yuylut~ L. Since, ideally, organs should be preserved in V55 (to avoid the need for high p}essures), this trend was uu~avvlàl)lc. However, a positive implir~Ation was that cooling injury might become negligible at . u~ AIill 1~
lower than 49%, so that cooling to t~,.UIJ~,.dLulc~ near -30 C might then be innocuous. This suggested the possibility of cooiing at a relatively low t~ r. IuAliml so as to avoid cooling injury and then raising the rr~nr~nrr~tir~mto a vitrifiable level at the lower te.llpcldLulc. Tbis approach would have the additional advantage of exposing the organ to vitrificatiûn solution at a Ltlll~ lLU~ at which its toxicity should be reduced. Thus, by avoiding cooling injury, toxicity might also be avoided.
A secondary point was that a variety of ~ .h~ lL on the pl,r ~ , of thermal shock in both c.y~lllu~ ts and kidney slices suggested that cooling injury below -30~C might be minimal even in the presenoe of V55 if cooling injury abûve -30~C were first prevented. Therefore, by first cooling to near -30~C in the presence of a r~v . .~nnliull that does not cause cooling injury, it was inferred that even V55 might not cause fatal cooling injury when the organ was loaded with V55 at the lu.. ~I~ d~Ul~ perfusion t,lll,u~,ldLul~ and was ~,,1,~,1. . .,lly cooled to below-30~C.
As noted in the preceding section, the first hypothesis was verified in that the two-step approach successfully avoided cooling injury and the toxicity of V55 at -25~C. As noted in the results section, the second hypothesis was also verified in that fatal injury did not occur upon further cooling to below ~6~C was also avoided.

21 ~7773 wo s610s727 Pf~r/Usss/10223 4. Cryol"asc/~ ;ol~ of the Organ The next step of any practical cryopreservation procedure, such as ir~ion, is to cool tl-e organ to cryogenic t~ JCIdLulcs using c~,u~up~
protocols1withorwithoutpriorplc~aLlli~dtiull. TilecryuL~lc~cl~Liollstepalso includes the storage of the organ. The present invention is not concerned with the actual ulyu~un~cl~ tion and storage of the organ, but only with the preparation of the organ for cryopreservation and the preparation of the previously-cryopreserved organ for tr~ncrl~n~-irn 5. Perfusion of fhe Organ in Preparat~on for i,fs Trl n~rl(~nfrt;~i In ,UIClJdldLiull for transplanting it, the organ is first warmed up from the storage t~ p.,ldLu.~ to an dl~,uru,ulidlc Lcllli-.,.dLulc for lc,u~lfLI~iull of the organ. The warming of the organ after its cryopreservation is presently not performed in the apparatus of this invention. The organ may then be piaced back into the perfusion apparatus of this invention to resume the type of perfusion protocol shown in Figures 5 and 6 at the beginning of phase 5 (the first cryul~luLe-,~d-lL washout platcau).
a) TL~..IJC~U~UIC during pl~ase 5: In the best mode method, the organ was warmed to a~J,Luw;illldt~,ly -3.0~C and placed into the perfusion apparatus to begin cryu,urutccLdllL washout at this te.. liJ-,ldLulc. The inventors l-nP~rP,rrPrlly found that this approach was superior when the two-step best mode method for introducing cryuylulc~,LdllL was used and was successful even when the final ~dLIirh,dtiull solution used was VS5. Given that the introductionof vitrifiable cr~nrPn~rAlionc was possible at Lc...~J~.IdLul~ near -25~C, the inventors had expected that it would be advantageous as well to remove part of the .,lyu~JIu~LdllL at this Lc"-l.crdtu.c in order to avoid the expected hightoxicity of fully vitrifiable concentrations at temperatures near -3~C. Instead,the dilution of vitrification s,olution at the low temperature perfusion l~.nl~..,,m~ was found to be detrimental. Withill the method limits, the t~,lu~,ldLul~; during phase 5 can range from -2û~C to +5~C.

.. . ... . . ... . .... ... ... .. _ . _ .. _ . . .......... ..... ...... . .. _ . .

2 1 ~77~
WO 96/05727 PCT/US95/lOZ23 b) Clyoprotectant concentration and duration of phase 5:
The rnnrPnlr~rion of ~,lyu~luic~LdllL during phase S in the best mode proLocol for the kidney and liver was 30% w/v (300 gramslliter; 4.6 M) to 33% w/v V49 solutes (D, F, and P in the usual proportions), acceptable variations being 20-40 % (w/v or w/w) ~,lyu~!luL~LdllL (roughly 3 to 6.0 M) . The ~ un~ f ~ (Jl lat this stage should not be less than 40% (2/5) of the ~:un~inildLiull of the ~iLIificdLiun solution in order to avoid osmotic damage; in the best mode, the conrrntr~linn at phase 5 was 3/5 of the highest collrPn~r~linn perfused.
The criterion for tc.lllhlaLilly, phase 5 and moving on to phase 6 was somewhat different from that previously employed. It was found that prolonged periods at phase S sometimes led to changes suggestive of cellular uptake of the LMW OBA that was generally present during this plateau and that should remain r~rArrlhll:~r for l":~h,l~ ,g the viability of the organ. It was '~ ly determined that the duration of phase 5 should be limited to what is required to allow the A-V concentration difference to begin to return to zero (in tlle inventors' experience, to return from an off-scale value to a value near -50 mM), rather than prolonged to the point that the A-V
c u, 1~ ~, .n ~n inn difference is no longer rapidly cllanging~ Note the shorter phase S time in Figure 6 as compared to thât in Figure 5, reflecting the ulJLillli~dLion required for success at the higher ~;o~ .,nrliu ~ used ill the protocol reflected in Figure 6. Note also the abrupt end to the recovery of the A-V
r~ ".li..ll gradient in Figure 6 as contrasted with the prolonged c-qnilihr~rinn of A-V cu~ n,uiull during phase S in Figure 5. For the rabbit kidney, the optimal time was determined to be 9 min. Within the method limits, durations of 0-30 min are acceptable.
c) OBAs and their use during phase 5: One or more OBAs (defined as above) were generally present during phase 5.
As previously defined, one way to categorize OBAs, for ease of discussion, is as LMW (Mr between 100 and lû00 daltons~ OBAs and HMW
(Mr between 1000 and 500,000 daltons) OBAs. However, there is in fact no sharp dividing line between LMW and HMW OBAs, and different Mr ranges Wo 96/05727 PcrlUS95/1/)Z23 have uniquely differen~ properties, and hel1ce different practical applications.Some of these key properties, whiclI give rise to the broader prh1ciples behind the usages described below, can be ~uuuurlli~d as follows:

Mr Range Membrane Osmotic Oncotic Viscosity Cost Permeability Effect Effect 180-342 Highest Highest Nil Lowest Lowest 343-1,000 Low to High- Nil Mod. Mod.-Mod. Mod. High 1,000- Low to Nil Low Nil- Mod.- Mod.
50,000 Low High >50,0000 Nil Lowest Low- Highest Mod.
High The inventors have ~ PYperri~ly discovered several new modes of OBA usage. For application at phases 5 and 6, illese new modes consist of i) combined LMW and HMW OBAs (for use with the highest-ron~ntr~ion protocols), ii) single midrange OBAs (for high and moderate-rl-nrentr~ion protocols), iii) very LMW OBAs (for lower-ronr~nt~tion protocols), and iv) specific OBA protocols for the liver. In this section, these usages and the principles on which they depend are discussed generally without reference to phase 6.

i) Combined LMW a~d HMW OBAs. For the kidney and most other organs, the best mode OBA usage was considered to be sucrose, for example about 300-350 mM, or other LMW OBA in rc.nlhin~tion with hydluA~,Lllyl starch (HES: relative molecular mass (Mr) of 20-500 kd (20,000-500,000 daltons)), for example 3-8% w/v, or other equivalent HMW OBA.
Two specific experimental examples illustrated below whicll yielded good results after perfusion with the Iwllllubdric vitrification solution V55 involved the use of 350 mM sucrose in romhin:~tirn with 3 % w/v HES of Mr 450 kd.
Other preferred LMW OBAs include maltose, rafhnose, potassium and sodium 21 q717~3 fructose 1,6-~lirhrsr~ P~ potassium and sodium l~rtrbir,n:~rP~ potassium and sodium glycerophospllate, potassium and sodium gluconate, m~itr.~rincP
rn~ltrlrentrlsp~ stachyose and mamlitol The preferred HMW OBA, HES, is sold by McGaw Corp. of Irvine, CA as a 200 or 450 kd chain, but is easily hydrolyzed to lower molecular weight forms. Particularly preferred are HES
molecular weights in the I to 100 kd range. Other preferred HMW OBAs include polyvinylpyrrolidone (PVP), potassium rafhnose 1."~
(available from Sigma Chemical Co., St. Louis, IL) and Ficoll (I to 100 kd).
The presence of a LMW OBAis required to counteract the otherwise fatal osmotic effects of a large stepwise drop in penetratillg ~,lyuplute.,~
ronrPntr Irion In protocol variations employing larger drops i n ~,lyupl u~
conrPntr~ir~n (e.g., lesser phase 5 crnrentr~tionc near 20% w/v yuplu~,lan~), more LMW OBAis required (to an upper limit of 750 mM).
In variations employing higher phase 5 ronrentrlltiorl~ (e.g., 40% w/v ~,lyupluLt~ ), less LMW OBAis required (to a lower limit of about 150 mM).
This best mode use of OBAsdurhlgtlle first ~,lyOpluv~ IL washout plateau (phase 5) applies particularly to protocols employing more than 7.5 M
V49 solutes, i.e., to protocols employing less than 500-1,000 .~llloa~Jlle.~
(atm) ûf hydrostatic pressure for vitrification. Exclusive use of the LMW
OBAs mannitol and sucrose were found by the inventors to be compatible with at best only a 30% kidney survival rate (2 survivors of 7 SO treated) when V52 was used in place of V49, vs. a 100% survival rate (14/14) when V49 was used. However, adding 3% w/v 450 kd HES during washout of the cryupllJt~Ldll~ raised survival to 75% when either mannitol or sucrose was used as the LMW OBA (6 survivors out of 8 kidneys treated) when the one-step ~d~lifi~ion solution addition method was used.
The concept of using HMW agents as OBs had not previously been contemplated, at least in part, because such agents have little osmotic effect in l;ulnpdliaull to lower molecular weight crmrol~n~i~ While not wishing to Wo 96/05727 Pcrlusssllo223 be bound by any specific theory, the following concide~tiolls led the inventorS
to use HES as a prototypical HMW OBA.
(a) In the presence of high concel1tratiolls of Cryul,luL~uL~,l, the ronn~nrr~tinn of HMW material was higller with respect to water than in the S case of traditional, dilute aqueous solutions. Therefore, the osmotic effect of the agent was enllanced.
(b) The oncotic function of a HMW agent could be crucial in protecting the vascular system from abrupt collapse upon sudden dilution of the cryu~l~Jt~,~,L.~ or could otherwise beneft tl~e vascular system.
(c) The HMW agent may reduce abnormal cellular uptake of LMW
OBAs by lowering interstitial volume (thus lowering the pool si~e of LMW
OBA available to penetrate cells) or by acting as a physical barrier to diffusion of LMW OBA to and/or through the cell membrane.
(d) The HMW OBA, by its oncotic action to dilate or prevent the collapse of the vascular Cu~.,Jdl Lll~ , should faci litate cryulJl u;~ L washout and thus reduce osmotic stress caused by lags in l,lyu~luL~L.IllL washout.
(e) Any abnormal increase in membrane permeability that may cause LMW OBAs to partly penetrate organ cells will not cause HMW OBAs to penetrate, thus the use of HMW OBAs will reduce the net amount of abnormal penetration per miliosmole of OBA that is used.
The best mode use of HMW OBAs was to use agents that have at least the osmotic or oncotic pressure of 3-6% 45û kd HES. However, lower M, agents than this may be better since a relative molecular mass of 50 to 200 kd should create equal or greater oncotic pressure and stili guarantee failure of the agent to penetrate a viable cell.
The .u~ on of HMW and LMW OBAs was preferred because the former offset the uptake of the latter and added to the latter's osmotic c .~ B,~,"c~, while LMW agents provided sufficient osmotic pressure to ~rromplich the primary job of preventing cellular water uptake during cryoprotectant dilution. In addition, the higil viscosity of HMW OBAs in 2 1 ~
Wo 96105727 PCr/US95/10223 wyu~uk~,Ldlll solutions supported the use of the less viscous LMW agents as the primary osmolytes to which HMW agents were added as adjuncts.
ii) Midrallge OBAs. For the kidney and most other organs, another preferred OBA usage method is the exclusive use of single OBAs in the molecular weight range of 360-lû,000 daltons, used at total I ~JIII . .lnAlirm~
of 2%-15% w/v. Examples of suitable OBAs in this application include maltose, raffinose,~Fotassium and sodium fructose 1,6-dilJllu~ d~e, potassium and sodium 1~ I(lb;~lllrtr, ~n~ltotrio~P, mAltrlpentrse~ stachyose, potassium raffinose ..,..1. ~ lm Ficoll and HES within the specified molecular weight range.
While not wishing to be bound by any theory, single agents in this weight range often adequately combine the properties of LMW and HMW
OBAs ineo a single agent. Osmolyte i",l,~.",~l,ility is the mûst important feature of an OBA and this i.,.l.~ u~ hility may approach a practically-relevantmaximum at molecular weights between 360-10,000 daltons or, more narrowly, of around 360-2,000 daltons. Solutes in this weight range are relatively osmotically effective while behlg also relatively low in viscosity and relatively high in solubility. This middle molecular weight range is therefore u~"l~ .,iy the ideal ûne when neither oncûtic eff~ "~ s nor cost is 2û critical. Some agents in this weight range will also be i.. ".~ ,al,lc to both kidneys and livers, thus eliminating at least part of the distinction between these organs.
(iii) Very LMW OBAs as tlle sole OBAs for "low"
concentration me~lzods. When the vilfirl~,dLion method was to involve the use of relatively low C~ r~ IAI;UI~C of clyu~ule~,L~L~ e.g., V4'~, the use of mannitol (M, = 180 daltons) as the sole OBA has yielded satisfactory results (see results section for pertinent data), and the low viscosities of mannitol solutions maintained better organ flOw than more viscous (higher Mr) solutions. Consequently, another P.mhorlimPnt of the best mode was the use of very LMW OBAs (OBAs with M, < 40û daltons) as the sole OBAs when wo 96/05727 2 1 9 7 7 7 3 PcrluS95/10223 vitrification methods are used thal employ elevated pressures and/or conren-rzi~ir,nc less than that of V52.
While not wishing lo be bound by any theory, lower cryoprotectant I~Jn~ . .,n,~ ."c were less stressful and mailltailled membrane permeability more effectively, and for this reason allowed lower M~ agents tO be effective.
Because mannitol was extremely inexpensive and universally non-toxic and because the cost of OBAs tends to rise sharply with Mr in the range from 180-2,000 or more daltons, mannitol and/or similar LMW OBAs (e.g., sucrose, maltose) will be the agents of choice hl these "low" rrnrPnrr~tior Pmhoniimf~nlc of the method.
(iv) OBA usage for flle liver. For the liver, the best mode OBA usage was the complete omission of OBAs. Two other preferred uses of OBAs are the use of HMW OBA alone (for example, 3-5% HES~ Mr 10,000450,000 daltons, or its equivalents as noted above) and the use of midrange OBAs (Mr about 350 to 10,000), particularly when the ~,lyu~lu~ dll~ washout rate is higll.
(a) Complete Omission of OBAs. E~ . h..~.~t~ with 4 control livers perfused with neither cryoprotectant nor the normal HES of modifed UW solution indicated that life support function could be obtained in three cases. When the experiment was repeated with the inclusion of V49 perfusion, and no LMW osmolyte was used, not only did about 50% of the livers support life after n,,~ inn but they did so after almost complete equilibration with V49, in contrast to livers perfused with V49 in the presence of HES~ which e~nL~ ihr~rfi poorly and had a survival rate no better t-h-an the livers perfused without HES. Therefore, neither LMW nor HMW osmolytes were mandatory for livers.
While not wishing to be bound by any theory, the arrer~ y and the desirability of omission of all OBAs for the liver were thought to be based on the liverts high permeability to both cryu~-lutc~Ldl,L~ and nominal LMW OBAs.
The liver is unique in that its ~Jal~nr~llyllldl cells are exceptionally permeable tO LMW solutes, includhlg cr~u~"utc~dnL~. This allows faster rates of WO 96/05727 PCT/US95/lOZZ3 -6~-~.lyuL)luLe~,LdllL addition and washout with less osmotic stress than occurs in other organs. For example, liver slices were found to withstand abrupt multimolarchangesin~.yu~,ut~Ld~ Jl~ ""~irmtllatwouldhavebeenlethal to most other types ûf tissue, including the kidney, and using smaller changes S in IO~ . ,u,.l.OIl did not produce improved survival in liver cells after yu,uluLe~LIIIL exposure and washout. With respect to the intact liver, note in Figure 10 the reasonably steady flow rates (suggesting no excessive osmotic cell swelling) during washout of V49 from the liver despite the absence of both LMW OBAs and HMW OBAs. Finally, since liver cells are somewhat permeable to sucrose, sucrose will be relatively ineffective as an OB during ~,lyu~JIui~,Ldnl washout, but its leakage into the cells might actually cause cell swelling upon LIA~ n~
(vJ Excll~si~e Use of HMW OBA. The above-described eAp~lhn~ta revealed one difficulty with the omission of HES, and that is the fact that only 3 out of 4 control livers (no Clyu~lut~,~,LduL) survived perfusion in the absence of HES, vs. higller survival whell HES was used. HES or its equivalent may therefore have to he present to adequately support hepatic viability regardless of the presence or absence of ~,lyulJIut~.LallL Because HES cannût be presen~ (except at minimal uulll . .,I"U;r"~c) during the loading of vitrifiable ~lyu~JluleuLr~llL rv~ ;o due in part to its unr.,vu.. ,l,le effect on viscosity, one way to maximize HES for ~ h ~ h.~, v;ability would be to add HES only when the ~;IyU~lUU,~,LGIIL is being washed out, simply because perfusion with HES will be more feasible from a physical standpoint (lower viscosity) when the u~yu~"ut.,.,L~-L .. u~ lc are low compared to the vitrif~able co ~r nrr~ionc~ and when these con~ r n~r~tir n~ are falling rather than rjSing, In this context, HES would not necessarily be acting as a true OBA
but only as an ordinary osmotic support agent. N~ lLhc~ a~ the HES would be used in essentially the same manner procedurally as it would be used if it were being used as an OBA, so from a practical point of view this would be the equivalent of using a HMW OBA as the sole OBA. Furthermore, it must be remembered that the liver consists of more than merely hc~ LuuyLea, and -2 1 ~7773 W O 96/05727 PC~rAUS95/10223 an osmolyte such as HES could act as a true OBA for these non-h~,~.d~u~"~t~,i,.
The decision to use HES or other equivalent HMW OBA during elution of l,~yu~rut~,~,Ld-~l from the liver can be made depending 011 the ability of the type of liver in question to withstand the absence of HES durhIg control perfusions and to withstand the absence of HES .during cryoprotectant elution.
Although the liver did not equilibrate well with cryoprotectant when perfused with a ~ hj~ . of ~,ryul,lul~vdl,L and HES in the above-described cAL~ lL, this problem can be overcome by using an osmolyte with a ~u[r~;c.llly low M, to control viscosity adequately, e.g., HMW OBAs equivalent to HES of M, = 2-50 kd.
(c) Midrange OBAs for rapid c,~vp,ute.tv~t efflux from the liver. OBAs ranging in M, from 350 to 10,000 daltons, being less permeable than sucrose, yet l.lJ~ I,ly less viscous than most HMW OBAs (hence, perfusable at a ~ufrlc;~ tly rapid rate), may protect liver cells other than ' ~,~ y;~,S from osmotic injury, especially during very rapid rates of change of cryc,p.uL~vdllL c~ ln~ u~ Therefore, either one such agent or a rr mhin~lion of two or more such agents falls within the method limits for the liver.
d) Phase 6: Cradual reduction of ~"~v,v,..~
; : ~; to zero with ' ' elel~ation of perfvsion ~ tu~;
In the best mode method for the kidney, the gradual reduction of ~,lyu~lULr~vdl~L C.. ~ n,ui"" to zero or virtually zero was carried out at a constant rate of about ~2 mM/min (acceptable variations being -31 to -75 mM/min for the kidney and most other organs, or -31 to -150 mM/min for the liver). Non-constant declining rclrrrn~rlllion schedules (rapid fall at high rrmrrn~rlri(~n~ slower fall at lower concentrations) are also an acceptable variation, e.g., a linear fall at 1.5. times the average linear raLe for the first third of the washout followed by a linear fall at 0.~6 times the average linear rate for the second two-thirds of the washout.
During clyu~JluL~vdlll washout, the tUIllp~ldlul~ was elevated to facilitate washout, reduce osmotic forces, and restore a perfusion tcll.~,e~dLu~

~ t ~7~
Wo 96/0s727 Pcrlusssllo22 d~/l./lU~JI ' for an organ contahlillg no cryul luk.~,Ldl,L. In the best mode method for the kidney, LtllllJ.,ldLlllt~ eievation began as the rnnn~n~r~li,nn fell to 4.7 M and continued linearly with ronnt~ntration drop until the initial perfusion ~ n}~ dLl~ was reached and aroerial concentration reached 1.3 to 0.8 M (1~C rise per 0.68 to 0.78 M decrease in concentration; total of 3.4-3.9 M nnnrPrltr~tinll change during warming) as illustrated in Figures 5 and 6.
Acceptable variations for the l ~ nAtioll at which the t~,ulu~,-dLul~ initially rises are 2.5-5 5 M and for the nnnnentrA~ion at which oelllLlGldlulci rise is completed are 0.5M-4.5M.
e) 013 washout dur7ng phase 6: The general method for OB washout during phase 6 was to incompletely wash out the LMW OBA
while I~lA;lllAillillg HMW OBA concelltration (when HMW OBA was present) constant or reducing HMW OBA ronren~rAtion by only 1-2% w/Y. More particularly, as penetrating cryoprotective agent ronn~ntrA~inns fell, the ~,.. ~ .UIAliun of LMW OBA also fell in proportion reaclling a final nonzero concentration of OB when penetrating l~yu~u~ute~ulla uulll,;,lllldLiull reached zero. This final nonzero u.~ uAliull of LMW OBA was 50 mM in the best mode method and may acceptably vary from 25 mM to 500 mM. As an example, in the best mode (Figure 6), in which 350 mM sucrose was brought to 50 mM sucrose while 5.0 M cryu!,ruoe~LdllL was reduced to 0.0 M
elyul~lu;~ LdllL at a rate of 42 mM/min, sucrose r..~ nAIiO~. dropped at the raoe of 2.5 mM/m;n.
While not wishing to be bound by any theory, during reduction of ~.lyuiJIut~L~llll n,"" ~ -nAIiu~ absolute UAl.~lll. ..lhlAnl' osmotic forces dlllibuLdlJlctotlle~lyu~uru~ Ldll~ldll~ ldl~econcentrationgradientbecame reduced, thus reducing the requirement for osmotic buffering. Reducing OB
c~ rAlinn during c~yulJ~u~euLdllL washout was therefore designed to minimize osmotic damage from the OB both during ~Iyul~ruLr~,LdnL washout and thereafoer and was further designed to reduce potential cellular uptake of nominally non-penetrating OBA. No previous perfusion technique of ~Iyu,ulutl_~,Ldll~ washout has ever made use of this "declinillg OB principle."

2l 97773 ~ W O 96105727 PC~rrUS95/10223 When LMW and HMW OBAs were used together, a differential decrease in OB was perforrned wherein the ~ u"~ "nAI;nn of the LMW agents declined while that of the HMW OBAs remained the same or nearly the same.
J7 0~ wasltorl~ pltase 7:
i) Standard mode. The final step in the method after removing all cryoprotectant is to continue to perfuse the organ to allow it to fully equilibrate with the ~Iyoplut_~,Ld~ free medium and, if desired, to continue or complete the washout of the OB. In the current best mode for the kidney, 50 mM sucrose and 3% w/v HES Mr 450 kd was attained at the end of clyulJIuLtcl.~llL washout, and no additional washout of these OBAs was undertaken prior to t~ncrlAmAtinn Although it is acceptable to leave such IOW wnc~llLldLi(Jll~ of OB in the organ during short holding times before nA~ ;On~ interstitial OB is expected to cause osmotic expansion of the interstitial space during blood reflow with a consequent temporary reduction in organ perfusion in vivo. This effect will become unacceptable at higher OB
feOII~ (2100-500 mM, or 2 3-7% w/v) and will necessitate at least partial OB washout before transplantation A further problem with leaving OB
in the organ for extended times before Lldll~lJhlllLdLiUn is the potential leakage of OB into organ cells with consequent cellular swelling and reduced perfusion upon nA.~LllAIIIA~iO~ In ;~ hll~,llLs with V49, the inventors typically washed out 50 mM mannitol over the course of 30 min with complete success upon ~,,.r.~pl-"lAIin" However, it was generally observed that leaving 50 mM
LMW OB in the kidney for short times before trAncrlAnl-Ation was beneficial at higher wy~Jplu~ldllL c~n~ Alionc~ in some cases representing the difference between organ survival or death It has never been observed that leaving 50 mM mannitol or sucrose in the kidney prior to Lld~ JldllL~Lion was more detrimental than entirely removing this final concentration, so the washout of OBA during phase 7 is primarily concerned with reducing LMW
OBA rJ~nrPntrrtinnc down to less than about 100-500 mM and with reducing HMW OBA l Ull~f .lll~lioll~ down to less thal1 about 5-8% w/v wo 9610s727 2 1 q ~ 7 ~ 3 Pcrlusssllo223 ~

While not wishillg to be bound by any theory, the retention of 50 mM
LMW OBA is belived to be beneficial because interstitial osmolyte will reduce cell and organelle swelling until the moment metabolism is restored in vivo, and that mf-~hf 1i7ing cells are capable of osmoregulation to cope with infrArPlbllAr leaked mannitol or sucrose provided the extracellular osmolyte can slow down passive cellular swellhlg long enough for osmoregulation to be restored. In addition, the use of higher Mr OBAs will preclude cellular uptake of OBAs, further increasing the ~Irreptlhility of leaving in the OBA.
Higher concentrations of OB (up to 500 mM) may be washed out over more extended times (30-90 min) tha~ depend on the perfusion resistance response to OB dilution. For clinical purposes, the duration of the post-washout perfusion period, comprising the OB washout, and the degree of OBA
washout must be adjusted to be compatible with the exposure times imposed by the logistic I~UhL~ IL~ of organ transportation and nA~ IAmAI;On ii) The three-osmolyte washout technique. In the inventors' early experience involving perfusion of 8.4 M ~yu~uL~uL at about -3~C (one-sLep addition technique), consistent control of vascular resistance during Iny~ ,Lf~l" washout and excellent appearance of the i~idneys 40 minutes after tr~ncplAnt~ ln were obtained when the following procedure was used, and only when it was used.
After perfusion with 8.4 M ulyu~Jlote~L-llL~ the nonrf~nfrAfion of the ~,lyu~l~Jt~,.,~uL was dropped to about 5.0 M with the cimnlfAnf-ollc introduction of 250 mM sucrose and 4% wtv HES. After a 9 minute phase 'i plateau, the standard linear sucrose washout technique was followed while holding HES
1~ U~ .... constant at 4% w/v. However, once all ~,lyv~luLt;~,~nL was removed, the HES nonrf-nfrAfinn was gradually reduced to 3% w/v while the sucrose ~Inll.,lnAli~lll was gradually reduced to zero and mannitol was f.u", O~ A~IIY introduced to a final concentratioll of 50 rnM.
Thus, the i"~n~v~lLions involved in the three-osmolyte washout technique were: 1) to combine a HMW with two LMW OBs resulting in a 3-OBA

Wo 96/05727 2 1 q 7 7 7 3 PcrluS95/10223 method, and (2) to replace one OB (sucrose) with another OB (mannitol) just prior to tr~nsrl~nt~ti~nn While not wishing to be bound by any theory, this approach was developed for the foilowing reasons. Sucrose is more effective osmotically than mannitol, i.e.~ it is less likely to leak into renal cells due to its higher molecular weight. However, unlike mamlitol, it does not have any ability to quench free radical reactions durhlg reperfusion of the organ with blood upon ~r~nsr!:~n~tion By using sucrose to carry out the primary osmotic buffering function and mannitol to maintain osmotic buffering at the end of the perfusion, the advantages of both agents were obtained and the disadvantages of both agents were avoided. Second, 4 % HES appeared optimal for balancingthetradeoffbetween~ viscositywlliiem:~Timi7ingosmotic and oncotic effectiveness during phases 5 and 6. Fhlally, 4% HES was reduced to 3 % just before ~Idu~yl~uldlion to minimize perfusate viscosity and the quantity of interstitial HMW species.
It is not to be construed that this method depends specihcally on sucrose, HI~S or mamlitol. The goveming prhlciple involved is a general one.

6. Treatrnent of Jhe Orl,7an and the Recipient at the Time of T, rh ' '-U and Thereafter It is important tbat the recipient receive aspirin (acetylsalicylate, 1-3 mg/kg) and heparin (100-250 units/kg) shortly before release of the vascular clamps and Icy~fu~;O~ of the n~n~y~-. d organ, both higher and lower rnnrPn~r rirJns of both drugs resulting hl vascular obstruction and failure. Thebest mode conrpn~r~inns were 2 mg/kg and 200 units/kg, respectively It may also be helpful to gradually infuse agents that reverse sulfhydryl oxidation(e g., culu~lliO~lucuac or ~i-acetylcysteine at serum levels of 0.1-10 mM), inhibit ~ ~tr~rcllnl~r (e.g, o!-2 macroglobulin, amiloride, tissue inhibitor of metalluy.utci-.~ (TIMP)) and intr~rP~ rr (leupepthl, glycine) proteases or facilitate endothelial cell adhesion (TGF,BI, 0.1-10 ~g i.v. per every 5 min for40-300 min). The inventors have found that dimethyl sulfoxide reduces the .. .... . , . .. .. .. . . . ... _ .... . . _ . . ... _ .

2 t ~77~3 ability of renal tissue ~o restore depleted tissue SH content and have found massive elevation of urinary urokinase after the ~ransplal1ta~ion of rabbi~
kidneys.

IIl. Method for the Perfusion of an Organ With I~on-Cryoprotectant Perfusates In addi~ion ~o ~he organ cryopro~ection perfusion protocols, the apparatus and methods described herein are capable of use hl a wide variety of protocols for .u..~.,.niolldl organ hypo~hermic and llullllulh~ ic preservation. In addi~ion, a wide variety of normothermic phdll"d~,ologi.,dl, physiological, and ~ hu~Jllyi~;OlOgicdl protocols are possible using the apparatus and methods of tbis inventiom The inventors indicated many of these possibilities earlier and in describing tlle steps required to carry out many of these protocols in Figures I I A and I I B, whicll are self-explana~ory.
IV. ~esults A. ~ cell protection with TGF,~I
TGF,~I allowed endotllelial cells to remahl properly attached to fibronectin medium or subs~ra~e hl a cul~ure flask when washed with UlyU,UlU~l,~ solu~ion Table 4. TGF~I is expected to have a similar effec~
on endothelial cells in vivo.

WO 96/05727 2 1 9 ~ PCT/US95/102Z3 Table 4 Protecdon Against Endothelial Cell 1~ ' by TGF,151*
.

Treatment ~ of Non-Detacbed p Value vs.
Cells Controts 37~C Controls 5.05 i 0.31 x 106 2~C Controls 4.33 i~ 0.38 x 106 n.s.
V52 (superfused according to wbole1.38 i 0.10 x 106< .00002 kidney protocol: V52 itself = 20 min exposure) V52 + TGF31 (same as V52 above hut4.95 i: 0.21 x 106 n.s culture pretreated with TGFj~l at 10 ng/ml for 22 hours) * Detachment was determined hy trypsinizing the flasks after each experiment, washing out the cultured endotbelial cells and counting them. Detached cells removed during the superfusion are not seen in this assay, causing the cell count to go down.

B. Rabbit Kidneys.
1. Suitability oJ V~9B-type Solutions.
Viability data from rabbit kidney slices after treatment with V49 or V49B are shown in Table 5.

WO 96/05727 2 ~ q ~ 7 7 3 PCT/US95110223 I'al)le 5 Viability of Rabbit Kidney Slices Treated With V49 or V49B

Treatment K/Na ratio of tissue-(mean +/- SEM) V49 3 .43 + /- 0.07 V49B 3.27 +/- 0.12T
Tp > 005 ~Phe K/Na ratio was measured aher wasl~blg out tlle cryoprotectants and incubating tlle cortical slices at 25~C for 90 mh~utes to permit active transport of Kt and Na+.

2. Suitability of V~9 and V57 for tlte Intact Kidney.
Figure 12 shows post-operative serum creath1ine levels of rabbiLs which had received transplanted kidneys that had been previously perfused with V49 in Euro-Collins solutiom Prior to procurement, the kidneys were treated in vivo with zero, IS, or 25 ,ug/kg of iloprost administered by systemic hlLl~ nuu~ infusion over a 20 minute period. Kidneys in these three groups were exposed to V49 (7.5 M) at +2', 0-2~ and -1~ to -6~C, respectively.
Initial and final perfusion L~.n~ dLI~cS were 2~C in ail cases. Rabbit survivals in these three groups were S/16 (31%), 6/10 (60%), and 10/10 (100%), respectively. Only data for rabbits surviving the first night after surgery are hlcluded. Rabbit survivals depended entirely on the function of the tr~ncrlan~pd kidney because a rontrrl~lpr~l nephrectomy was performed at the time of L~u~l~k.llLdLion, and no support by dialysis was attempLed.
Histology in these rabbits was poor at long-term follow-up witl1out iloprost treatment, marginal with the lower dose of iloprost, and normal with the higher dose of iloprost and the lowest perfusion tell~ d~ulcs. The resl~lts of control (no ulyuplu~ecLdnL) perfusions with Euro Collhls are hlcluded in Figure 8 as well (bottom curve). Aitllougll damage hl the best V49 group is greater , . _ . ... ., .. . , .. .. ........ ... . , . . , , _ WO 96/05727 2 1 ~ 7 7 7 3 PCTIUS95/10223 than in the controls, all damage appeared to be fully reversible within a shon time postoperatively.
Table 6 shows that whell an attempt was made to extend the success at 7.5 M ~Iyo~ tc~,ldnt to 8 M cryoprotectan~, the result was nearly uniform S failure unless 3% HES was incollJul~ted into the solutions used to wash out the 8 M conrpntration The use of HES allowed the survival of 75 % of rabbit kidneys after ~Idn~lJldllLdLion. Leaving the LMW OBA in the rabbit kidney was also beneficial to the kidneys after their transplantdtion (Table 6).

Table G

Recovery of Whole Rabbit Kidneys Perfused with 8 M C-,1u,u- . ' ' ~c Life Treatment Support Function A. Standard Protocol witb Either Mamlitol or Sucrose Washout 0 B. Modified Protocol with Ist Plateau Raised to 30% and 3rd 8 Plateau Raised to 33% w/v to Reduce Osmotic Stress C. Same as B, but Lowered Perfusion Temperature from -1.5~C 29 to -3~C
D. Same as C, but Used 3% HES During Wasbout of 75 Gyo~ Jtc~ t and left 50 mM Mannitol in the Kidney Until T- I ' E. Same as D, but Used Sucrose vs. Mannitol 75 F. Same as D, but Removed All Mannitol Before T. , ' 0 G. Same as E, but Removed All Sucrose Before T . ' . 33 wo 96/os7z7 2 ~ ~ 7 7 7 3 PCTNSgS/IoZ23 C. Overcoming Cooling Injury at 46~C and Toxicity at 8.4 M
Cryoprotectalzt When kidneys were treated with either 7.~ M or 8 M cryoprotectan~
using 3% HES dur.ing washout as in Table 6, tlley were still unable to withstand cooling to -30~C (Figure 13). The 100% survival rate at 49%
~,~yu~ute-Ldnt fell to just over 50% as a result of cooling, and the 75%
survival ra~e of the 8 M group fell to 0%.
Although some of this injury was due to the greater time required to allow coolhlg and warmhlg to take place, tissue slice evidence indicated that cooling per se was actively detrimenLal. As seen in Figure 14, exposure of slices to 8 M cryoprotecLive agent a~ 0~C was considerably more damaging than exposing them to 6.1 M cryopro~ectant at ~lle same temperature (cf. bars 2 and 4), and cooling these 8 M slices to -23~C caused additional injury (cf.
bars 2 and 3). Interestingly, llowever, cooling 6.1 M slices to -23~C did not cause additional injury (cf. bars 4 and 5). Even more hlLc~Lillgly, when slices loaded with 6.1 M cryoprotectant were transferred to a -23~C solution of 8 M, or even 8.4 M cryu~u~e.L~"L, there was still no damage associated with cooling, nor was there damage associated with cooling, nor was there damage assûciated with exposure tû these higlle m u"~c ~ (cf. bars 4 and 5 to bars 6 and 7). In fac~, slices exposed to 8.4 M at -23~C according to the two-step approach (flrst cool, then expose to higher noncPntr ~ti~nc, bar 7) hadmore viability than slices simply exposed to the lowest ~onrPntr~tion of 8 M
at 0~C withou~ cooling (bar 2, p=0.033). These results showed that, at least in slices, both coolhlg injury and cryoprotectan~ ~oxicity were preventable by cooling first in a low conr~ntr~tion and introducing higher conrPntr~tionC only in a second step at the lower temperature, hl this case -23~C.
Re-examinationofFigure 13 suggested thattllesameph~ u,.applied to the intact kidney. Recovery was higher at the lower conrPrtr~ti~-n of u~yu~luLectall~, and if one drew a Ihle connec~ing the 8 M cooled point and the 7.5 M cooled poin~ ex~rapola~ed to 100% survival at some concentration below 7.5 M. Because the Ihlearity of such an extrapolation was not known, , . , , ., .. . ... , .. , . . , .. . ...... , .,, .. , ... , . .... , _ _ . _ .

2 1 ~7~3 the inventors elected to try an experiment witll a ~t)n~elltr~tioll comfortably below 7.5 M i.e. with 6.1 M as in the slice experiment.
The results of this experhnent are indicated in Figure 15. 100% of the kidneys loaded with 6.1 M cryuplu~e-idllL~ cooled to around -22~C and warmed up (protocol indicated in the insert) supported life giving excellent mean serum creatinine levels after 14 days (Cr,4) and acceptable peak creatinine values (pCr). Figure 16 shows the results of loading 6.1 M
cryoprotectant at -3~C cooling to -23~C and thelI perfusing the kidney with 8 M wyul)lùLt~Ldnt until equilibrium was achieved. As in the previous t,~l,e,i",e"L~ the 8 M cryoprotectant was washed out using 35'o HES. In stark contrast to the results of the one-step ~-yu~-uLe~.LdllL addition method followed by cooling to -30~C (Figure 13) the 8 M kidneys of Figure 16 had an excellent survival rate of 7/8, and the kidneys that did survive were not different from the 6.1 M kidneys in terms of their Crl4 and pCr values in full agreement with the siice results of Figure 14. Ful~ .lllolt as shown in Figure 17, when kidneys were perfused with 8 M cryupluLt ~Ld-lL at -22~C
they could then be cooled anotller 10~C to -32~C (colder than in Figure 13) with 100% survival upon warming and with Cr,4 values identical to those of slices exposed only to 6.1 M ~ Iyu~ t~L~ulL again in complete agreement with the predictions of Figure 14.
The inset of Figure 18 shows that the injury associated with cooling increases between -30 and -60~C but does not increase with further cooling to near the glass transition t~ JCl~lLUlr~. The main portion of Figure 18 shows an attempt to more precisely determine where between -30~ and -60~C
cooling injury stops increasing. Although the magnitude of the drop was somewhat small in this t~t~ Ihll~ ." it appeared that slices cooled to 45~ to -50~C t;~.p~ n~ ed a maximum amount of cooling Injury.
Using the hlru"-,dtio., of Figure 18 as a guideline two additional " u~ were done witll intact kidneys. After spending approximately 9 months optimizing the procedure for hItroducing and removing V55 the following optimum method was identified. The first step of the two-step 2l ~ 77~3 wo s6/0s727 Pcr/usg~/l02~3 approach was to perfuse 44% w/v cryoprotectant (6.73 M) at -3~C and then cool to -25~C for perfusion with V55 (55% cryul lut~ L1 8.4 M). The kidneys were then warmed back to -3~C and were washed out with 3% w/v HES and 350 mM sucrose as described above. This protocoi resulted in a survival thus far of 2 out of 3 kidneys so treated. These kidneys looked excellent after 40 min of blood refiow in vivo and, as shown in Figure 19, they were able to return serum creatinine levels to near or below 2 mg/dl, an excellent result. F~ n~ulc, one kidney perfused with V55 at -25~C by this procedure was cooled to 46~C prior to warming and washed by the same procedure used in the non-cooled VSS kidneys. The result for this kidney, also shown in Figure 19 (dashed line), was similar: the kidney looked excellent upon L~ ,la,.Ldtioll and, at the thne of submission of the patent ~ppljr~irn was restoring serum creathline to a value near 2 mg/dl. The kidney showed a peculiarly delayed recovery, ll.Ahl~ lg creatinine at values near 15 for an ~ ,;ie.. t~,;i amount of time, but the peak creatinine and the rate of return of serum creatinine back to baseline after this long delay were not different than what was observed for the other two VSS kidneys.
Taken together, the slice results of Figure 18 and the intact kidney data of Figure 19 indicateci that rabbit kidneys can now be cooled tO the glass transition t~ withoutlosing viability. Furthermore, since Figure 19 employed a ~Onr~ ;ul. of u~yu~lvtl,uLalU that vitrifies without applied pressure, the implication is that high pressures are no longer mandatory for organ ~;t~ih~tio".

D. Pert~nence of Animal Datn to Human Kidney C,.~u~,, L.~LI ~uhon 1. First Human l~idney A 232 gram human kidney was perfused according to the method of this invention and was then vitrihed. Digital data from the method was captured using a BASIC program and was edited and plotted using a sigmaPlot 5.0 2 1 9~73 W O 96/05727 PC~rrUS95/10223 graphics package (Jandel Scientific, San Rafael, CA) to generate the data in Figures 20A, 20B and 21.
The data in Figure 20A show ~hat the method of the invention produced the expected results in this human kidney. Althougll the measured molarity S was slightly greater than the target molarity and the first step change in c~mnentr~tion slightiy overshot the target, the data follow the protocol reasonably well.
The data in Figure 20B from the same human kidney show that resistance (expressed as mm Hg divided by fow) and fow (ml/min/gm of kidney) behaved in a way that was qualitatively similar to their behavior in rabbit kidneys.
The data from the subsequent v;tlir~cdLioll of this human kidney ~n."~.n~n,.lrd that this method performed adequately. The data in Figure 21 provide no indication of freezing of the kidney which would have been represented by a t~,.U~.. dLulc; plateau followed by a relatively rapid fall in t~ ,U~.Id~UI~. After an initial thermal lag above 0CC which l~ ".;ud the time for the external t~.,u~J~"dLulc front to penetrate through the mass of the kidney to the L~u~ ,la~ul~ probe in the middle, the Lelll~J.,I.ILUI~ dropped rather smoothly, revealing virtually no evidence for ice formation.

2. Second Human Kudney This human kidney was a pediatric kidney from a four month old donor.
This kidney was stored for about 79 hours after it was collected but before it was perfused with V55 ulyu,ulut~,L~ulL according to the method of this invention. The data in Figure 22 show the perfusion of this kidney with V55 (ascending portion of the curve), and the removal of V55 ~,lyuplut~,~,L~IllL from the kidney (descending portion of the curve). The dotted and solid lines in Figure 22 show theachieved and target V55 ~ u~liùl~, respectively. The perfusion pressure was set at 35 mmHg in this experiment.
The discrepancy between the measured and target concentrations was merely a matter of calibration rather than a ~rue limiLatioll of the method. The 2 1 q7773 wo s6/0s727 Pcr/UssS/10223 pressure spikes which occurred when a conr~n~ on of 8.4 M was quickly approached or retreated from reflected software that was not speciFcally designed to prevent these spikes and has since been corrected This was not a limitation of the method. Since this kidney was unloaded, a cooling curve S was not generated. Resistance, flow and t~ UI~: are not shown in gure 22.

E. Kidney Slice Vrab~llty Data Viability data from rabbit (Figure 23A) and human (Flgure 23B) kidney slices and normalized data for rabbit (R) and human (H) kidney slices (Figure 23C) show nearly identical responses of tlle human and rabbit kidney slices to V49. Although the data showed a slightly lower recovery of human kidney tissue compared to rabbit tissue after cooling to -30~C, this recover,Y was within dhe variability seen widh rabbit kidney slices. The human kidney was seYeral days old before the experiment was carried out, whereas the rabbit kidneys were "fresh". The absolute human KlNa ratio was depressed about as much as would be expected for rabbit slices stored for a similar time.
These data in uulllbil ~don with the perfusion data in this section showed that human kidneys can be loaded with ulyulJIut,~ according to the method of this invention and can be essentially vitrified on cooling -- e.g., be cooledbelow the glass transition t~ /.,.dlUI~ with minimal or no ice formation in the organ. These data also showed that the l,lyul)lute~,~lll can be removed from the human kidneys using the method of this invention. Lastly, the similarity of the Yiability data of the rabbit and human kidney slices combined with the fact that rabbit kidneys actually surviYed and maintained the lives of rabbits into which dley had been ll~ .. ~d, suggest similar results will be obtained when human kidneys are treated using the methods of dlis hlYention.

2 ~ ~77~3 WO 96105727 PC~rrUS9~10223 . Applicability to Otf~er Organs: Tlze Rat l,iven Modei Rat livers were perfused using the protocol as shown in Figure 10. The perfusion fluid did not contain either HES or LMW OBs. The data in Table 7 show total bile production at 5, 10 and 15 minutes after transplantation and 5survival at 7 days after liYer transplantation into host rats. These data f~r, ~ nAIr(l that rat livers perfused with the solutions supported the lives ofhost rats into wbich they had been Llan~yLII\t~,d after their perfusion.

Table 7 Functional Recovery and Life Support Function of Rat Li~ers 10Perfused with Vehicle or V49 Liver Total sile Produc~ioll a~ s,Rat Survival 7 Experimeul weigllt 10 and Is Ulill ~I/g i SD)days after (% change) after T , ~ ' T , ' CoLtrol P&l*-9.9 I.G85.19 9.32 6/6 (lOOf~o) w/UWlt (HES)i2.1 i.94i2.33 i3.65 Control Pfu#-8.7 2.034.62 7.67 516 (83%) w/UW2t (I~oiO.5 i.75i 1.50 i2.48 HES) V49 Pcrfusioll 8.7 0.66 1.62 3.14 214 (50%) w/UWlt (HES)iO.7 i .50iO.82 i 1.38 V49 Perfusiou -6.3 1.20 2.56 4.43 214 (50~) wGUW2t(110 i3.0 i.98il.92 i3.18 HES) *P&l = Perfusion t Uwl = Illodified Uw Solutioll I ~see Table 3) ~ UW2 = modihed UW Soluholl 2 (see Table 3) Taken together, tlle data from kidneys and livers implied that the herein-2 1 q~773 disclosed methods for preparing organs for cryopreservation and of preparing organs for transplantation after u~yu,u~r~ v~L;on are broadly applicable.
While various emhorlimr-l-tc of the present hlventioll have been described above, it should be understood tllat they have been presented by way of example, and not limitatiom Thus the breadth and scope of the present invention should not be limited by any of the above described exemplary e.l.hrJ-II,,,. r,~, but should be dehned only in accordance with the following claims and their equivalents. Sh1ce it will be understood by those of skill in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the prh1ciples of the inventiol1 and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features h.,.~h~ rul~ set forth and as follows in the scope of the appended claims.

Claims (35)

What Is Claimed Is:

1 . A method of preparing a biological organ for cryopreservation, comprising:
(a) perfusing said organ with gradually increasing concentrations of cryoprotectant solution to a first predetermined concentrationwhile concurrently reducing the temperature of said organ;
(b) maintaining the concentration of said cryoprotectant for a sufficient time to permit the approximate osmotic equilibration of said organ to occur; and (c) increasing the cryoprotectant concentration of said solution to a higher second predetermined concentration and maintaining the cryoprotectant concentration of said solution at said second concentration for a time sufficient to permit the approximate osmotic equilibrium of said organ to occur.

2. The method of claim 1, further comprising perfusing said organ without cryoprotectant before perfusing said organ with gradually increasing concentrations of cryoprotectant.

3. The method of claim 2, further comprising perfusing said organ with iloprost or transforming growth factor .beta.1.

4. The method of claim 1, wherein said second predetermined concentration is not permissive of vitrification and step (c) further comprises cooling the organ before the introduction of a vitrifiable concentration of cryoprotectant to said organ.

5. The method of any one of claims 1-3 or 4, wherein said organ is a kidney or a liver.

6. The method of any one of claims 1-3 or 4, wherein said cryopreservation is by vitrification and said final cryoprotectant concentrationpermits vitrification.

7. A method for preparing an organ for transplantation after its cryopreservation, comprising:
(a) warming said organ to a temperature which permits reperfusion of said organ, wherein damage to said organ is minimized;
(b) perfusing said organ with a non-vitrifiable concentration of cryoprotectant for a time sufficient to permit the approximate osmotic equilibration of said organ to occur; and (c) perfusing substantially all of said cryoprotectant out of said organ while concurrently increasing the temperature of said organ to render said organ suitable for transplantation.

8. The method of claim 7, wherein said cryopreservation is by vitrification.

9. The method of claim 7, wherein said cryopreservation is by freezing.

10. The method of claim 7, further comprising:
in step (b) perfusing said organ with a non-vitrifiable concentration of cryoprotectant in combination with an osmotic buffering agent.

11. The method of claim 10, wherein said osmotic buffering agent is a low molecular weight osmotic buffering agent.

12. The method of claim 11, wherein said low molecular weight osmotic buffering agent is selected from the group consisting of: maltose, potassium and sodium fructose 1,6-diphosphate, potassium and sodium lactobionate, potassium and sodium glycerophosphate,raffinose,maltopentose, stachyose, sucrose and mannitol.

13. The method of claim 11, wherein said low molecular weight osmotic buffering agent is sucrose.

14. The method of claim 11, wherein said low molecular weight osmotic buffering agent is mannitol.

15. The method of claim 10, wherein said osmotic buffering agent is a high molecular weight agent.

16. The method of claim 15, wherein said high molecular weight osmotic buffering agent is selected from the group consisting of hydroxyethyl starch (~450,000 daltons), polyvinylpyrrolidine, potassium raffinose undecaacetate and Ficoll (1,000 to 100,000 daltons).

17. The method of claim 15, wherein said high molecular weight osmotic buffering agent is hydroxyethyl starch.

18. The method of claim 17, wherein the molecular weight of said hydroxyethyl starch is approximately 450,000.

19. The method of claim 10, further comprising perfusing said organ with said non-vitrifiable concentration of cryoprotectant in combination with one or more low molecular weight and one or more high molecular weight osmotic buffering agents.

20. The method of claim 10, further comprising perfusing said organ with said non-vitrifiable concentration of cryoprotectant in combination with one low molecular weight and one high molecular weight osmotic buffering agent.

21. The method of claim 11, wherein the concentration of the low molecular weight osmotic buffering agent is gradually reduced to a nonzero value while the concentration of said cryoprotectant is also being gradually reduced to less than 200 millimolar.

22. The method of claim 21, wherein the concentration of said low molecular weight osmotic buffering agent is reduced to between 150 mM and 1,000 mM.

23. The method of either claim 21 or 22, wherein the concentration of said cryoprotectant is reduced to zero.

24. The method of claim 11, wherein the concentration of said low molecular weight osmotic buffering agent is gradually reduced after the concentration of cryoprotectant has been reduced to less than 200 millimolar.

25. The method of claim 19, wherein said low molecular weight osmotic buffering agent is selected from the group consisting of mannitol and sucrose, and said high molecular weight osmotic buffering agent is hydroxyethyl starch (HES).

26. The method of claim 25, wherein once all of said cryoprotectant is removed from said organ, the HES concentration is gradually reduced to a non-zero level while the sucrose concentration is gradually reduced to zero and mannitol is concomitantly perfused into said organ.

27. The method of any one of claims 15, 16, 18, 19 or 20 wherein said organ is the liver.

28. The method of any one claims 10, 19, 20, 25 or 26 wherein said organ is a kidney.

29. The method of either claim 7 or 8, wherein said organ is a liver.

30. The method of claim 7, wherein said temperature in step (a) of said claim is -3,0°C when said organ is a kidney or a liver.

31. The method of any one of claims 7, 10, 19 or 20, wherein said non-vitrifiable concentration of cryoprotectant is from 20-40 % weight/volume.

32. A composition of matter for perfusing an organ, wherein said composition maintains the viability of said organ, said composition comprising:
NaH2PO4 ~ H2O; potassium gluconate; magnesium gluconate; glucose;
glutathione; adenosine; HEPES; adenine; ribose; and calcium chloride.

33. The composition of claim 32, further comprising hydroxyethyl starch.

34. The solution of claim 31, wherein the concentrations of the components of said solution are: NaH2PO7H2O (3.45 g/l); potassium gluconate (23.42 g/l); magnesium gluconate (0.21 g/l); glutathione (0.92 g/l);
adenosine hydrochloride (1.34 g/l); HEPES (2.38 g/l); adenine (0.17 g/l);
ribose (0.15 g/l); and calcium chloride (0.0056 g/l).

35. The solution of claim 33, wherein the concentration of said hydroxyethyl starch is 50 g/l and of the glucose is 0.90 g/l.