CA2147626C - Cell encapsulating device - Google Patents

Abstract

This invention provides cell encapsulating devices capable of maintaining large numbers of viable cells. The devices are comprised of an inert,substantially cell-free core, a permeable membrane and a zone for maintaining cells. The permeable surrounds the core such that the zone of cells is bounded by the core and the permeable membrane. The devices of the invention are suitable for implantation into an individual in need of treatment and are capable of supplying therapeutic substances to such individuals.

Description

~ WQ95/~21 2 1 4 7 6 2 6 PCT~S94/07190 ~F~.T. ~CAPSUT~TT~G D~V~

~ CAT FTF~n OF T~ v~h~lON
Thi8 invention relates to devices usQful for maint~; n i ng cells in a discrete space while permitting r~r~ge of cell nutrients and wa-te products in and out of the device. The devices of thir invention are suitable for implanting in an individual who would benefit from e~o_u.e to products pro~lc~ by the cells which diffuse out of the device. ~he invention also relates to purification of cell products from the n vitro growth of cells.

BA~A~UNU OF T}~ lNV l'h ~lON
Transplanted cells provide the potential for treating various ~;~6~g~ bec~use of their ability to detect and Le_~ol,d to physiologically important substAnc~c in the host.
Cell implantation therapy is particularly desirable bQcause the cells can provide subst~nc~s to replace or supplement natural substAncec which, due to their insufficiency or Ah-enc~ causQ ~;r--re. The release of therapeutic substAnce~ from the transplanted cells may also be properly regulated provided the transplanted cells have the n~ceccAry L~ ors and ability to respond to endogenous regulators.
Patients having ~i~e rQ as a result of the loss or deficiency of hormonQB ~ neuL U~L ~nsmitters, growth factors or other physiological subct~nces ~re con_idered to be among thorQ who would achieve significant henefits from transplant therapy. For example, implantation of pancreatic islet cells could provide in ulin as ne~e~ to a diabetic.
Adrenal chromaffin cells or-PC12 cells implanted in the br~in may provide dopamine to treat patients with Par~in~on's ~ re. Several other hormones, growth factors and other subst~ces have been identified and are discusced in P ~ application WO 92/19195, ~ ' SUBSTITUTE SHEET (RULE 26) ' WOgS/~21 - PCT~S94107190 o as potential therapeutics which could be administered to an individual usinq tr_nsplanted cells.
~ ecause cells which are implanted may be foreign to the host it is n~cor~ry to ~L~ the host immune system from attacking and thereby causing the death of the implanted cells. In addition, cells which secrete such therapeutic substA~coa may have been derived from transformed cells or have been infected with viru~e~ and may therefore present a potential threat to the host in the form of increasing the likelihood of tumor formation. At least four methods are possible to attenuate the host immune L f, ~,~VJ~? for the pur~e~ of protecting the transplant cell viability. one method involves immuno~u~ession to prevent transplant rejection. Immuno~u~lcssion may be _ccomplished through a variety of methods, including using immunG_~p~essive drugs such as cyclosporins. In another method, immunomodulation, the anti~nicity of the implanted cells is altered. This could involve att~chi~7 antibody fraqments to the implanted cells. The third method involvQs modulating the host immune system to obtain toler_nce to the implanted cells. In a fourth method, the cells to b~ implanted are contained in a device which effectively isolates the implanted cells from the immune sy~tem. The ability of cont~i n~ cells to manufacture and secrete subst~ce~ of therapeutic value has led to the development of implantable devices for m_intaining cells within an individual in need of treatment.
A common feAture of i~olation dQvices is a colony of livinq cells ~.o~.ded by a permeable membrane. The tran~port of nutrients, waste _nd other products _cross the membr_ne is driven by pre~sure and/or diffusion gradients.
This movement of substAnce~ across the membrane is limited by the permeability of the membr_ne and the distance through which th-~e substA~e~ must travel. If in~ufficient trAn~port of these substA~ is provided for either the number or volume of cells, cell vi_bility and function m~y be dimin iSh~ .

SUBSTITUTE S~lEET (RULE 26) WO95/W521 2 1 ~ 7 6 2 6 PCT~Sg4107190 o Dionne, has reported that a dense metabolically active cell mass must not ~-~ ee~ certain maximum dimensions if the viability of the entire cell mass i~ to be maintained.
"Effect of Hypoxia on Insulin Secretion by Isolated Rat and C~nin~ I~lets of Tanqerh~ns", D~h~tes, Vol. 42, 12:20, (January 1993). When large ~hELoidal cell agglomerates receive nutrition from an external source, cells at the center of the cell mass may not receive sufficient nutrition and die.
Most encApculation device~ feature larger cell chambers than will allow diffusion of a sufficient flux of nutrients to ~PGL- a viable full density cell ma~. A full density cell mass is the maximum number of cells which can be maintai n~~ in a fixed volume if the entire space available for cells is occupied by the cells to achieve a minimum of cell-free space. $his number is approximated by dividing the total available volume for cont~ ng cells by the volume of a single cell.
In the cylindrical devices referred to by Aeb;~ch~r in WO 92/~9595, the diameter is larger than the maximum diameter which would !.~.p~r~ a viable full density cell mass. Accordingly, the cells of the device described in WO
92/19595 must be in a diluted sl~sp~cion at a lesser cell density. The diluted cell s~p~cion has lower overall nutrient requirements per unit volume and thus maintains es~entially full viability with the available nutrient tran~ported through the permeable membrane. The larger than optimum cell container allows for easier manufacture and s~h-Qquent manipulation than would be pos~ible if this device were made small eno~gh to ~ o~L an optimum, full density cell pack. Aebi~-h~r also refers to the use of a gellinq substance in the cell suspension to immobilize the cells into a uniform dispersion to ~ e.,~ ay~e~ation of cells into clumps. Such clumps could otherwise become necrotic due to localized depletion of nutrients within these clump~.

SUBSTITUTE SHEET (RULE 26) - - -WOg5/~521 214 7 6 2 6 PCT~S94/07190 o Several immunoisolating devices have been developed for implanting cells in a host. U.S. Patent 5,158,881, refers to a device in which cells are ~tated to be encA~ Ated within a semipermeable, polymeric mem~rane by co-extruding an agueous cell suspension of polymeric solution through a common port to form a t~ Ar exL~ate having a polymeric outer coating which en~Ap~~lates the cell s~r~ncion. In one embodiment described in the 5,158,881 patent, the cell suspension and polymeric solution are exL.u~cd through a common extrusion port having at least two co~ L~ic bores, such that the cell ~ r~n~ion is sxtruded through the inner bore and the polymeric solution is extruded through the outer bore. The polymeric solution is stated to coagulate to form an outer coating. In another embodiment of the 5,158,881 patent, the t~ Ar extrudate is sealed at intervals to define separate cell compartments col.l.ected by polymeric links.
A different approach to supply nutrients to an isolation device is to route a flowing blood supply or other physiologic fluid tl~ou~l. one or more CQ~lt; ts within the cell mass. This internalized source of nutrient mimics the structure of the circulatory system of almost all complex organisms, by providing nutrient to the center of a cell mass or tissue. These nutrients then diffuse radially outward. In one such internally fed device described in Wo 91/02498, the transplanted cells are contained in-between two ~v..~l.L~ic tubes. One end of the inner tube is grafted to an artery while the other end is grafted to a vein. A
common problem with internally fed devicea is the potential for thrombosis formation or clotting of blood within the artificial conduits which O~UL-- in relatively short periods of time. The formation of such obstructing masses cut off the flow of nutrients to internally fed devices.
In another device described by Goosen, United States Patent Nos. 4,673,566, 4,689,293 and 4,806,35S, the cells are contained in a semisolid matrix which is enc~p~ulated in SUBSTITUTE SHEET (RULE 26) WO95/W521 ~ 21 ~ 7 6 2 6 PCT~S94/07190 o a biocompatible semipermeable electrically charged membrane.
The membrane is stated to permit the pA~--ge of nutrients and factors while excluding virU~eC~ ant~hoAie~ and other detrimental agents pre~ent in the external environment.
WO84/01287 refers to devices for e~c~reulating genetically ~Lvy-~mmed living organi~ms. One of the devices referred to compri~e~ a nutrient material ~UL - ~ by an inner membrane wall which i8 ~u..v~.ded by a layer of organisms auL,o~ by an outer m~mbrane wall. The organisms are stated to provide therapeutic subst~n~sfi.
0 These organism~ receive nutrients from the inner layer.
Including a nutrient layer in the center of the device makes the manufacture of such devices difficult and eYren-eive.
For implanted devices to be therapeutic, eno~yh cells must be ~ ?nt and viable within the device to manufacture and secrete therapeutically effective amounts of a therapeutic substance. If too many cells are consuming nutrients within the device, the local ~ el.~Lation of these solute~ will drop below the minimum level required for cell viability. Cells which are located near the outer surface of the cell mass will typically receive ample nutrition, while cells located in the interior will be the first to die or otherwise become disabled. Factors which may have a negative effect on the viability of cells contained within a device are: device dimensions which position cells far from nutrients; cells with high metabolic demand; and any re~istance to diffusive tr~ o.L resulting from thick or impermeable membrane~ or unstirred fluid layers. Cell masses which become too large may inhibit diffusion of nutrients or gasses into the depths of the cell mass, re~ulting in the death of such cells and a ~oL,~_Io~linqly decreased substance ~uL~L. This phenomenon is ~e~o.Led by Schrezenmeir, et al., in "The Role of O~y~el.
Supply in Islet Transplantation~, Transmlantation ~ lin~s~ Vol. 24, No. 6, pp. 292S:2929, (1992), which Le~L Ls a ce.,L,al core of n _.oBis in islets greater than SUBSTIME SHEET (RULE 26) WOg5/~521 ~ PCT~S94/07190 o about lS0 microns diameter after culturing of the islets in an ~ncaps~lating device. Additi Q~- 1 ly, the 6ecretion of other factors associated with lysis of dead cells may be harmful to the host or adjacent cells.
Another method for maintaining the viability of cells within an ~ncap-ulating device is to make the device sufficiently narrow to keep the cells sufficiently clo~e to the permeable membrane in contact with the environment.
Decreasing the device diameter however, results in a finer, more fragile stru~L~e which is increasingly hard to manufacture and use.
Another requirement of enCAp-~l 1 Ation devices is that the device must have sufficient mech~nical sL~e~,~Lh and a geometry suitable for allowing the device to be manip~lated by a S~l~eG~. during implantation. Mech~nical integrity allows immunoisolating devices to be manipulated as a unit.
SL.e~.~Lh requirements will vary with the size, weight and shape of the device, but in general, the longer, heavier, or larger the device, the ~L~G..Yer it will have to be. As the number of cells reguired to provide therapeutic benefit increases, the size of the device and the amount of structural material must also increase. The additional structural material n~ ry to manufacture a larger device may interfere with the function of the device if it r~ c~
cell viability or the trA~-I,Y.L of therapeutic subst~nce .
Suitable geometries for implantation might include a size and shape which can be ~An~le~ aseptically using gloved hands and surgical instruments, and which will fit in the intenAD~ implant sites within the host.
Various methods have been described for filling devices with living cells. Some devices are filled after the permeable membrane is formed by fl~hi~g a cell s~spen~ion into the device, while other devices are proAl~ce~ by forming a membrane around the cell mass using a chemical ~Locess which causes the membrane to form without killing cells. In the former, the device is easier to load with viable cells SUBSTITUTE SHEET (RULE 26) WO95/~ PCT~S94/07190 1~762B' o if the dimensions of the device are large ~o~gh to allow low shear flow of the cell su~pen~ion. In the latter example, larger device dimensions al~o en~n~-manufacturability as a greater ~o~Lion of cells remain viable becau~e they are protected from the membrane formation ~L~ by the ~L~ nc~ of an un~tirred fluid layer and are more di~tant from the site of membrane formation. A disadvantage of larger device dimensions G~ when cell~ near the surface are triggered to ~e_~G-.d to a stimulus which may not reach cells situated more internally therefore dimini~ing the release of the therapeutic substance from the internally situated cells.
It is therefore n~-eF~-ry to develop a device of suitable geometry and ~L.c..~Lh which can provide an adequate number of viable cells and which may be in~erted in an individual.
S~ RY OF T~ Yl~ r-l - ON
This invention provides an implantable device for providing therapeutic subst~n~-a to an individual in need of treatment. The invention maximizes the ~.o~G.Lion of cells in clo~e proximity to a membrane in contact with the environment while maintaining a geometry which is practical for implantation in the individual. This is accomplished by providing a device comprising a core su.~ou,.ded by a permeable membrane wherein the outer surface of the core and the inner surface of the permeable membrane define a ho~ ry for a zone in which cells may be contained. The maximum distance LcL~ - the outer core surface and the permeable membrane is sufficiently narrow to provide conditions suitable for survival and function of the contained cells, whereby the viability of a large proportion of the contained cell mass is ~U~G~ Led. Preferably the core i~ substantially cell-free.
In one embodiment of the invention, a device for providing substa~-6 derived from cells containe~ in the SUBSTITUTE SHEET (RULE 26) WO95/0~21 PCT~S94/07190 o device comprises a core having a an interior region and an outer hol~nA-ry ~ul10l~ lin~ the interior region. A zone for containing cells substantially ~u~,v~.ds the core and extends from the outer ho~ y of the core to an inside surface of a permeable membrane. The permeable membrane ha~
inside and out~ide surface~. The distance from the outer ho~nA~ry of the core to the inside surface of the permeable m mbrane is sufficiently thin to ~ L the viability of cells in a cell layer located clos-~t to the outer ho~nA-~y of the core and most distant from the inner surface of the permeable membrane.
In another embodiment, the distance from outer hol~nA-ry of the core to the inside surface of the permeable membrane is defined by a diffusion length parameter of less than about 500 microns.
In another embodiment, the core and the permeable membrane are ~v~Lioned such that a diffusion length parameter (DLP), which is mea~ured on a crosa-section of the device taken perpendicular to a long axis of the core and passing t~u~1. the core, cell zone and permeable membrane at a point along the long axis of the core where the cell zone i~s sufficiently thick to contain at least one cell layer, is less than _bout 500 microns. The DLP is defined a~ the ratio of the total area of cell zone of the cross-section divided by the perimeter of the cell zone. The perimeter of the cell zone is defined as the length of the permeable membrane of the cro~s-fiection.
The device~ may be implanted directly into a host to provide substAnc~s proA~c~ by the cells contA i n~ within the device.
0 In a preferred embodiment of this invention an intern_l core is provided in the device which allows for a greater number of viable cells to be maintain~ within the device than would be possible if the core were ab~ent from the device.
The device of this invention also provides for a more SUBSTITUTE S~lEET (RULE 26) WOg5/~21 PCT~S94/07190 21~Y626 g o rapid rise to a plateau level of released substAncec by decreasing the transport delay of environmental stimuli to the cells, and by decreasing the c~L-e~ ng delay aR~ociated with diffusion of therapeutic substAnc~ out of the device.
In another embodiment of thi~ invention, a method of treating patients in ne-d of supplemental or replacement therapy is provided by implanting into ~uch individuals the device~ of this invention con~ain;nq cells capable of providing therapeutic substan~
Another embodiment of this invention provides a surface within the zone for contAining cells which increases the number of attachment sites for the ~u~v~ of Anchnrage dep~nA~nt cells.
This invention also provides a method of producing the cell contAining devices. This method comprises providing an exterior membrane comprising a lumen wherein the membrane is impermeable to cells but permeable to both nutrients and the therapeutic substance proA~ A by the cells. The method further compri~es providing a cell displacing core and i~ vducing the core into the lumen of the membrane to create a zone for maintAinin~ cells. The zone for maintaining cells is defined by the s~rface of the core and the inner surface of the membrane. Cells are i.,~ol~ceA
into the zone for maintAining cells and the membrane is s-aled so as to contain the core and cells within the device.
Another embodiment of this invention provides a method of separating cells from a bioreactor to facilitate the purification of cell products.
An ob~ect of this invention is to provide an implantable device which maintains cells in a viable state.
Another object of this invention is to provide device~
having a space filling core which enables a greater number of viable cells to be ContA; n~A within the total volume occupied by a cell ~ncAp~ ation device.

SUBSTITUTE SHEET (RULE 26) WO 95/04521 PCT/us94/07190 ~47 6~6 lo -o R~TFF ~CRIPTION OF THF FTGU~
Figures lA and lB. Transverse (lA) and longitl~Ai~a 1 (lB) cross-sectional diagram of cylindrical device with a core illustrating the permeable membrane (l), the zone for sustAining cells (2), cells (3), core (4), and ~u,-~Lictive S ~als (5).
Figure 2. Diagram of cylindrical devices including coils (6 ) ~ longit~A i na 1 ridges (7), and bumps (8) which act to center the core within the per~-hls ~embrane.
Figure 3. Diagram of cylindrical device including the following surfaces ~uLLo~ g the core to provide an increa~e in the number of surface sites available for attachment by a~c~nrage ~erenAsnt cells: a miuLG~G~ous eYpan~e~ polytetrafluoroethylene (PTFE) (9), an a~e~ation of cell culture mi~-o,l~h~res (l0), and a fibrous matte (ll).
Figure 4. Diagram of cylindrical device wherein core material (12) is distributed tl~ù~gl~o~L the volume enclosed by the permeable membrane (l), and wherein cells (3) pqp~late the more peripheral ~p~l-e 8 provided in the core material.
Figure 5. Graphical illustration of the increase in cell pop~lation over time for four devices (labelled +, ~, and x, re_rectively), ~ A with CGT-6 cells. Cell number is based on measurements of glucose consumption.
Data from a first device is omitted because that device was cultured under no~ an~ard conditions.
DF~AT~Fn D~RTPTION OF T~ .V~ ON
The devices of this invention are comprised of a core ~ULL.~ by a permeable membrane and a ~pace bounded by the core outer surface and the permeable membrane inner surface. The space in between the core and the permeable membrane is a zone capable of maint~ ng cells. The device may have any geometry which allows for the mainten~nc of the cu~ ace-permeable membrane relationship. This geometry may include but is not limited to spheres, SUBSTITUTE SHEET (RULE 26) WO95/04521 21~76,26 PCT/USg4/071gO

o cylinders or sheets. Cy~ ors and spheres are preferred.
Mo~t preferred are devices wherein the permeable membrane and the core _re both cylindrical having longit~A i nA 1 aXQ8 which are sub~tantially parallel to each other. Figures LA
and lB illustrate a cylindrical device of the invention and shows the perme_ble m~hrane (l) ~UL~ ling the device, the zone (2) for contAinin~ cells (3), and the core (4).
Cells contained within the device~ of this invention obtain nutrients from the environment outside the device.
The devices of this invention provide greater oYrh~q of nutrients and wa~te~ LcLwaen the cells within the device and the external environment by locating the cell mass in close proximity to the permeable membrane in contact with the outer environment. This close proxi_ity of cells to the permeable momhrane is achieved by displacing cells from the inner portions of the device by the ~f~n~o of the core.
The core of the device is inert in that it is not prim~rily intenAo~ or reguired to provide diffusible nutrients to be u~ed by the cells in the device. Al~ho~yh the core i~ not primarily designed to ~upply nutrients, it m_y be treated to provide a surface which promotes adhesion or provide substa~c~ which promote the ~urvival, growth, or function of the cells providing therapeutic cell products.
Preferably; such cell promoting subst~nce~ are adsorbed on - the outer surf_ce of the core. Collagen, poly-L-lysine, l~minin, fibronectin, and porous PTFE are among the sub~tanroa which may be adsorbed to the core to promote cell growth and/or functions. In addition, the core may have an outer porou~ layer which serve~ as a matrix for attachment and maint-nanc~ of Anchorage deprn~nt cells.
The cores of the device of the invention may be considered to have an interior region and an outer ho~ln~ary 0 .1ing the interior region. Di~ferent regions of the core may have the same or different porosities and may be porous or no~rorous. However, the porosity of the core should ~ve~.~ the migration of cells into the interior SUBSTITUTE SHEET (RULE 26) W095/04521 2 1 4 7 6 2 6 PCT~S94/07190 o region of the core which should be substantially cell-free.
~ esides displ ACi ng cells from the interior of the devicQ, the core al~o ~ervQs to provide rigidity to the devicQ which facilitates manip~lation during manufacture and implantation and retrieval by the health care provider. The S core may be any shape. In one embodim nt, the shape of the core is substantially the same a~ that of the permeable m~mbrane. In another e~bodiment, the core may be spherical or cylindrical with ~Lo~L~sion~ ex~-~A~n~ out from the core ~urf_cQ. The~e ~ro~ ions, for ex_mplQ, and a~ illustratQd in ~igure 2, may be coils (6), ridges (7), or bumps (8).
The height of each ~L~LLusion may be de~igned to define the minimum space in between the core surfacQ and the permeable membrane inner surface and may aid to center the core within the device.
The space hot~nAeA by the core and the permeable mQmbr_ne, optionally m_y contain subst~nc~ to promote cell growth. Such subs~Ar:f~ may include m_terial to provide a ~ rL on which ~ r~ge depQnd-nt cells may adhere.
Preferably, a ~L 0~-~ nQtwork iS provided throughout the ~pace provided for cell growth in ~eL~aLn the core and the permeable membrane. Suitable material for the ~u~GLL are solids including but not limited to eypAnA~A or porous PTFE, dextran, collagen, polyester, poly~LyL~ne and other natural or synthetic polymers which promote cell attachment. T~e !~Ll~L may be ~e~nt in the cell zone in various forms including, among other~, random or trAh~ ar networks, micro~pheres and fibrous matte~. Figure 3 illustrates examples of solid ~ ~v~Ls such as mi~ ous eYr~A~A PTFE
(9), an a~Le~dtion of cell culture mi~ rh~res (10), and a fibrous matte (11). The solid ~u~ in the cell zone may also contribute strength to the device and aid in maint~i n i n~ the shape of the device.
In a preferred embodiment, the geometry of the device maintains the viability of cells in cont~ct with, or in clo~e proximity to the core. Cell viability may be assessed SUBSTITUTE SHEET (RULE 26) W095/~1 ~; 2 1 ~ 7~%~6 PCT~Sg4/07190 O using various indicators of cell function. For example, the ability of cells to exclude certain dyes, such as trypan blue, which aceumulate in dead cells may be used to asse~
cell viability. Evi~sncs of cell viAhility may also be ba~ed on ~ ~3ments of basal metabolism or cell proliferation. The d~monstration of synthe~is of cell produets is also indicative of cell viability. A conclusion of cell viability may be ba~ed on the detection of any one indicator of cell vi~h~lity.
Cell viability may be asse~ed prior to or after implantation. H~we~er, it i8 preferred to a~ - viability prior to implantation l-ec~l-e of the potential for interactions between the device and the environment which compromise cell viability in a manner which is unrelated to the geometry of the deviee.
In a preferred method of a~sessing cell viability, the device containinq cells is cultured in vitro for a period of time sufficient to allow the por~lation of cells to reach a plateau or steady state. The medium for culturing the deviee ~h9~ eontain sufficient-c ~..L.ations of nutrients to maintain the viability of the number of cells at the plateau level if such cells were grown in eulture without the device. In addition, the medium ~o~ be sufficiently repleni-~ to avoid cell death due to depletion of nutrients from the medium outside the deviee. Evidence of havinq r~a~h~ a plateau may be based on a stable metabolic rate.
To a~ c cell viability in the device, the number of viable cells most distant from the permeable membrane and forming a perimeter -UlLo~ lin~ the substantially cell-free core is determined. The perimeter of cells which are a~ for viability will be e~sentially the first cell layer ~Lo-lling the substantially cell-free region of the core. Preferably, at least about 10% of the cells in the first cell layer will be viable. Nore preferably, at least about 50% of the cells in the first cell layer will be SUBSTITUTE SHEET (RULE 26) WOg5/0~21 21~ 7 6 2 6 PCT~S94/07190 o viable. Most preferably, at least 80% of the cells in the first cell layer will be viable.
If a vital dye is to be used to a8se8s viability, the dye is added to the culture medium, or injected directly into the cell space of the device, when the number of cells has rD~h~ a pl_teau. To a~ cell viability, the device i~ removed from the medium after a sufficient time to allow the dye to diffuse into the device, the device is cross-sectioned, and the number of viable cells in the first cell layer -u..o~-linq the core io determined as described above.
Another method of a~r~~Fing viability may consist of pulsing the cells at the plateau phase with a radioactive y~e_~Oor for a metabolic product and determining the pec~e-~L of cells in the first cell layer which i-l~GL~o~ate the yLF_~OoL into a product. Autoradiography may be used to lor~lize the radioactive ~,G~u~L. BQcause the r_dioactive pLe_~k~or and the yLG~L may be y~-ent in the same vicinity, the analysis may be done in conjunction with immunolabelling of the newly synthesized cell products.
The fir~t cell layer closest to the core may be irregular in shape due to the core geometry or the substance uOed as the core material. In addition, and as illustrated in Figure 4, the core material may be comprised of the same material u~ed to provide a ~ ol~ for cells in the cell zone. If the core material is the same material as ~LI~-ent in the cell zone, the porosity of the material comprising the core preferably ~Oltl~ be ~uch as to ~Le~e,-L cells migrating to a distance from the permeable membrane where they will not receive sufficient nutrition to remain viable.
Another parameter for determining dimensions of the preferred devices of the invention is the diffusion length parameter (hereinafter, "DLPn) which is y.G~LLional to the thickness of the zone for maintAining cells. DLP is determined by ~A~i ng a planar section through the device, where such section is taken perpendicular to a longest axis SUBSTITUTE SHEET (RULE 26) WO95/~521 21 ~ 7 62 6 PCT~S94/07190 o of the device and p~R-e~ through the core, the permeable membrane, and the cell zone ~t a point sufficiently thick to contain at lea~t a single layer of cells. The total are~
available for cells L~ .. the substantially cell-free region of the core and the inner perimeter of the permeable m~brane i~ determined from direct or mi~L~--opic observations. DLP i5 then calc~lAted by dividing the total area available for cell~ by the length of the inner perimeter of the permeable me~brane. In calculating the DLP
for devices con~ni ng cell displacing subst~nces in the cell zone such as a solid ~ Y~L, the entire area of the cell zone is used to calculate DLP, without subtracting the area occupied by the ~u~o~-.
St~n~-rd methods may be used to determine the area beL~-En the core and the permeable membrane available for cells. In one method, the planar cross-section of the device is photographed. The area for cell growth is then cut out and weighed. To determine the area, the resultant weight is then-divided by the average weight per unit area of the photc~ 1,ic paper, and scaled by the a~ iate magnification factor of the mi~&~~o2e and camera.
DLP values are preferably less than about 500 microns.
More preferably, DLP values range from about 25 to 250 microns, and most preferably from about 50 to lOO microns.
Preferably, the thickness of the cell zone and the cG~ on~ing DLP value is inversely ~Lv~G~ional to the square root of the metabolic activity of the. cells to be contained within the device. In addition, the thic~ne~ and DLP values are directly ~LV~O~ Lional to the square root of the concentration diffe~ _e between available nutrients and ro~ LL ations of nutrients required for survival.
Accordingly, the~e relatic -~ipe may be used to design devices of various geometries which are particularly well suited for use. with a particular cell type.
By determining a preferred geometry for one cell type, other devices may be constructed for other cell types by SUBSTITUTE SHEET (RULE 26 214762~

o comparing the metabolic activities of the cells and modifying the design in accordance with the relation~ e described above. For cells having a metabolic rate, of about 1 mg glucose/(ml of cells ~ min) in a medium cont~ining glucose at 5 mg/100 ml, the most preferred DLP
value ranges from about 50 to 100 microns. Preferably, ox~_.. con~umption would be used to de-ign devices having preferred geometries. For example, the preferred DLP range for cells utilizing oxygen at a rate of about 4.6 x 10 moles ox~e.-/(ml of cells ~ sec) is also about 50 to 100 microns. (Values for glucose and oxygen consumption are based on a cell volume of 2000 femtoliters~cell.) This value assumes the diffusion coefficient of ox~.. in culture medium to approximate that of water, the partial pressure of ox~e.. to be 120 mm of meL~.y, and that the permeable membrane contributes negligible resistance to the diffusion of nutrients.
Al~ho~gh the p~enre of any core will provide the advantage of increa~ing the p~o~o,Lion of cells near the permeable membrane, the preferred devices have cores which increase the number of cells which may be packed in a given volume compared to devices without cores. In devices without cores, larger diffusion di~tAnce~ result in lower ~c. ~ ation gradients, which in turn result in lower net flux of nutrients and lower cApacity for viable cells.
In addition, cell~ in the interior of the device which do not receive ~no~gh nutrient_ may die and release Qubsta~c~c toxic to the cells in clo~er proximity to the permeable membrane thereby causing the death of even a greater number of cells. Accordingly, a greater number of cells may be viable in the An~ Ar space or zone created in between the outer core surface and the internal surface of the permeable membrane than would otherwise be viable if the core was not pre_ent. A limit will be reached where increasing the core size no longer im~o~3 viability, and ~imply displaces viable cells.

SUBSTITUTE SHEET (RULE 26) o A core of any size which displaces cells from the ce...... ...LLal portion of the device is suitable for use in the devices of this invention. Preferably, the core occupies sufficient volume to limit the dirfusion length parameter (DLP) of the device to a value le~8 than or equal to the maximum thic~nen~ to which _ given cell ma~s can be sustained by diffuQion tl,Lo~l. a given permeable membrane.
The maximum thickne~ will depend on several factors including the metabolic rate of the cellg, ~ pAr~i ng efficiency of the cells, permeability characteristics of the permeable membrane and the cell ma~s, the nutritive content of the ~uLL.~ ng medium, and any volume displaced by a porous cell growth substrate. Preferred values of maximum thickne~s for a den~e cell mass in which the volume of the zone available for cells is m_ximally pAc~eA with cells -15 based on the cell volume, range from about 50 to 100 microns for pancreatic cells and cells having similar metabolic rates and thus similar nutrit~onAl requirements, to about 500 microns for cells which are ~ ent in dilute s~lsr~n~ion or which have low metabolic d~mand. The. maximum thickness value~ will be relatively small, 1-~ thJn about ten microns, in the case where the metabolic demand of the cell is high or where nutrients are sc_rce due to unfavorable environmental conditions or poor membrane permeability.
As di~ above, to determine the preferred 25 dimensions of the device it may be ~ ~e-~ry to first determine the consumption requirements for certain critical nutrients, for example o~ .. or glucose. Method~ of determining ox~e.. and glucose consumption rates are well known to tho~e skilled in the art and are commercially available. From the determinations of the minimal cpnditions n~ces-~ry to maintain the cells in an adequate state of nourishment device geometry may be optimized.
Fick's ~QconA law of diffusion may be applied to the design of the devices of the invention. Using a cylindrical device as an example, it can be shown that i..~oducing a SUBSTITUTE SHEET (RULE 26) W095/~1 PCT~Sg4/07190 21~7626 0 core that is 90% of the perme_ble membrane radial dimension remarkably increafies the li n~r carrying capacity of a coreless device tenfold. ~hese devices of the invention have several advantages over coreles~ device~. By increa~ing the size of the device, the diameter of the device become~ larger and ~a~iPr to man;r~late. For a given cell rap-rity~ the estimated length c_n decrease further ~~rin~ manir~lation. Ea~e of ma~irllation will be im~Lo~ad during all device h-~l in~ step~, ;nCll~Ain~ manufacture, storage, implantation, and ultimately retrieval.
In a preferred embodiment, a cylindrical device i8 provided with an An~ll 1 ar cell zone defined as the space between the inner surface of the permeable membrane and the outer surface of the core material, ~uch that the average distance between the two surfacea is le~6 than about 500 microns. More preferably the space Le.~_en the core and the inner surface of the permeable membrane averages about 25 to 250 microns. Most preferably, the space between the core and the permeable m~mbrane a~ e~ about 50 to 100 microns.
An empirical method for optimizing the core size for a given devicQ is to build ~everal devices of varying core dimension, culture the devices in their intenA~ environment or analogous medium, and çhso~q the device which provides the greatest steady state mass of viable cells.
Alternatively, the device may be optimized by selecting a core which results in the greate~t production of therapeutic ~ubstance. In addition, devices of a desired diameter may be ~6.~_ LL ~cted without a core, filled with cells and cultured to obtain a constant cell mas~. If a necrotic cell mass is ob~erved upon ~ Lion of the device after it has been cultured for a sufficient length of time, a device may then be produced contai n j n~ a core. Preferably, the core diameter is about equal to, or less than, the diameter of the ~C_LO~iC cell mass.
A further advantage of the devices of this invention is the im~ovc~ e..yLh of the device. Devices without a core SUBSTITUTE SHEET (RULE 26) WO95/~521 PCT~S94/07190 21q7~ 6''-O derive all of their aL~e~Lh from the ~c~rc~ ting membrane. Long coreless devices which must be thin in order to maintain cell viability require t~icker membranes to provide sufficient -L.e..~Lh. Increa~ing membrane thi.X.~
al~o increases resistance to diffusion, and therefore decreases the number of viable cells which can be contained within the device. Tha ~LE~~~9 of a core in the devices of this invention provide the ability to add significant ~ L~ Cl~ h to the de~ice without increa~ing membrane thickness.
Another advantage provided by the ~r~-~nce of the core is a more rapid ~e~ to environmental stimuli by the cells con~ain~ within the device. ~ec~r? the core requires the cells to reside close to the encapculating membrane, the distance and thus the time required for transient diffusion to occur to the cells is r~nc~. This increase in speed is valuable in the ca~e where the therapeutic cells function by ,~-ponAi~ to a chemical signal such as a changing ~.,. e..LL~ting of a physiologic substance. The faster the si~nall~n~ substance can diffuse to the cells, and the fa~ter the therapeutic product can diffuse out of the device, the better the device performs.
Any material which acts to displace cells from the space defined by the perimeter of the permeable membrane is suitable for use as the core material. Suitable core materials may include but are not limited to porous or ~Ypan~ polytetrafluG~&~Lhylene, polydimethysiloxane, polyurethane, polyester, polyamide, or h~L~e1S derived from polysaccharides, alginate or hydrophilic polyacrylonitrile such as Hypan~. The core is preferably a flexible polymer or elastomer. More preferably, the core may be manufa~.ed from poly6~ccharides, hy~ u~hilic copolymers of polyacrylonitrile, or other polymer - components. Most preferably, core compositions such as Hypan~ comprise a copolymer of polyacrylonitrile and acrylamide. When hyd~G~el such as Hypan0 is used the water SUBSTITUTE SHEET (RULE 26) WO 951~21 2~4~ ~2~ PCT~S94107190 o content of the hydrated gel should be sufficient to provide flexibility while not PYcee~i~q a water content which allows cells to enter the core. Preferably, the gel comprising the cores is hydrated to betw-en 35 and 95%. Most preferably, the water content is about 68%. Further details of the preferred core composition and the mQthod of manufacturing the core materials are di~closed in the art, such as, United Stat~ PatQnt~ 4,379,874, 4,420,589 and 4,94~3,618.
Reaction conditions are rhr ~~ which provide a 38% ~G,.veLaion of acrylonitrile ~u~5 to acrylamide.
Manufacture of the core may be by any method known to those skilled in the art of manufacturing polymer stru~Lu,es. The core i5 preferably formed as a cylindrical rod by extruding the polymer th~ h a round die.
Preferably the core diameter when it i~ a cylinder is between about o.2 to 10 mm. More preferably the core diameter is about 1.5 mm.
The permeable membrane may be manufauLu.ed from any biologically compatible material having the appropriate permeability characteristics. The permeable membrane should permit the p~ e therethrough of cellular nutrients, waste products, and therapeutic substAncec secreted by cells cont~inP~ within the device. The permeable membrane should not allow the passage of cells and virus-s. Preferably, the permQa~le m~mbrane should serve to isolate the cells contained within the device from ~ec~J~ition and attack by cellular components of the host immune system. More preferably, the permeable membrane ~olllA serve to isolate the cells cont~i~e~ within the device from contact with molecules of the host immune syatem which function to ~o~.~ize foreign cells, to direct an attack ~g~in~t such foreign cells, or to directly exert toxic effects against foreign cells.
Ex~mples of polymers having suitable selective permeability ~u~e~Lies and which may be used as the SUBSTITUl~ SHEET(RULE 26) , ~,, permeable membrane may be selected from the group consisting sodium alginate polyhydrate, cellulose acetate, panvinyl copolymers, chitosan alginate, polyacrylates such as Eudradit RL~ manufactured by Rohm & Haas, GmbH, agarose, acrylonitrile, sodium methylyl-sulphonate, polyvinyl acrylates such as those available as XM50 available from W.R. Grace and Co., and porous PTFE.
Most preferably the permeable membrane is prepared from a polyacrylonitrile copolymer of the type described in U.S.
Patents 4,379,874, 4,420,589 and 4,943,618.

In a prefered embodiment, the permeable membrane is a hydrogel such as that available from Kingston Technologies Inc. and sold under the tradename Hypan~. Preferably the hydrogel suitable for the permeable membrane has a water content of between about 35 and 95. Most preferably the water content of the hydrogel is about 68%.
Manufacture of the permeable membrane is also preferably accomplished by extruding the polymer through a die wherein a hollow tube is formed having the appropriate dimensions. The extruded polymer, which serves as the permeable membrane, should be of sufficient diameter to allow the insertion of the core into the permeable membrane.
Extrusion of the polymer material to prepare the permeable membrane is accomplished using standard extrusion techniques. To prepare the permeable membranes for making the devices of this invention, polymer material is dissolved in a suitable solvent to result in a solution of sufficiently low viscosity to allow the solution to be extruded through a thin walled annular die orifice.
Preferably, the annular orifice is formed from a die having a 0.105 inch O.D. and a 0.095 inch I.D. to produce a tube of permeable membrane having a lumen of the desired size.
Polymer solution may be fed through the die with a controlled flow rate using a syringe pump. Coagulation of the polymer solution may be achieved by having the die WO gS/~l 2 1 47 6 2 ~ ~us94~07lgo o immersed in a coagulation bath of room temperature deion; 7~
water. Coagulation of the polymer solution G~ upon contact with the water at the die exit. Ths resultinq solidified tube may be taken up by speed ~G"LLolled capstan rolle~s and a storage spool. Tubing is then rinsed of residual solvent and stored immersed in water.
Preferably, very t,hin walled t~hing is obt~in~~. This may be accomplished by selecting a take up speed which a the rate at which tubing exits the die to cause tube stret~ing. The die exit velocity is calculated by dividing the polymer feed rate by the die annulus cross sectional area.
The wall thir~nec- of the permeable membrane is of a thir~n~ which permits the pacr~ge of nutrients and waste products and allows for the viability of the cells contained within the device. Preferably the thickness of the permeable membrane is b~,r-~., 2 and 100 microns. More preferably the ~ ness is bc~r~Qn about 5 and 50 microns.
Most preferably the thi~ne~s is between 15 and 25 microns.
The permeable membrane -~o~l~ have a molPcvlAr weight cut off (MWC0) range sufficient to ~e~e~L cells from moving into or out of the device but large e~.o~JI~ to allow the pA-~~ge of nutrients, wastes and the.a~cuLic substances secreted by cells contained within the device. The precise NMCO range will vary ~epe~ing on the membrane material, type of cells contained within the device and the size of the therapeutic cell product to be released into the D~l-o~ ing environment. Accordingly permeable membranes having a MWC0 of between 10 kD to 2000 kD may be suitable for use with the devices of this inventions. A MMC0 range of between 30 kD and 150 kD is par~icl~lArly preferred in applications where it is desired to isolate the contained cells from contact with molecules of the immune system capable of ~-0~'31~i 7ing or de_~.oying the contained cells-In a preferred method of manufacturing a device of this invention a core and permeable membrane are separat~ly SUBSTITUTE SHEET (RULE 26) WOg5104521 PCT~S94/07190 21~7626 o prepared and the lumen of the permeable membrane is PYran~P~ with liguid or gas to allow for the insertion of the core component. Once the core is inserted into the lumen of the permeable membrane various methods may be used to ~eal the device.
The core may be inserted into the permeable membrane b~fore or after the device is ino~lAted with cells. Cores may be loaded into the permeable me~branes inco~pletely hydrated or swelled. In a preferred method of preparing the device a substantially dry, or incompletely hydrated core is inserted into the lumen of the permeable membrane. Cells may be added before, with or after the insertion of the core. After sealing the device, the device is placed in an environment that allows the core to ~well to the a~v~.iate size. In another method of preparing the devices of the invention, cells are prepared in a slurry comprising cells, media and a hydrated Hypan~ core. This slurry is then injected into the permeable m~mbrane which has beQn previously rAnn~lAted with a stainless steel ~tlh;~g and ~teril~zed. Once the core is located inside the tubing, the stainless steel tube is removed and the ends are sealed.
The ends may be ~ with surgical ligatures clips or by other suitable means. In a preferred embodiment, a short length of biocompatible tubing is placed over the ends of the device to provide a cu.,~ LL ictive seal. Figure lB
illustrates cG,.-LLictive ~eals (5) which seal the permeable m~mbrane to the core. Such seals may be made of substAnc~e such as silicone rubber or PTFE. S~-l ing of the ends may also be accomplished by allowing the core to swell into a re~trictive collar.
The devices of this invention provide a source of therapeutic substance by virtue of the ability of the cells within the ~nc~p~ ting device to manufacture and secrete such therapeutic subst~nce . Accordingly, the device should contain a sufficient number of cells to provide a therapeutically effective amount of substance. The SUBSTITUTE SHEET (RULE 26) WO95/04521 PCT~S94/07190 21~762~

o advantage of the geometry of the devices of this invention is that the ~L ~ ~en~ of the core material increases the ~ oL~ion of cells in proximity to the permeable membrane.
This increa~ed proximity re~cea the ~Lv~vLLion of cells which do not receive sufficient nutrients because of their distance from the permeable membrane.
Another advantage of the devices of this invention is that cells can be encapsulated in a m~n~ge~hle device such that the cells are in sufficient quantity to provide therapeutic amounts of cell products and are sufficiently close to the permeable membrane to avoid deleterious effects of cell ne_LG_is which may occur if the diameter of the permeable membrane is so large that the cells in the inner portions of the device are not able to CY~Ange nutrients and waste. This invention avoids such effects by the ~L~7~n~e of a volume displAc;n1 core cont~in~ within the permeable membrane which effectively ~Le~-..Ls cells from becoming too distant from the permeable membrane surface.
An added advantage of this configuration is that the device is kept sufficiently large to be easily manip~ ted during implantation and removal.
Various types of prokaryotic and eukaryotic cells may be used with the devices of this invention. Preferably the cells secrete a therapeutically useful substance. Such subs~Ance~ may be hormones, growth factors, trophic factors, n.~L~LL~nsmitters lymp~o~i n es, an~iho~ies or other cell du~Ls which provide a therapeutic benefit to the device recipient. Examples of such therapeutic cell products include but are not limited to in~-l;n, nerve growth factor, interle~ki~C, parathyroid hormone, erythropoietin, albumin, 0 transferrin, and Factor YIII.
The devices of this invention may be used to provide trophic substAncec to treat various nc~odegenerative disorders. Such factors include but are not limited to nerve growth factor (NGF), and other members of the NGF gene family including brain derived neuLoLL~hic factor, SUBSTITUTE S~tEET (RULE 26) WO95/~1 PCT~S94/07190 21~7626 ~ 25 o neuLG~ophin-3 and neurotrophin-4; ciliary nch~uLLuphic factor; and basic fibrobl_st growth factor.
Cells such as PC12 rh~schromocytoma cells may be implanted in the devices of the invention to provide neuLG~ansmitters such a8 dopamine to provide therapy for Par~in-on~S ~ Fe. Cells providing other ~nsmitters may be used ~ well.
m ose skilled in the art will L~eO-J~i ze the wide v_riety of cell products u~ful for treating various disorders which may be pro~vce~ by the cells used to seed the devices of this invention. PCT application W092/19195 of Dionne published November 12, 1992, - -describes various cell types suitable ~~
for use with immunoisolating devices and their application.
Cells which have been genetic_lly altered to contain at least one additional nucleic acid sequence related to the expression of a therapeutic substance may be particularly useful to be included in the cell zone of the devices of this invention. These qenetically altered cells are distingll~shAhle from naturally o~ ing cells which do not contain the additional nucleic acid sequence. The additional nucleic acid seq~lencP~ may be heterologous or homologous ~o the cells expressing the therapeutic substance. In addition, the additional nucleic acid seql~ences may code for the therapeutic substance itself and/or comprise nvn co~ling seq~ ces, e.g. regulatory or _nti-ense -se~lences which modify the e~p~ession of endogenous genes. Among the form~ of nucleic acid seql~Pnces which may be useful for having been inserted into the genetically altered cells are intronless co~ing se~n~es (i.e. cDNA), copies of genomic genes, and regulatory se~lenc~s. The additional nucleic _cid seq~lences may be comprised of sequences obta i n~ from other cells, viruses, or synthetic sequences.
The size of the device to be implanted will vary dependinq on the number of cells nec_ssAry to provide - SUBSTITUTE SHEET(RULE 26) . ', ~_J

- -WO95/~521 PCT~S94/07190 o sufficient amounts of therapeutic subst~ncec and the location of the device. Preferably, the device to be implanted in a human would be cylindrical and have an over~ll length of be~ een about 0.5 cm to about 3 meters.
Multiple devices may be implanted in a singlQ individual.
Devices may be implanted anywher- in the recipient which allow the device to receive the necessary environmental stimuli to re~pond by releasing therapeutic sub_tA~c~ and which provide the neC~ ry nutrients to maintain the viability of cells within the device. Suitable locations for implanting devices include but are not limited to the perito~~-l cavity, cerebral ventricles or inside blood veQ--lc. Devices may also be located subcutaneously or intramu~c~ ly.
To treat indivi~ in need of treatment in accordance with the method of this invention, at least one device of the invention is ---A~~ with cells which will provide the r~C~ ry therapeutic substance. After establ~in~ the device in culture, the device is transferred into the individual in need of treatment. As ~i~C~Q--~ above, the devices may be inserted in various sites throughout the body, by means such as intrava~ lar suspension, subcut~neo~c or intraperitoneal insertion, through ~- O 'ed~L es now known or later developed.
Sufficient numbers of cells are inserted into an individual to produce therapeutically effective amounts of the therapeutic substance. One or more devices may be used to achieve the requisite number of cells. The amount of therapeutic substance pro~ce~ by the cell cont~ini~ device may be estimated based on the production of the therapeutic substance by the device in t;~ e culture outside the individual. Ba~ed on such in vitro measurements, the ~vL.e~L size and number of devices cont~inin~ the a~v~-iate number of cells may be determined.
~he devices of this invention may also be used to 3 prepare cell products such as therapeutic subst~nces. For SUBSTITUTE SHEET (RULE 26) W095/~1 21 ~ 76~ 6 PCT~Sg4107190 o such applications, the devices of the invention are ~
with cells and cultured in v;tro for a sufficient time to allow for cell products to diffuse out of the device and into the cell medium SUL~O~ ; ng the device. The therapeutic substan~-- may then be isolated from the culture medium without being contaminated with cells. Having the cells contained within an easily removable device facilitate~ substance purification by eliminating the burden of ~L. -~res n~ r-ry to remove cellular components from the culture medium.

Devices were made and tested with cells ~a vitro to demonstrate that a stable viable cell pop~lAtion could be achieved.
.15 A. ~Ytru~ion Of T~hnlAr MerhrAn~
Hypan0 tubing (HN-68 obtained from Kingston ~ .Glogies, Inc.) for use as the permeable membrane was prepared by dissolving polymer (10% w/w) in a solvent consisting of an aqueous solution of 55% NaSCN resulting in a polymer solution of sufficiently low viscosity to be extruded through a thin walled Ann~lar die orifice. Using a syringe pump, the polymer solution was fed through a die at a ~ olled flow rate of 10 ml per hr. The die used for prQA~ ng the permeable membrane consisted of a 0.105 inch OD, O.09S inch I.D. Ann--lAr opening and wa~ immersed in a coagulation bath consisting of room temperature deionized water. Coagulation of the polymer solution G~ upon contact of the polymer with the water at the exit of the die. The resulting solidified tube was taken up by capstan rollers at a ~G..L~olled speed of 3.5 feet per minute and t~e ttlhi ng was rinsed of any residual solvent. The resulting t~hinq was stored immersed in water. Stretçhinq the tube as it coagulated by the above t~ellp speed resulted in very 3 thin walled tubing. The resulting permeable membrane was SUBSTJTUTE SHEET (RULE 26) WO95/~1 PCT~S94/07190 o measured to have an outside diameter of 1.95 mm and a wall thic~es~ of 15 microns.
A Hypan~ core of HN68 was ex~uded with a re~ulting diameter of 1.5 mm.

B. S~ina The Device W~h C~llc CGT-6 cells, a genetic~lly engineered murine cell line described in ~l-ghPa SD et al. Proc. N~-l. A~. Sci., USA;
89:688-692 (1992), grown to confluency in T75 flasks, were prepared for ~e6~ ~ nq into the device. Cells were trypsinized, centrifuged and resuspended in 5 ml of medium (DMEM contAininq 450 mg/dl of glucose). A slurry consisting of cells, media, and hydrated Hypan~ core was injected into five lengths of Hypan~ tubing each of which had been precAnnl~lAted with a length of stainless steel tubing and steam sterilized. Once the core was located inside the tubing, the ends of the tubes were ~ with surgical ligating clips, ra~ by a small square of silicone rubber.
The five devices were each placed in a separate well of a 6 well ti ~"? culture plate. Unloaded cells were placed in the sixth well to determine the viability of this por~lAtion of cells after the lo~ing ~o~el re. All wells were filled with 4~ml of 450 mg/dl glucose growth medium. The medium was replaced every day with fresh medium. The exhausted medium from each well was ret~i~e~ and frozen for subsequent analysis. An estimate of the por~llAtion of the devices was determined by measuring the glucose consumption. Previous experiments determined that 1 million CGT-6 cells consumed glucose at a rate of 3 mg/day.
Devices were c~e~ with cells at day zero and glucose consumption was continuously monitored through day 83.
Between day 83 and day 114, the medium continued to be changed althol~gh less frequently and glucose consumption was not measured. At day 114, the devices were removed from the medium, examined, measured and the cells harvested.
Harvested cells were treated with trypsin and counted using SUBSTITUTE SHEET (RULE 26) -WO 9S/04521 PCT/US94tO7190 7,6~?C

o a Coulter counter.
Immediately after s~e~ the devices with cells, the devices had a uniformly milky App~rance. Within one day and for approximately 3 weeks thereafter, the cells colonized the lowest part of the device to form a line of agglomerated cells parallel to the length of the device.
Over time, the cell number increased until the cells were confluent and the cells appeared to have grown around the core completely o~ ing the ~nn~lAr space formed between the core and the permeable membrane. In the final months of culture, the devices appeared fully packed, having a superconfluent AppeArance. Altho~gh the core was obscured from view, its outline could be approximated at the center of the cell mass.
Due to the ~L ~ ~enc~ of cells outside of the device, some of which adhered to the silicone rubber pads, the devices were rinsed daily to remove cells not cont~i n-~within the device. After the rinses, the devices contAin-~a large superconfluent cell mass with trace clumps of cells observed on the silicone padQ.
Measurements of the device dimensions were determined from photomi~rGyL~hs taken of the devices immediately prior to harvesting. Table 1 shows the dimensions of the five devices. Precise measurements were not obt~in~ for device no. 5 which did not photograph with sufficient clarity to allow for length determinations.
TART.F 1 Approximate Approximate Device # ~ eter nPnnth 1 1.8 mm 22.9 mm 2 1.8 22.0 3 1.67 31.5 4 1.67 18.5 1.47 >12.8 (lenqth uncertain) SUBSTITUTE SHEET (PIULE 26) WO 95104521 2 1 ~ 7 6 2 6 PCTtUS94tO7190 o Figure 5 shows the relationship between days in culture and glucose consumption. Data between day 1 and day 43 is omitted because cells were found to be y~-~nt in the medium outside the device and contributed significantly to glucose conQumption. An increa~e in the gluco~e consumption over time up to a plateau level was observed in all of the deviceQ tested.
Viability and cell number for the five devices at the termination of the experiment (114 days in culture) is shown in Table 2. Viability for the five devices averaged about 85% with cell numbers ranging from 5.4 x 105 to 1.6 x 106 cells per device.
TARr~ 2 A~oxLmate Device # Cell N~hDr ~x lo6) Vi~hil;tY (%) 1 1.6 80 2 0.94 89 3 1.3 8g 4 0.54 84 0.81 84 F~MPT-F~ 2 Another encapsulation device proAl~e~ to demonstrate viability was constructed utilizing a thin walled (15 micron) tnh~ r permeable membrane with an external diameter 25 of 1.95 mm cG-Llucted of a polyacrylonitrile-acrylate copolymer as described in U.S. Patent NOQ. 4,943,618, 4,379,874 and 4,420,589. This device was prepared by injecting into the permeable membrane a liquid slurry comprising a suspen~PA quantity of viable CGT-6 cells and a porous PTFE space filling core 2.5 cm in length and 1.75 mm in diameter. The ends of the device were sealed using st~n~rd surgical ligating clips with a small (5mm x Smm) square of silicon rubber used to augment the clip function by limiting stress on the tube membrane.
The CGT-6 cells were prepared for slurry injection into SUBSTITUTE SHEET (RULE 26) WO95/~21 ~ ~ 7 PCT~S94/07190 o the device using trypsin, followed by centrifugation and p~n~ion in cell culture media.
Immediately following assembly, the device was placed in a tissue culture well of a 6 well plate, immersed in 4 ml media and inc~lhAted under st~n~rd conditions a~L G~L iate S for th- cells in free culture. The initial appearance of the device was translucent. S~p~n~~~ cells were observable L~o~}. the clear permeable membrane.
Within one day, the cell slurry had settled to form a curvilinear agglomeration approximately defined by the lowest portion of the device as it lay in the cell culture well. Over the next three weeks, this conformation changed as the cell colony multiplied and occupied larger areas of the An~nlAr space, forming a translucent amorphous mass.
Glucose uptake was measured as an indication of cell viability and proliferation of cells in the device using the same co..vL.sion factor described in Example l. Glucose uptake was mQasured as the diffe~e~.~a betl3en glucose levels mea~ured in the media ~Lo~ inq the device over a 24 hour period, and the glucose level in a ~..LLol well. Glucose uptake of the device was initially low, and increased over the term of the device until it stabilized at a level which cGLLe r~on~~~ to the ~L~-ence of 2 x lO6 cells in free culture. Insulin production of the device during the period when the device population was stable was consistent with a 2S p~p~lAtion of 2 x lo6 cells.
At many times during the culture of this device it was n~c~ ry to manipulate the device in an aseptic manner with surgical instruments. This handling consisted of grasping the device with forceps, bathing it with a stream of media, and placing it into another contAi~r.
A final evaluation of this device was performed after a term of 56 days. The intact device was treated with a viability stain which stAin~ living cells a green color and dead cells a red color ("Live/Dead TM Viability/cytotoxicity assay, Mol~c~lAr Probes Inc.). Subsequent gross examination SUBSTITUTE SHEET (RULE 26) W095/~521 PCT~S94/07190 21~76~6 o of the stained device revealed a high degree of viability as indicated by a generally green hue. After evaluation of the intact device the device was cut opened and the cells were prepared for Coulter counting by trypsinization. Viability was also evaluated. Cell counts of 1.8 x lo6 cells obtAi~
from the Coulter counter substantially agreed with the 2 x 106 cells ectimated from the glucose uptake estimates. Cell viability was determined to be 82%.

FX~MPT.F~ 3 Another device having the following dimensions was constructed essentially as described in Example 1:
permeable membrane inner radius: 800 microns core outer radius; 725 microns permeable membrane thi~n~sF: 25 microns This device was 6ee~~~ with approximately 1.4 x 106 cells and was cultured for 51 days with daily media changes.
The number of cells ~L~qnt in the device after 51 days was e~tim,ted at 1.6 x lo6 cells based on daily glucose consumption measured in the media.

Flrl~ ~ 4 Four devices similar to those described in Example 3 were prepared for implantation into dogs.
To specifically adapt the devices for implantation into the va~c~lar system, a small bullet ~~peA piece of stainless steel was attached to one end of each device using an elastomeric silicone tube to aid in fluoroscopic and radiographic localization of the devices. Also, a continl~o~ loop of ~u~uLe waB pa7-~~ through the opposite end of each device to act as a tether.
A device was surgically i~,LLo~ ce~ into the external jugular vein in each of four adult Greyhound dogs. The tethering suture was used to affix the device to the wall of the vein. At seven days post-implantation, the device~ were SUBSTITUTE SHEET (RULE 26~

WO95/~521 1 ~ ~ ~ 6 PCT~S94/07190 o retrieved.
One of the devices retrieved from the animals yielded the following viability estimate~ by three different methods of assessment: 56% by glucose consumption, 62% by Live/Dead~
Viability/Cytotoxicity Assay, and 58% by insulin production.
Overall, this particular device ~hc~e~ after seven days ~n v vo a viable popl~lAtion of cells that was 50-60% the size of its pre-implantation value.
Any viability demonstrated after seven days of m v vo implantation is indicative of proper functioning of the device as defined by tra~ olL of nutrients to the cell mass. It has been previously noted that the viable popl~lAtion which can be sustained within a device d~p~n~ on the conc~ntrations of nutrients in the environment external to the device. This may explain the decrease in viable porl~lation observed when the device was removed from tissue culture media and implanted in the animal. Alternatively, this viability estimate may be spuriously low as the device was expoQed to adverse conditions during retrieval from the animal. ~ 3ment of viahility in the other three devices wa~ compromised by experimental difficulties, and the re-Qults are not inte-y~eLable in a me~ningful fashion.
While we have her~inheforé described a number of embodiments of this invention, it is apparent that the basic construction~ can be altered to provide other embodiments which utilize the methods and devicea of this invention.
Therefore, it will be appreciated that the scope of this invention i5 defined by the claims App~nAeA hereto rather than by the specific embodiments which have been yL~-ented hereinbefore by way of example.

SUBSTITUTE SHEET (RULE 26)

Claims (42)

WE CLAIM:

1. A device for providing substances derived from cells contained in the device, said device comprising a core having an interior region and an outer boundary surrounding the interior region;
a cell zone for containing cells, said cell zone substantially surrounding the core and extending between the outer boundary of the core to an inside surface of a permeable membrane, the permeable membrane having inside and outside surfaces and surrounding the cell zone and the core, and wherein the cell zone has a thickness such that the viability of cells in a cell layer located closest to the outer boundary of the core and most distant from the inner surface of the permeable membrane is supported.

2. The device according to claim 1 wherein the cell zone thickness is such that at least about 10% of the cells in the cell layer located closest to the outer boundary of the core remain viable.

3. The device according to claim 2 wherein the cell zone thickness is such that at least about 50% of the cells in the cell layer located closest to the outer boundary of the core remain viable.

4. The device according to claim 3 wherein the cell zone thickness is such that at least about 80% of the cells in the cell layer located closest to the outer boundary of the core remain viable.

5. The device according to claim 1 further comprising a support means for providing a site for cell attachment in the cell zone.

6. The device according to claim 5 wherein the support means is comprised of a porous PTFE material, a dextran material, a collagen material, a polyester material or a polystyrene material.

7. The device according to claim 5 wherein the support means is a random network, a trabecular network, microspheres or fibrous mattes.

8. The device according to claim 5 wherein the outer boundary of the core is porous and the interior region of the core is substantially cell free.

9. The device according to claim 1 wherein the outer boundary of the core is comprised of a material which promotes cell adhesion.

10. The device according to claim 9 wherein the material to promote cell adhesion is collagen, poly-L-lysine, laminin, fibronectin or porous PTFE.

11. The device according to claim 1 wherein the permeable membrane and the core are both cylindrical having longitudinal axes which are substantially parallel to each other.

12. The device of claim 1 wherein the core is substantially centrally located within the permeable membrane.

13. The device according to claim 12 wherein the cell zone thickness is less than about 500 microns.

14. The device according to claim 13 wherein the cell zone thickness is from about 25 to about 250 microns.

15. The device according to claim 14 wherein the cell zone thickness is from about 50 to about 100 microns.

16. The device according to claim 3 wherein the cell zone thickness is from about 50 to about 100 microns.

17. The device according to claim 16 further comprising a support means for attachment of cells to the core.

18. The device according to claim 17 wherein the support means is a random network, a trabecular network, microspheres or fibrous mattes.

19. The device according to claim 18 wherein the support means is made from porous PTFE, dextran, collagen, polyester or polystyrene.

20. The device according to claim 1 wherein the permeable membrane is comprised of porous polytetrafluoroethylene (PTFE) or hydrogel.

21. The device according to claim 20 wherein the permeable membrane comprises a hydrogel with an average thickness of between about 15 and about 25 microns.

22. The device according to claim 1 wherein the core comprises an inert, cell-free material made from polytetrafluoroethylene (PTFE), polydimethylsiloxane, polyurethane, polyester, polyamide or hydrogel.

23. The device according to claim 16 wherein the cells comprise insulin secreting cells.

24. A device for providing substances from cells contained in the device, said device comprising a core having an interior region and an outer boundary surrounding the interior region and a long axis;
a cell zone containing cells, said cell zone substantially surrounding the core and extending from the outer boundary of the core to an inside surface of a permeable membrane, the permeable membrane having inside and outside surfaces and a long axis substantially parallel to the long axis of the core, and surrounding the cell zone, and wherein the core and the permeable membrane are proportioned such that a diffusion length parameter (DLP), measured on a planar cross-section of the device taken perpendicular to the long axis of the core and passing through the core, cell zone and permeable membrane at a point along the long axis of the core wherein the cell zone is sufficiently thick to contain at least one cell layer, and wherein the DLP is defined as the ratio of an area of the cell zone wherein the area is the total area of the cell zone of the cross-section, divided by a perimeter of the cell zone of the cross-section wherein the perimeter is defined as the length of the inside surface of the permeable membrane on the cross section, is less than about 500 microns.

25. The device according to claim 24 wherein the diffusion length parameter ranges from about 25 to about 250 microns.

26. The device according to claim 25 wherein the diffusion length parameter ranges from about 50 to about 100 microns.

27. The device according to claim 24 wherein the cell zone thickness is such that viability of cells in a cell layer located closest to the outer boundary of the core and most distant from the inner surface of the permeable membrane is supported.

28. The device according to claim 27 wherein the cell zone thickness is such that at least about 10% of the cells in the cell layer located closest to the outer boundary of the core remain viable.

29. The device according to claim 28 wherein the cell zone thickness is such that at least about 50% of the cells in the cell layer located closest to the outer boundary of the core remain viable.

30. The device according to claim 29 wherein the cell zone thickness is such that at least about 80% of the cells in the cell layer located closest to the outer boundary of the core remain viable.

31. The device according to claim 26 further comprising a support means for attachment of cells.

32. The device according to claim 31 wherein the form of the support means is a random network, a trabecular network, fibrous mattes or microspheres.

33. The device according to claim 31 wherein the material for producing the solid support means is made from porous PTFE, dextran, collagen, polyester or polystyrene.

34. The device according to claim 33 wherein the cells comprise insulin secreting cells.

35. The device according to claim 24 wherein the permeable membrane is made from porous polytetrafluoroethylene (PTFE) or hydrogel.

36. The device according to claim 35 wherein the permeable membrane comprises a hydrogel with an average thickness of between about 15 and about 25 microns.

37. The device according to claim 24 wherein the core comprises an inert, cell-free material and is made from polytetrafluoroethylene (PTFE), polydimethylsiloxane, polyurethane, polyester, polyamide or hydrogel.

38. The device according to claim 24 wherein the core and the permeable membrane are substantially spherical.

39. A method of producing a device for encapsulating and culturing cells to produce a therapeutic substance the method comprising providing an exterior membrane impermeable to cells but permeable to both nutrients and the therapeutic substance produced by the cells and wherein the membrane forms a lumen;
providing a cell-impermeable core to be inserted into the lumen of the exterior membrane;
introducing the core into the lumen of the exterior membrane to create a cell zone for maintaining cells defined by the outer surface of the core and the inner surface of the exterior membrane;
introducing cells into the cell zone; and sealing the exterior membrane so that the exterior membrane surrounds the core, the cell zone and the cells therein.

40. The method according to claim 39 further comprising a swelling step to enlarge the core to a final volume.

41. A method of preparing cell products comprising culturing in vitro, cells capable of producing the products, the cells being contained in a device wherein the device comprises a core having an interior region and an outer boundary surrounding the interior region;
a cell zone for containing cells, said cell zone substantially surrounding the core and extending between the outer boundary of the core to an inside surface of a permeable membrane, the permeable membrane having inside and outside surfaces and surrounding the cell zone and the core, and wherein the cell zone has a thickness such that the viability of cells in a cell layer located closest to the outer boundary of the core and most distant from the inner surface of the permeable membrane is supported, and culturing the device to allow cell products to diffuse out of the device and into culture medium surrounding the device; and purifying the cell product from the culture medium.

42. The method of claim 41 wherein the cell product is insulin.