CN101258237A - Apparatus and methods for preparing tissue grafts - Google Patents

Abstract

The invention provides automated apparatus and methods for manufacturing implantable tissue grafts in an operating room setting, preferably utilizing autologous adipose tissue cells for preparing vascular grafts. The automated apparatus includes media and tissue dissociating chemical reservoirs, filters, a cell separator and a perfusion flow loop through a biochamber which supports a graft substrate. The present invention further provides methods for using the tissue grafts and cell samples prepared by the devices described herein in a multitude of therapies including revascularization, regeneration and reconstruction of tissues and organs, as well as treatment and prevention of diseases. The invention also provides methods biochamber configurations for applying a sustained low pressure gradient across a permeable scaffold material using a media containing cells to be deposited on the scaffold for the production of tissue grafts, including, but not limited to, vascular grafts.

Description

Be used to prepare the apparatus and method of tissue grafts

The application requires No. the 60/697th, 954, U.S. Patent application series submitting on July 12nd, 2005 and the right of priority of No. the 11/314th, 281, the U.S. Patent application series submitted on December 22nd, 2005, with above-mentioned both all be incorporated into this paper with way of reference.

About the research of federal government's subsidy or the statement of exploitation

The present invention makes under the subsidy of being authorized by septic yanks' medical research and material headquarters (United StatesArmy Medical Research and Material Command) number is supported for the government of #W81XWH-04-1-0503 and #W81XWH-05-1-0620.

Technical field

The present invention relates to the organizational project system, it relates to mammalian cell and the cellular products that is used to make anatomic implants and graft, and wherein anatomic implants and graft include but not limited to artificial blood vessel and arteriovenous graft.The invention particularly relates to that (the persistent pressure cell is sodded method by level ground, persistent pressure cell shop method, sustained pressure cell sodding) will be derived from the mesenchymal cell of fat or other cytoadherence (adhesion) method and automated installation to permeable timbering material, it comprises utilizing and comprises the medium of waiting to be deposited on the cell on the material and pass permeable material and apply lasting pressure gradient (sustained pressuregradient).Method and apparatus of the present invention promotes acceleration adhesion and the maturation of cell on timbering material.Utilize the automatization compatible of persistent pressure shop level ground method with Operation theatre, the preferred embodiments of the present invention make can gather in the crops patient's oneself cell from the sample of patient's fatty tissue, washing, separation also deposit these cells, with the suitable graft of preparation for clinical use.Such embodiment relates to cell extraction, then is level ground, persistent pressure shop method automatization, in aseptic, the closed system.

Background technology

Organizational engineering just develops towards the clinical application direction of the repair and reconstruction that are used for impaired or illing tissue and organ.Especially, in heart and peripheral vascular field, the research and development of blood vessel graft are major objectives.In the first world, cardiovascular disorder is the major cause of mortality ratio and sickness rate.The standard of nursing, autograft is not have serious sickness rate.The patient's (it does not stay suitable autograft material or has stood autograft, only in the U.S. 100,000 patients is arranged every year) who suffers from systemic disease does not almost have autograft to select.

Therefore the investigator studies synthetic graft above 30 years.Main challenge provides such graft material, it is biocompatible, promptly, non-thrombus gene, non-immunogenic, anti-mechanical (hard, mechanically resistant), and have acceptable wound healing and physiological response (for example, vasoconstriction/stretching reaction, solute transportcapacity etc.).In addition, the tissue grafts material should be easy to handle, store and transportation, and commercial be feasible.

Conduit has two kinds of main failure modes (failure mode): machinery with biology, it is that inwardly growth is caused by the thrombosis in the conduit and obstruction subsequently and/or cell.Synthetic conduit with the material property that can bear arterial pressure is common, makes the material of seeking non-thrombus gene become main research interest.The endotheliocyte that obtains from the patient has presented thrombogenicity (thrombogenicity) (Williams et al., 1994, J.Vasc.Surg., the 19:594-604 that can reduce implantation catheter; Arts et al., 2001Lab Invest 81:1461-1465).

(the cell liner, endotheliocyte is very important in process celllining) to set up non-thrombus gene cell liner in synthetic graft.Therefore, hope about several minutes or in several hours by means of instrument in permeable base, support or other permeable cell based bottom material or on realize cytoadherence fast, wherein instrument is suitable for operating room environment, keep sterile barrier, be easy to use, and produce consistent graft result.

At present, there are four kinds of main modes to satisfy these requirements (but having limited success): (i) to make to spend the cell organization material; (ii) use self-assembly mechanism, wherein cell is cultured in the tissue culturing plastic in the substratum, and its inducing cell epimatrix (ECM) is synthetic; (iii) use the synthetic biodegradable polymer, afterwards inoculating cell and in the mimic physiological environment, cultivating thereon; And (iv) use biological polymer, as regeneration type i collagen gel, its by apply that mechanical force is formed and and histocyte compress with simulate physiological environment (referring to, for example, Robert T.Tranquillo, 2002, Ann.N.Y.Acad.Sci., 961:251-254).

The pressure gradient that relates to instantaneous pressure be used for by sieving action with cell deposition to permeable support, that is, bulk flow is provided and utilizes matrix or the timbering material that has less than the hole of cell colony, thereby (for example in matrix, catch cell, United States Patent (USP) 5,628,781; Williams et al., 1992, J Biomed Mat Res 26:103-117; Williams et al., 1992, J Biomed Mat Res 28:203-212.).These captured cell have demonstrated and have been adhered to timbering material subsequently, but owing to can not satisfy the prerequisite of above-mentioned successful graft fully, that is, and biocompatibility, physical strength and necessary physiological property, thereby only have limited clinical application.

Since the later stage seventies 20th century, endotheliocyte is seeded in the opening (patency) that experimentally just is used for improving minor diameter polymer blood vessel graft with the opposing untoward reaction.Since then, make progress at this target, wherein the great majority progress concentrates on the biological or biological hybridization graft of design.

Endotheliocyte is more more complicated than what think at first, because they not merely produce unicellular liner on the surface of internal cavity of blood vessel.Endotheliocyte also discharges the molecule that is used for regulating solidification, platelet aggregation, leukocyte adhesion and vascular tone.Do not having under the situation of these cells, for example, under the situation of the inner chamber of the synthetic polymer blood vessel graft of implanting, host response develops into and proves an abortion.Open forfeiture is to result from acute thrombus disease in the junior three after implantation ten days.Early failure is the result of intrinsic thrombogenicity of the blood surface in contact (it is by non-endothelialization) of biomaterial.So far, only known complete non-thrombus genetic material is an endothelium; Any other material that contacts with blood flow tends to platelet deposition and thrombosis subsequently.The chronic frustration pattern of minor diameter polymer blood vessel graft is to cause the anastomose property hyperplasia (anastomotic hyperplasia) of open forfeiture.Causing anastomose property hyperplasia accurate mechanism behind still has to be determined; Yet, may relate to endotheliocyte and smooth muscle cell dysfunction and unsuitable communication.

Early stage worker in the minor diameter graft development field makes every effort to promote the graft endothelialization, thus before implanting by the general in various degree to blood vessel graft, increase opening from the body transplanted endothelial cell.This method has been known as endotheliocyte inoculation method (part covers, and it depends on p cell propagation) or level ground, cell shop method, and (cell is sodded method, cellsodding) (covers fully).Basic hypothesis is quite simple: promptly, endotheliocyte by promoting patient oneself is at artificial blood vessel (blood vessel prosthesis, vascular prosthesis) foundation on the blood surface in contact, " normally " endotheliocyte liner (cell lining) will be formed on the graft with relevant basement membrane (being called neointima together) and resist rheological force, physiology power and biomaterial power, and these reactive force synergies lost efficacy to encourage graft.After 30 years of this field research (comprising animal data likely), this simple hypothesis does not also produce clinical device.

The failure mode of endothelium inoculation graft is same as untreated polymer grafts, i.e. thrombosis and intimal hyperplasia.Failure mode lacks functional endodermis (neointima) and/or unusual endotheliocyte is direct with smooth muscle cell and a connection communicates relevant at least in part with on the surface, chamber of graft.These lost efficacy and can take place in people's test in early days, grew (cell lining development) although the successful example of inoculation graft is just developing into the cell liner.These data show, in animal model, to form be by taking place from the endothelial cell proliferation of arteriae anastomotica on every side for lip-deep neointima in polymer blood vessel graft chamber, and closely spaced capillary blood vessel of graft or circulation origin endotheliocyte be not strictly from the cell of inoculation.

The potential source of endotheliocyte inoculation is capillary endothelium (MVEC).People such as Williams in their laboratory, at first adopt (open up, pioneer) people, dog, rabbit, rat, ox and pig endotheliocyte, the especially MVEC of fresh separated and cultivation, with the research cell function.The source of people MVEC is the aspirate tissue from the beauty treatment lipsuction.The end-use that depends on cell colony has been used two kinds of independent alternatives to be used for people's fat MVEC and has been separated.The difference of these schemes is separate complex: from the simple Operation theatre inclusive routine on the level ground, shop immediately (immediate sodding) that is used for the human or animal graft to more meticulous program (if will cultivate MVEC subsequently).

By using lipsuction to strengthen the separation of people MVEC to obtain the sample of people's fat.Method by lipsuction intubate (liposuction cannula) suction of fat is separated into small pieces with subcutaneous lipids, and it can strengthen the effectiveness of digestive process.Can be at 37 ℃ down with fatty 20 minutes of collagenase (4mg/cc) digestion, its release＞10 ⁶Individual cell/gram fat.These MVEC can separate with fat by gradient centrifugation.MVEC will form rounded grain (pellet) and can be resuspended in the substratum after abandoning supernatant subsequently.These cells have experienced conventional the sign with the cell of determining main isolate and have formed.By means of the isolating most cells of this program is endotheliocyte, this be since they have the expression of Feng's von Willebrand antigen (von Willebrand antigen), the form synthetic and micella drink blister capsule synthetic, basilar membrane collagen that lacks the keratic expression of mesothelial cell's specific cell, angiotonin (Angiotensin) saccharase, prostacyclin and prostaglandin E2 is expressed.

Carried out people's clinical trial to be evaluated at the transplanted endothelial cell among the patient who needs the periphery bypass.At duration of test, a large amount of endotheliocytes directly is placed on the surface, chamber of ePTFE graft.In order to improve cell deposition, in comprising autoserous substratum, all grafts are carried out wetting in advance.2 * 10 ⁵Individual cell/cm ²Under the density of graft cavity area, cell suspension is in identical substratum.This solution is remained on striding under wall (cross-wall) or the transmural pressure power gradient to force cell to the surface of 5psi, and this method is called pressurization shop level ground method (pressure sodding).After system is got permission, 11 patients are registered and accept to test graft.Between the standby period, the patient stands lipsuction to take out about 50 gram stomach wall fat in surgical operation.Utilize said procedure fat is handled and with level ground, the cell colony that obtains pressurization shop to desirable graft, implant immediately then.After following up a case by regular visits to more than 4 years, these grafts keep being similar to the open speed (patency rate) of saphena blood vessel graft.

Before used the pressure gradient that relates to instantaneous (＜1 minute) relatively high pressure (250mmHg) come by sieving action with cell deposition to permeable support, promptly, bulk flow is provided and utilizes matrix or the timbering material that has less than the hole of cell colony, thereby (for example in matrix, catch cell, United States Patent (USP) 5,628,781; Williams et al., 1992, JBiomed Mat Res 26:103-117; Williams et al., J Biomed Mat Res 28:203-212).Yet although above-mentioned progress is arranged, clinical so far coronary artery suitability is restricted, and this is that therefore research concentrates on the other maturation in vitro time because conduit does not keep viscous enough non-thrombus that the surface takes place.

Endotheliocyte has important effect in setting up non-thrombus generation cell liner (non-thrombogenic cell lining).In addition, still need effective and reliable method, therefore the invention provides a solution to produce the endotheliocyte liner on the synthetic graft in the Operation theatre device.Hope in permeable matrix, support or other permeable cell matrix materials or on realize cytoadherence fast at several minutes or in several hours by means of instrument, wherein instrument is suitable for operating room environment, keeps sterile barrier, is easy to use, produce consistent graft product and cheap.The invention enables the level ground, quick cell shop that from fatty tissue, to separate a large amount of endotheliocytes and carry out synthetic graft, and make and to be equipped with automatization and the adhesion that instrument is realized cell with turn key (turn-key), Operation theatre, thereby spread the level ground graft fast.Except the liner of the graft that is used to implant, the present invention can have other purposes.

Summary of the invention

The invention provides apparatus and method with during organizational project, be implemented in matrix, support or other cell matrix materials or on quick cytoadherence, wherein relate to a kind of automatic mode and device come to obtain patient oneself from the sample of patient's fatty tissue cell, and washing, separate and these cell depositions are supplied clinical application on the graft that suits.

The present invention is the discovery that is based in part on the inventor: preferably in the cell filling system, pass that graft support or substrate material form about 5 minutes up to about 24 hours lasting low pressure gradient can provide than the method for using previously known significantly the adhesion of cell on substrate material more fast with and subsequent ripe.Adopt to continue the bigger control that low pressure comes also to provide at level ground, permeable material upper berth cell pair cell activation and differentiation pathway, and lack the effort that above-mentioned control has stoped the prior structure engineering.The present invention also provides method and apparatus a kind of automatization, aseptic and safe to supply clinical application to form cell at short notice on suitable graft, and the method and apparatus that is used to collect the cell sample that is suitable for therepic use is provided.The present invention further provides the method that will be used for multiple therapy by the tissue grafts and the cell sample of device preparation described herein, wherein said therapy comprises the regeneration and the reconstruction of revascularization, tissue and organ, and treatment of diseases and prevention.

The invention still further relates to level ground, cell shop biotron, it provides controlled lasting wall to flow, thereby produces the lasting differential pressure gradients of passing porous material, so that prepare anatomic implants and graft by means of adhesion, growth and the differentiation of cell.

Description of drawings

Fig. 1 illustrates the synoptic diagram of an embodiment of automated installation of the present invention.

Fig. 2 illustrates the skeleton view of an embodiment of apparatus of the present invention.

Fig. 3 shows according to conduit biotron of the present invention, and it has the nearside regulating cock (stopcock) that is used to introduce cell, and Y-connection, and this Y-connection is convenient to apply transmural pressure power (transmural pressure), then is to flow in the chamber.

Fig. 4 A-4D shows the view according to another kind of biotron of the present invention.This embodiment adopts tubular structure.Utility appliance, as be used to introduce cell regulating cock, be used to apply wall and saturating chamber mobile Y-connection and relevant automatization perfusion equipment, then not shown.Such equipment can be any suitable equipment, and it includes but not limited to equipment as shown in Figure 1.

Fig. 5 provides the skeleton view of an embodiment of the device that comprises cellular segregation unit, Pu Ping unit and graft chamber.

Fig. 6 shows the durable and disposable unit of cellular segregation module.

Fig. 7 provides the skeleton view of cellular segregation module, and wherein disposable unit is loaded on the cellular segregation durable components.

Fig. 8 shows the durable and disposable unit of level ground, graft shop module.

Fig. 9 shows level ground, graft shop module durable components, and it is connected in the cellular segregation unit that is loaded with for the disposable unit that uses.

Figure 10 shows the major parts in the durable and disposable unit in level ground, graft shop.

Figure 11 shows the durable and disposable unit of cell harvesting module.

Figure 12 shows cell harvesting module durable components, and it is connected in the cellular segregation module that is loaded with for the disposable unit that uses.

Figure 13 shows mounted bar code scanning device.

Figure 14 shows the sectional view of an embodiment of centrifugal bowl (centrifuge bowl).

Figure 15 illustrates the total system flow process.

Embodiment

The invention provides the apparatus and method that cytoadherence or " level ground, shop " prepared various anatomic implants or graft on any suitable graft support or other permeable base material by exert pressure (be preferably continue low amplitude pressure).In specific embodiment, tissue is a tubular tissue, as vascular tissue.Yet, the present invention can also be applied to the tissue grafts of any kind, these tissue grafts relate to cytoadherence to support or other substrate material, and it includes but not limited to skin, cartilage, bone, marrow, tendon, ligament, gi tract, genitourinary tract, liver, pancreas, kidney, suprarenal gland, mucous epithelium and nerve graft.This method is well suited for tubular tissue especially, and it includes but not limited to the unify tubular tissue of urinary system of cardiovascular system.

When this paper uses, term " continues low amplitude pressure " and is meant such pressure, it has the manometric head of about 10mmHg, about 15mmHg, about 20mmHg, about 25mmHg, about 30mmHg and about 55mmHg, time cycle is about 5 minutes, about 20 minutes, about 30 minutes, about 40 minutes, about 50 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 4 hours, about 5 hours or about 6 hours, to strengthen adhesion, growth and/or the differentiation of cell.According to the type and the given instruction of this paper of cell, tissue grafts, substrate material, those of ordinary skill in the art can select felicity condition to apply the lasting pressure of concrete low amplitude.

The cell for the treatment of adhesion can comprise, for example, inoblast, smooth muscle cell, pericyte, scavenger cell, monocyte, plasmocyte, mastocyte, adipocyte, tissue specificity parenchyma (parenchymatous cell), endotheliocyte, urothelium cell (urothelial cell, urothelial cell) and the cell of various other types that in organizational project is used, run into, it comprises from various tissue-derived undifferentiated adult stem cells.In a preferred embodiment, adherent cell is an endotheliocyte, more preferably available from the human microvascular endothelial cell (mvec) that is rich in from the microvascular fatty tissue of body, as referring to United States Patent (USP) the 4th, 820, No. 626 (Williams etc., be published on April 11st, 1989), the 5th, 230, No. 693 (Williams etc. are published on July 27th, 1993) and the 5th, 628, No. 781 (Williams etc. are published on May 13rd, 1997) is incorporated into this paper with the full content of all above-mentioned patents with way of reference.Adherent cell can be from body, allochthonous or xenogeneic, and origin goes up from body but be preferably.

The implant matrix of using among the present invention (" support ") material can be any preferred permeable material of various size and geometrical shape.This material can be natural or synthetic materials, and it includes but not limited to polyethylene terephthalate, polyurethane(s) or expanded polytetrafluoroethyl,ne (ePTFE).In another embodiment, the graft support can be a biological polymer, as collagen.This material can be before the blood coagulation and/or elastin or allograft conduit, as the refrigeration vein, remove cell vein or artery.In another embodiment, support can be a matrix material, as (for example lip-deep electrospinning silk, elastin support electrospun) is to improve mechanical property to have polymeric coating.Can use protein (for example, albumin) or blood plasma that material is carried out pre-blood coagulation or pre-treatment, it can be used for further strengthening the adhesion of histocyte on substrate material, sprawls and grow in some specific embodiment.Implant matrix or support can be constructed by any suitable method, and wherein said method includes but not limited to those at Liu, T.V.et al., 2004, Adv.Drug.Deliv.Rev.56 (11): 1635-47; Nygren, P.A.et al., 2004, J.Immunol.Methods 290 (1-2): 3-28; Hutmacher, D.W.et al., 2004, TrendsBiotechnol.22 (7): 354-62; Webb, A.R.et al., 2004, Expert Opin.Biol.Ther.4 (6): 801-12; And Yang, C.et al., 2004, BioDrugs 18 (2): the method for mentioning among the 103-19.

When using in this article, term " transmural pressure power or flow " be meant the wall that passes the graft support from a side of graft support to opposite side pressure or flow.At the graft support is under the situation of gillies' graft support, and this term is meant the outside or extracapillary (EC) the spatial pressure from (IC) space in the inner chamber or kapillary of graft to graft or flows.

When using in this article, term " saturating cavity pressure or mobile " is meant by the pressure of the inner chamber of gillies' graft or flows.Term " flowing in saturating chamber " and " perfusion of saturating chamber " can be used interchangeably.Though do not need the chamber perfusion for cytoadherence in the present invention, for example after saturating wall flowed, it can be used to provide training or cleaning effect.In this case, up to and the flow velocity that comprises physiology flow velocity (about 160ml/min) be preferred, be enough to provide the physiology mobile that can bear subsequently cytoadherence usually though be low to moderate the flow velocity of 5ml/min.

When using in this article, term " nearside " is meant the reference point (referring to Fig. 4) that flows into a side with respect to the center of biotron conduit at medium.

When using in this article, term " distally " is meant the reference point (referring to Fig. 4) that flows out a side with respect to the center of biotron conduit at medium.

Term " in the kapillary (IC) " is meant the inner chamber or the internal space of gillies' graft support, and can be called " saturating chamber " or " in the chamber " interchangeably.

Term " extracapillary (EC) " is meant the space outerpace of gillies' graft support and can be called " blood vessel is outer " or " outside the chamber " interchangeably.

Referring now to Fig. 1.Fig. 1 illustrates an embodiment of system 120, this system comprises optional disposable bags (disposable bags, disposable bag) 100, its can be loaded in the suitable hard device be used for Operation theatre or other gnotobasis or near, thereby results will deposit to the cell on the suitable graft.Disposable bags 100 comprises separate tissue chemistry reservoir 20, medium reservoir 10 and waste container 30.In use, can carry out preload to reservoir 20 and medium reservoir.Preferably, separate tissue chemistry reservoir 20 comprises collagenase.

System 120 further comprises steeping cell and well heater 50, pump 60, buoyancy separator 40 and biotron 70.Pump 60 can be any suitable pump or the combination of pump, its include but not limited to toothed gear pump, peristaltic pump, surge pump, impeller pump and by fluid column produce by kinetic head.In one embodiment, biotron 70 is a kind of conduit biotrons, as hereinafter and the conduit biotron of being discussed in No. the 11/314th, 281, the common unsettled U.S. Patent application series of submitting on December 22nd, 2005, above-mentioned patent application is incorporated into this paper with way of reference.

Preferred systems 120 can be used for from patient's tissue isolated cell and the desirable fraction of these cells deposited on the graft material.This can be in the Operation theatre device (time frame is finished in timeframe) in the automatization mode and in clinical feasible time range.Clinical feasible time range is considered to about 30 minutes to about 24 hours usually, and it depends on various factors, and is ripe for organizing the needed time etc. as amount, the cellular layer of cell type, raw-material amount, the transplanted cells that needs.Technician in the association area can easily understand these factors.

Preferred systems provides persistent pressure level ground, shop and has separated the automatization of clinical procedure of the desired fraction of patient's cell from tissue, and following one or more: filtration, washing, heating, dipping, proteolysis release, separation, resuspending and the cell pressurization spread the level ground to permeable graft.

More particularly, still with reference to Fig. 1 exemplary embodiment, preferred preload separate tissue chemistry reservoir 20 and medium reservoir 10.By enter the mouth 90 will be to be transplanted cell input system 120.Preferably from patient's fatty tissue, it is by means of relative Noninvasive device such as Tulip to cell ^TMSystem is collected.At first wash and filtration cell in the strainer 18, from cell (as red blood corpuscle), to remove small-particle.By valve 14, small-particle is filtered to waste container 30.By valve-off 34 and 14 and make medium flow through remainder that conduit 76 and 38 comes cell mixing and medium from medium reservoir 10 from medium reservoir 10.By by pump 60 via conduit 28 and 24 and via opening that valve 12 and 16 pumps and mixing and medium is mixed with cell with proteolytic enzyme in the separate tissue chemistry reservoir 20.By this operation, mix cell, medium and proteolytic enzyme through washing.

By flowing through conduit 26, dipping and heating mix products in steeping cell and well heater 50.Can realize the dipping organized by the mode of any increase organization table area, thereby under the situation of less cell injury, allow structural higher protease activity.Steeping cell and well heater 50 can be merge or separate, and can comprise any suitable device, as centrifugal impeller, mixing tank, helical mixer etc.

The slurries of organizing in the steeping cell are preferably maintained in the range of from about 35 to about 40 ℃ temperature, and preferred about 37 ℃, so that keep high cell survival and make proteolytic enzyme have maximum activity.Can (comprise for example resistance heating element, electricity-Re piece (peltier blocks) etc.) in many ways and realize heating, and can heat be delivered to by any suitable conductive medium (comprising water-bath) and by any suitable heat conduction reservoir material and organize slurries.

Preferably, in the dipping slurries or what its desirable or reasonable time in office, the user can serum input system with the patient in.By input terminus 80 serum is imported in the exemplary system 120.Serum flows in the medium reservoir 10 via conduit 78.

Jin Zi slurries pass conduit 42 to cell separator 40 then.Separator 40 can be, for example, and buoyancy separator, whizzer or any other suitable device.Slurries can be remained in the separator 40 by valve-off 36 and 52.In separated slurry, the serum in the medium reservoir 10 and medium flow via flow loop (flow loop).Valve 12,14,16,32,36,52,62 and 68 is closed and valve 34,46,56,66 is opened to produce flow circuits.Pump 60 pumps serum/medium mixture via flow circuits, flow circuits comprises conduit 76,38,44,54,58 and 72.Serum in the flow circuits: the ratio of medium mixture is preferably about 1: 6.Flow to provide by the saturating chamber of conduit in serum/medium mixture preferred streams via flow loop, simultaneously separated slurry in separator 40.At the fixed time, valve-off 56 is also opened valve 62 and is flowed to biotron 70 so that wall to be provided, thereby the proteic coating of patients serum is placed on the timbering material, and then improves cytoadherence.Separation can be carried out about 2 minutes to about 30 minutes, and preferably carries out about 5 minutes to about 10 minutes.Being used for the chamber mobile scheduled time is preferably about 1 minute to about 10 minutes, more preferably about 3 minutes to 5 minutes.

In separator 40, separate then by fractionation (fraction) pair cell.Handle cell by strainer 48 with desired fraction.By being added in the cell, medium/serum comes the deactivation proteolytic enzyme relevant with these cells.In case from slurries, isolated cell, then open valve 52 so that desirable fraction can forward biotron 70 to, so that cell is transplanted on the surface of graft material, wherein graft material has the aperture size less than cell size usually.Can will flow for 74 within a short period of time by the waste line (not shown) or by conduit at first and deliver to waste container 30 can remove somatomedin.Aforesaid operations can carry out about 2 minutes to about 30 minutes.Thereafter, flowing through conduit 54,58,72,76,38 and 44 changes its course and enters the successive flow circuits that comprises biotron 70, medium reservoir 10 and pump 60.During flowing through this flow circuits, valve 12,14,16,32,36,52,56 and 68 is closed and valve 46,66,62 and 34 is opened.Make material cycle through flow circuits, preferably continue level ground, low amplitude pressurization shop, be transplanted on the graft material up to cell in order to provide.This can be about 5 minutes to about 24 hours, and it depends on condition.System 120 can comprise that visual indicator (as optical indicator) or sound telltale or warning howler be transplanted on the graft materials with showed cell.

During 5 minutes to 24 hours, 10mmHg and the lasting low pressure gradient that is not more than 100mmHg can be used for cell deposition from the teeth outwards or in the graft material, it depends on the normal pore size of graft at least.Can realize pressure gradient with any combination of positive pressure and/or negative pressure, so that clean gradient causes flowing by graft material.

Separating and spread the level ground medium can be commercially available medium, this medium comprises DMEM, F12, α-MEM, University of Wisconsin (university of wisconsin) solution etc. or its any combination, do not have or do not have the other factor, it can comprise heparin or other factor that adapts to desired cell type.

As mentioned above, biotron 70 holds graft material and can be so that medium flow is crossed permeable graft.Preferred biotron 70 has positive sealing member (positive seal) as two o ring sealing-rings and be easy to load and can separate with the rest part for the one-trip container 100 of Operation theatre use.Preferred biotron 70 also is easy to unloading so that graft is delivered to Operation theatre and comes load or unload without any need for instrument.It can be the groove of tube designs, and it makes do not have clean masterpiece to be used for biotron 70 by means of offsetting from all pressure of fluid flow in the system.Such design is convenient to place easily for example gillies' graft.

Under the situation of gillies' graft, cell is deposited on the surface, chamber of graft and biotron holds graft with by means of along the uniformly penetrating of graft major axis and allow uniform cell deposition.Such gillies' graft can also comprise the hole of opening graft material along its major axis by the perviousness of preload with the change graft.Substantially be under the situation of plane graft, cell is deposited on the surface of graft.

Referring now to Fig. 2, the view of its graphic extension system 120 wherein shows removable biotron 70 and system's housing 110.System's housing 110 has cell separator access device 112, is used for access separator 40.System's housing 110 also has access device 114, is used for inserting and removing disposable bags 100.

The example system of Fig. 1 and Fig. 2 will generally include microprocessor and related software to import Controlling System based on the user and one or more steps are operated automatically.By control pump and valve, controlled temperature and control cell separator and steeping cell device, software for example can allow, and controlling flow is through the mobile automatization wholly or in part of tubular conduit.Preferably, make system's fully automated, but can be reconfigured based on one or more input parameters.This system may further include various transmitters with detection or measuring system parameter (for example will show the pressure that blocks), and described system parameter is notified to microprocessor or user.In one embodiment, system is a kind of hand held system.

Though can in any suitable device, implement automatic mode of the present invention, but the present inventor has been found that, with regard to cell adhesion unanimity and uniform and user's convenience, special biotron design especially is well suited for obtaining optimum.

Conduit biotron according to an embodiment of the invention is shown among Fig. 3.This biotron comprises branches such as two, and it comprises top duct 201 and bottom conduit 202, has near-end and far-end separately.Place conduit mutually to limit internal space 203 by means of two O shape ring sealing-rings 216.(IC) double hook junctor 204 is positioned in the space that is limited by conduit in the kapillary.Junctor 204 is suitable for keeping graft support 205 at each near-end of biotron and far-end.Nearside pipeline 206 is connected to the IC junctor and flows so that the IC with respect to graft support 205 to be provided.By means of Y-connection 208, distally pipeline 207 connects the distally IC and the distally EC fluid-space.After entering biotron, by means of the ring of the pair of O shape on each side 214 sealing IC pipelines 206 and 213.This biotron can comprise other blood vessel external port 215.

By means of junctor 204, permeable timbering material can be mounted to IC nearside pipeline 206 and distally pipeline 213.In specific embodiment, biotron comprises the regulating cock 209 that is connected in nearside pipeline 206 by means of T shape junctor 210, so that injection cell is gone in the biotron.In another specific embodiment, as described below, biotron further comprises at least one anchor clamps (clamp) or valve 211, and it can close distally EC pipeline 212 or distally IC pipeline 213, producing transmural pressure power or saturating cavity pressure gradient, or between change.In a preferred embodiment, each distally IC pipeline 213 and distally EC pipeline 212 have valve or the sliding clamp of oneself.

Preferably, the top of biotron and bottom conduit are made by optically transparent material (for example, polystyrene or polycarbonate), flow so that can visually monitor the inside and outside of cavity.This biotron is preferably made by material autoclavable, gamma or gaseous sterilization.In addition, biotron can hold a plurality of silicone O shape loop systems, provides the dual seal contact for conduit connects, so that after between two hooks that sample are placed in the biotron, can regulate catheter length and position, angle.In addition, can use metal threaded inset need not the parts that cutting thread is made, and the potential of having eliminated plastic whorl lost efficacy.

The structure of distally pipeline 207,212,213 (it connects the distally IC and the distally EC fluid-space) makes the user can utilize sliding clamp 211 or switches between transmural pressure power gradient and saturating cavity pressure gradient in the automatization mode in bio-reactor.This switching will typically take place to provide cavity pressure later at cytoadherence.In specific embodiment,, cell is guided to nearside pipeline 206 by regulating cock 209 or partition (septum) by connecting by means of T shape junctor 210.Closing distally IC sliding clamp 211 then flows out from the EC space only allowing, thereby set up from nearside IC to distally EC spatial transmural pressure power gradient, and the low discharge medium that passes through permeable support, AC is deposited on the surface, chamber and/or in the wall of graft simultaneously.Can be by producing positive pressure in nearside IC side, producing negative pressure or the combination that produces positive pressure and produce negative pressure in EC space, distally at nearside IC comes the build-up pressure gradient in distally EC side.If desired,, can close distally EC sliding clamp and open distally IC sliding clamp allowing by the flowing of conduit cavity, thereby guarantee cytoadherence under the situation that has shearing stress to exist after the surface, chamber at cytoadherence, it simulates physiological environment.

Pass saturable timbering material controlled, continue differential pressure gradients and can produce by any suitable structure, described structure include but not limited to toothed gear pump, peristaltic pump, surge pump, impeller pump and by fluid column produce by kinetic head, as long as pressure is fully continued and has enough sizes to realize advantage of the present invention.In particularly preferred embodiment, utilize the medium comprise endotheliocyte, with the pressure head of about 50mmHg and in about 5 minutes time length, to the saturating wall of blood vessel graft support exert pressure.

Another embodiment that is used for the preferred biotron device on level ground, cell shop is the tubulose biotron shown in Fig. 4 A-4D.Hook is assemblied in the inner of two portions pipeline 303 (preferred hard silicone pipeline) to hook junctor (barb-to-barb connector) (not shown), and this pipeline is placed in " groove " 302 of inner sleeve 301.Blood vessel graft is connected in two hooks, thereby limits flow in the internal blood vessel (IVF) space or the chamber fluid-space.This space seals from the outside in the groove 302 that is produced by inner sleeve 301 and by means of the interference fit between hard silicone pipeline 303 and the inner sleeve I D.Can cause that IVF flows by two portions silicone pipeline 303 being connected to any flow system then by lumen port 305.So that being in, two pairs of O shape ring sealing-rings 307 seal biotron by making inner sleeve 303 slip into outer sleeve in the outer sleeve then.Vascular outflow moving (EVF) is finished by a pair of threading hook (not shown) that is connected in exocoel port 308.Two O shapes ring 307 by means of each end of inner sleeve seals EVF from the outside.Simultaneously EVF is opened to blowback road (by means of any one or both of pipe connection) to finish wall mobile by clamp cavity space via inflow chamber space, silicone pipeline nearside ground and distally ground to exocoel port 308.Unshowned is the hook and the graft of two hard silicone pipelines in threading hook junctor on the outer sleeve of exocoel port and the interior groove that is connected in inner sleeve.

Preferred biotron of the present invention has: 1) be used for two moving individual flow spaces of chamber and vascular outflow, 2) silicone O shape ring anchor clamps, be used to provide the dual seal contact, 3) minimized number threaded fastener, it minimizes treatment time and production cost, 4) optically transparent material, being used to observe sample and inside and outside of cavity flows, 5) can use material manufacturing autoclavable and gaseous sterilization, 6) embedded many silicone O shape loop systems, be used for conduit and connect, so that after sample is placed, can regulate catheter length and position, angle, and 7) metal threaded inset, thus need not the parts that cutting thread is made, and eliminated the inefficacy of plastic whorl.In addition, can provide automatization shaking table or other swivel arrangement during level ground, pressurization shop, to promote the uniform distribution of cell in conduit cavity.Shaking table can be fixed in the automatization filling system and by it power is provided, and biotron can be assemblied in the shaking table.Yet, do not need complete 360 degree of shaking table to cover graft.

The tubular structure of Fig. 4 has listed above being beneficial to, and comprise following benefit in addition: no tool to open/close, minimum volume, convenient length and the rotation of graft are regulated, are vertically increased in proportion and equilibrated pressure, so that seepage can not take place by means of O shape ring sealing mechanism.

Method of the present invention and biotron device can be used in combination with various medium filling systems.By using the automatic cytological culture apparatus, can should be used for optimizing advantage of the present invention at some organizational project, preferably as described in U.S. Patent application series the 09/967th, No. 995 and the 10/109th, No. 712.

Above-mentioned patent application provides automatization perfusion culture platform so that the mass transfer that controlled medium flowing, shearing stress, nutrition sent, refuse is removed and improve to be provided.These can solve many shortcomings of traditional culture systems by following aspect: the sterile barrier to polluting is provided, be that cell and tissue keep more all even controlled physiological environment simultaneously, to user's sampling access and data, and provide available reproducibility and reliability for data tracking and record.

Such automatization filling system can comprise durable shell (cartridge), and this shell comprises pump, valve array, under meter and user interface.It preferably has embedded microcontroller and pre-programmed flow pattern (having mobile state able to programme).A plurality of perfusions loop shell can be placed in the single dock framework (docking station rack), and this single dock Frame Design becomes to be placed in the thermostat container of laboratory.Disposable perfusion flow path combines and can have the medium reservoir of one, the pipeline that is used for gaseous interchange and control agent mobile valve matrix with shell.According to the present invention, biotron can cooperate with flow path.Periodically reflux and to be used for reducing the medium composition difference that exports from entering the mouth to.The user can also provide the automatic sampling system so that can obtain the dielectric sample that is used to analyze.Flow velocity can change to 120ml/min or bigger from for example 1ml/min; Can monitor mobile (flow) by the optics staetometer.

Since be used for of the present invention to flow saturating wall normally of small part, so flow velocity depends on the perviousness of graft material, and along with cell is applied in the surface, chamber and reduces.Depend on graft material, saturating wall flow velocity can be 5ml/min-50ml/min before the introducing cell, and is reduced to 1ml/min-10ml/min later on usually at the introducing cell.Preferred endotheliocyte number comprises 120,000-2,000,000 cell/cm ²Chamber surface-area, more preferably from about 250,000 cell/cm ²

In another specific embodiment of the present invention, apparatus system is modular, thus the device tissue digestion and separate part can with interchangeable module use with cell is applied to blood vessel graft or with cell harvesting in syringe.Fig. 5 shows the device with modular system (modular system) assembling.Because the cellular segregation of device partly is placed in unique independent unit,, this embodiment is used to handiness that the cellular segregation unit is cooperated with other system so also providing.Preferably, this device is divided into three different modules: cellular segregation module, graft shop level ground module and cell harvesting module.

The cellular segregation module

In one embodiment, the cellular segregation module is the separate piece of equipment, and it comprises all necessary electronic instruments and parts with cutting, heating, digestion and isolated adipose tissue.In a preferred embodiment, the cellular segregation parts comprise whizzer.Supply with the single-cell suspension liquid of isolated cell from the outlet of cellular segregation module, to be connected to graft shop level ground module or cell harvesting module.Fig. 6 shows cellular segregation module durable components and disposable parts.Fig. 7 shows the cellular segregation module, and wherein disposable unit is loaded on the durable components of cellular segregation module.

In specific embodiment, all electronic machines that storing apparatus operation in the durable unit of cellular segregation module is required comprise computer plate, software, power supply and user interface.In a preferred embodiment, user interface comprises the lcd screen with button, and it is by the setting and the Operating Guideline user of device.The durable unit further of cellular segregation module is held necessary pinch valve, motor, transmitter and cutting, heating, digestion and centrifugal curee tissue other required durable components of (subiect tissue).In a preferred embodiment, curee's tissue is a fatty tissue.Pinch valve stretches out so that valve can engage disposable fluid channel from the shell on the top plan.In a preferred embodiment, electronic machine is positioned at what fluid channel of leaving one's post and has maximum distance apart.

In another embodiment, device comprises that installable suspension hook is to hang medium bag and litter bag.Preferably, the bag suspension hook is mounted to graft shop level ground durable components or cell harvesting durable components so that the distance maximization between medium bag and the electronic machine (being contained in the cellular segregation durable components).This separation has reduced fluid and has overflowed the danger that electronic machine is caused damage.

In specific embodiments of the invention, all elements of cellular segregation module flow path are disposable.In one embodiment, these disposable units can be assembled on the rigid disk, and wherein rigid disk is loaded on the cellular segregation module durable components.The user can aim at and dish is placed on the plane of durable components with the valve isolating switch in the disposable dish by making pinch valve, loads disposable dish.Then the user forward slider disc to be bonded on the pipeline loop in the pinch valve and disposable dish be locked in the appropriate position.All disposable units are positioned in the dish to separate the durable components of the necessity in the durable components with zygoneure by this load operation aligning.Owing to need not the independent loading of many pipe connection and many disposable units, the dish design minimizes user's the setting and the burden of processing.After loading disc, the user can be loaded into disposable centrifugal bowl in the groove that is arranged in the durable components and will be from the entrance and exit pipe connection of disposable dish to whizzer, medium bag, litter bag and level ground, shop or collector unit.

Level ground, graft shop module

Level ground, graft shop module is meant and is used to utilize level ground, pressurization shop technology will be applied to durable and disposable unit required on the porous implants support by the cell that the cellular segregation unit provides.Level ground, graft shop module is durable to be shown among Fig. 8 with disposable unit.Fig. 9 further shows level ground, graft shop module durable components, and it is connected in the cellular segregation unit and is loaded with for the disposable unit that uses.

In an embodiment of the present invention, level ground, shop module comprises two durable components: Pu Ping unit durable components and graft chamber durable components.These durable components physically cooperate with the cellular segregation durable components electric power to be provided and to communicate to connect.In another embodiment of the present invention, control level ground, shop module durable components by the electronic machine in the cellular segregation module durable components.Graft chamber durable components provides fixed installation for disposable graft biotron and holds required parts are heated in the chamber.Level ground, shop durable components comprises hardware (for example, pinch valve, transmitter), and it is particularly to need by flowing of graft chamber in order to operation, uses needed as level ground, pressurization shop.In one embodiment, level ground, shop durable components has the top plan that has outstanding durable components, and level ground, shop disposable unit can be loaded on this top plan.Figure 10 shows the major parts in the durable and disposable unit on level ground, graft shop.

In another embodiment, level ground, shop disposable unit comprises the disposable dish of disposable graft biotron and level ground, shop.Usually preload has support or other substrate material in disposable graft chamber, and it provides sealed environment, prevents that simultaneously all other gases, liquid and the solid matter that carry out with surrounding environment from exchanging so that liquid is delivered to graft.In a kind of embodiment, three ports on the graft chamber are connected so that the inlet from the graft chamber, saturating wall outlet and chamber outlet to be provided with pipeline from the disposable dish in level ground, shop.In one embodiment, the graft chamber is placed on the inside of chamber durable components, and wherein the chamber durable components has door close in the level ground operating process of shop the chamber is sealed.

In one embodiment, the disposable rigid disk in level ground, shop comprises needed all disposable units of level ground, shop operation and is connected material.By make pinch valve aim at the valve isolating switch in the disposable dish and to front slide with the pipeline in the gripper valve, and dish is loaded on the plane of shop level ground durable components.The user connects cellular segregation disposable unit, shop level ground disposable unit and graft chamber disposable unit, to be formed for spreading the complete flow path on level ground.

Collection module

Collection module is meant and is used for from the cellular segregation unit of syringe collecting cell for the durable and disposable unit of the usefulness of cell therapy.The cell harvesting module is durable to be shown among Figure 11 with disposable unit.Figure 12 shows cell harvesting module durable components, and it is connected in the cellular segregation module, wherein is loaded with for the disposable unit that uses.In one embodiment, the collection module durable components physically matches with the cellular segregation unit electric power to be provided and to communicate to connect.Collect durable components and hold linear actuators, the cellular products that it is connected with syringe and produces with in the automatic collecting cell separating unit.

In another embodiment, the disposable unit in the collector unit is to be used for the syringe of collecting cell product.In keeping syringe in position by the clip on the collector unit durable components.With the top-loaded of syringe in durable components, thereby can draw syringe piston by moving of actuator.The user will be connected to the tip of syringe from the outlet conduit of cellular segregation module.

System operation

In one embodiment, the user installed the needed durable components of current application (that is, graft shop level ground durable components or cell harvesting durable components) in the past at switching on device.When switching on device, whether it imports, detects durable module and suitably engage, diagnose at first, and enters standby mode.The user presses near the button of indicating meter with the apparatus for initializing setting then.The OR test kit enters a kind of pattern to allow to install disposable unit.The prompting user utilizes the bar code scanning device that is installed on the cellular segregation durable components that each disposable unit is scanned.When the scanning input disposable unit, the OR test kit will verify that correct durable components is in the suitable position, then guides user by each step to load disposable unit and to carry out necessary pipe connection.This device is suitably loaded the sensing disposable unit and guarantees has installed all disposable units that need for current application.In a preferred embodiment, the bar code scanning device is positioned on the device, so that the scanning of disposable unit can not disturbed the loading of disposable unit.The bar code scanning device of installing is shown among Figure 13.

After finishing device was provided with, the user pressed " OK " button with beginning.Device carries out the air purge operation, and its medium and serum are pumped by flow path, shift air onto with venting port waste collection point, and this venting port can be discharged in the atmosphere air.The graft chamber is bypassed, so that graft will never be exposed to air.

The user is prompted then, and fat is injected in the port on the whizzer disposable unit.When fatty tissue passes stator blades and enters whizzer, the dipping fatty tissue.The user presses " OK " to continue.The user is prompted then, introduces protein enzyme solution by identical inlet.In a preferred embodiment, protein enzyme solution is collagenase/PBS solution.The user presses " OK " to continue.User interface display indication beginning cellular segregation process.

In a preferred embodiment, from this moment, do not need customer interaction up to finishing whole OR test kit process.User interface display provides the continuous renewal on the process, the estimated time that it is indicated ongoing concrete operations, finishes the estimated time of this operation and finish whole process.In one embodiment, can also obtain other important technical parameter (temperature, pump speed etc.) by means of display user.

In one embodiment, the graft support is put in the indoor ethanol of disposable graft or other the suitable sterile substance.Graft preparation and below the cellular segregation step that provides carry out simultaneously.Following steps are relevant with the preparation of the graft that is used to spread the level ground.(1) ethanol is removed-removed from the graft chamber by making medium flow cross that graft chamber and guiding liquids are discharged to refuse to ethanol; (2) support pre-treatment-make medium recirculation by the graft chamber up to the cell suspending liquid that can obtain to be used for level ground, graft shop.Medium can include but not limited to M199, M199E, PBS, salt solution or not have the DPBS of dication (no divalent cation, Di-Cation Free).In a preferred embodiment, medium is M199E and from 6: 1 mixtures of patient's serum.

For shop level ground operator scheme and cell harvesting operator scheme, the cellular segregation process is identical.In one embodiment, the cellular segregation step comprises: (1) fatty tissue digestion-with Centrifugal Machine Control is in about 37 ℃ temperature and low speed mixing effect (keeping mixing reasonable time to guarantee sufficient digestion) is provided; (2) centrifugation-, fatty tissue is separated into its constituent materials with high RPM rotary centrifuge; And (3) endotheliocyte separates and resuspending-isolating inclusion is pumped in the thin transparent pipeline position and the volume of optical pickocff detection endotheliocyte there.Unwanted material is directed into waste container, and the endotheliocyte of specific volume is returned to whizzer.6: 1 mixtures of M199E and serum are pumped in the whizzer.Whizzer is suspended in the mixture isolated cells by the low speed mixing effect.Then with cell suspending liquid from the whizzer pumping by 30 micron filters and be directed to graft Pu Ping unit or the cell harvesting unit, be used for collecting syringe.Figure 14 shows the sectional view of an embodiment of centrifugal bowl.

In the process on level ground, graft shop, liquid passes between separation module and graft module via level ground, shop module.Preferably, the temperature with graft is controlled at about 37 ℃.In specific embodiment, level ground, graft shop step comprises that cell is spread the level ground and " charging and outflow " flows.In the step of level ground, cell shop, endotheliocyte suspension is introduced in the recirculation flow path, makes cell suspending liquid at one end flow into graft and passes through the graft wall and flow out.At first, the liquid mixture that leaves the graft chamber is directed into waste container and has entered re-circulation path up to whole volumetrical cell suspending liquid.Cell suspending liquid carries out recirculation up to finishing level ground, graft shop then.Micropore ePTFE allows medium/serum mixture to pass through, but cell is encapsulated among the ePTFE.In this process, monitor transmural pressure power by the pressure transmitter in the module of level ground, shop.In " charging and outflow " flow step, when reaching specific transmural pressure power, graft is flowed and is switched to the chamber and flows, thereby shows that the level ground, shop finishes.In this process, flowing alternately is directed to waste container and pump, is used for recirculation.Flow be drawn towards waste container during, the medium and the serum that replenish from the reservoir pumping.Keep " charging and outflow " process reasonable time.

In an embodiment of cell harvesting process of the present invention, cell suspending liquid is pumped into syringe the collection module from separation module.Linear actuators pulling syringe piston, thereby with the cell suspending liquid inhalation syringe.

When operation is finished, provide vision indication and/or audible indication to the user.The user is prompted, and removes product (graft or the whole syringe on level ground, shop).When removing product, the user selects " OK " on user interface.Device enters the pattern that is used to remove disposable unit then.In this pattern, can remove disposable unit from the unit that is used for disposing.The user selects " OK ", and device enters standby mode then, waits for another process of operation or closure systems.Figure 15 illustrates system's flow path.

The present inventor is verified, and continuing pressure head (pass permeable timbering material and put on the liquid medium with suspension cell) provides the advantage of quick cytoadherence, and does not need as employed bigger pressure gradient in the technology of level ground, instantaneous pressurization shop.By means of the device design of any number, and at all cell types, timbering material and geometrical shape, those skilled in the art can easily implement the present invention.Utilization is no more than the experiment of normal experiment, and those skilled in the art will understand that maybe can determine specific embodiment of the present invention described herein many are equal to replacement.The replacement that is equal to like this is intended to be contained by claim.

Treatment is used

Tissue grafts and cell suspending liquid by the said apparatus preparation can be used for various treatments application.For example, in one embodiment of the invention, provide by with at least a tissue grafts of any said apparatus preparation or cell suspending liquid implanting tissue or the organ and to tissue that is in the curee in needing or the method that organ carries out revascularization.

In one embodiment, tissue grafts or cell suspending liquid comprise the cell that is selected from the group of being made up of auricle, lung, mesentery or the fatty tissue of skin, skeletal muscle, cardiac muscle, heart.Fatty tissue can be from fat, perirenal fat, pericardial fat, subcutaneous lipids, chest fat or epididymal adipose tissues before nethike embrane fat, the peritonaeum.

In certain embodiments, tissue grafts or cell suspending liquid further comprise suitable stroma cell, stem cell, relevant cell or its combination.When using in this article, term " stem cell " is used in a broad sense and comprises traditional stem cell, progenitor cell, preceding progenitor cell, replenishes cell etc.Exemplary stem cell comprises that embryonic stem cell, adult stem cell, multipotent stem cells, neural stem cell, liver stem cells, flesh stem cell, flesh precursor stem cell, endothelial progenitor cells, bone marrow stem cell, cartilage form stem cell, lymphoid stem cell, mescenchymal stem cell, hemopoietic stem cell, central nervous system stem cell, peripheral nervous system stem cell etc.Description (comprising the method that is used for separating and cultivating them) to stem cell can be found (except other place) at following document: Embryonic Stem Cells, Methods and Protocols, Turksen ed., Humana Press, 2002; Weisman etal., Annu.Rev.Cell.Dev.Biol.17:387403; Pittinger et al., Science, 284:143 47,1999; Animal Cell Culture, Masters, ed., OxfordUniversity Press, 2000; Jackson et al., PNAS 96 (Shepherd BR et al.Rapid perfusion and network remodeling in a microvascular constructafter implantation.Arterioscler Thromb Vasc Biol 24:898-904,2004): 1448286,1999; Zuk et al., Tissue Engineering, 7:211228,2001 (" Zuket al. "); Atala et al., especially 3341 chapters; And United States Patent (USP) the 5th, 559, No. 022, the 5th, 672, No. 346 and the 5th, 827, No. 735.Description (comprising the method that is used for separating them) to stroma cell can be found (except other place) at following document: Prockop, Science, 276:7174,1997; Theise et al., Hepatology, 31:23540,2000; Current Protocols in Cell Biology, Bonifacino et al., eds., John Wiley﹠amp; Sons, 2000 (comprising) to the renewal in March, 2002; And No. the 4th, 963,489, United States Patent (USP).The technician will understand, selectedly be included in the desirable application that stem cell in tissue grafts or the cell suspending liquid and/or stroma cell are generally suitable for constituting thing.

In specific embodiment, tissue grafts or cell suspending liquid comprise endotheliocyte, and these endotheliocytes can be divided into (but being not limited to) neurone, the myocardial cell, the chondrocyte, pancreatic acinar cell, pancreatic endocrine cell (comprising the Langerhans pancreas islet), liver cell, renal epithelial cell, the parathyroid gland cell, Leydig cell, sustenticular cell, gonocyte, ovocyte, blastocyst, Kupffer cell, lymphocyte, inoblast, the myocyte, sarcoplast, satellite cell, adipocyte, preceding adipocyte, osteocyte, scleroblast, osteoclast, the chondrocyte, the courage epithelial cell, Purkinje cell, and pacemaker cell.

In another specific embodiment, tissue grafts or cell suspending liquid comprise at least a stem cell, progenitor cell or relevant cell, it can be but be not limited to neurone, the myocardial cell, the chondrocyte, pancreatic acinar cell, pancreatic endocrine cell (comprising the Langerhans pancreas islet), liver cell, renal epithelial cell, the parathyroid gland cell, Leydig cell, sustenticular cell, gonocyte, ovocyte, blastocyst, Kupffer cell, lymphocyte, inoblast, the myocyte, sarcoplast, satellite cell, adipocyte, preceding adipocyte, osteocyte, scleroblast, osteoclast, the chondrocyte, the courage epithelial cell, Purkinje cell, and pacemaker cell.

When using in this article, term " relevant cell " is meant such cell, and based on the desirable purposes of tissue grafts or cell suspending liquid, these cells are suitable for adding by in the prepared tissue grafts or cell suspending liquid of device of the present invention.For example, the relevant cell that is suitable for reparation, revascularization or the repopulation of impaired liver can include but not limited to liver cell, courage epithelial cell, Kupffer cell, inoblast etc.The exemplary relevant cell that is used to add tissue grafts or cell suspending liquid comprises neurone, myocardial cell, myocyte, chondrocyte, pancreatic acinar cell, Langerhans pancreas islet, osteocyte, liver cell, Kupffer cell, inoblast, myocyte, sarcoplast, satellite cell, endotheliocyte, adipocyte, preceding adipocyte, courage epithelial cell etc.Can separate and cultivate the cell of these types by routine techniques known in the art.Typical technology can be referring to (except that other place): Atala et al., especially the 932nd chapter; Freshney, Culture of Animal Cells A Manual of Basic Techniques, 4th ed., WileyLiss, John Wiley﹠amp; Sons, 2000; Basic Cell Culture:A PracticalApproach, Davis, ed., Oxford University Press, 2002; Animal CellCulture:A Practical Approach, Masters, ed., 2000; And United States Patent (USP) the 3rd, 516, No. 681 and the 5th, 559, No. 022.

The technician will understand, can during preparation or after the preparation such stroma cell, stem cell and/or relevant cell be joined in tissue grafts or the cell suspending liquid.For example, but be not limited to, can realize cell suspending liquid, stem cell, relevant cell and/or stroma cell are incorporated into liquid dimensional culture thing, in collagen, fibrin etc., perhaps in tissue grafts or on inoculation or shop level ground stem cell, relevant cell and/or stroma cell.The example combinations that is used for adding suitable stem cell, stroma cell and the relevant cell of tissue grafts or cell suspending liquid comprises: Langerhans pancreas islet and/or pancreatic acinar cell in tissue grafts or the cell suspending liquid are used for the revascularization of impaired pancreas; Liver cell in tissue grafts or the cell suspending liquid, hepatic progenitor cells, Kupffer cell, endotheliocyte, endodermis stem cell, liver inoblast and/or liver are replenished cell, are used for the revascularization of impaired liver.For example, but be not limited to, be used for tissue grafts or cell suspending liquid (is used for vascularization, repair, and rebuild impaired or ill liver) suitable stem cell or stroma cell can comprise that liver replenishes cell, hepatic progenitor cells, as but be not limited to the liver inoblast, embryonic stem cell, liver stem cells, the myocardial cell, Purkinje cell, pacemaker cell, sarcoplast, mescenchymal stem cell, satellite cell, and/or bone marrow stem cell, be used to reproduce impaired or ischemic heart (referring to, for example, Atkins et al., J.of Heart and LungTransplantation, December 1999, and at pages 1,173 80; Tomita et al., Cardiovascular Research Institute, American Heart Association, 1999, at pages 92 101; Sakai et al., Cardiovascular Research Institute, American Heart Association, 1999, at pages 108 14) or the like.

In one embodiment, tissue grafts or cell suspending liquid further comprise a kind of preparation, said preparation be selected from by cytokine, chemokine, microbiotic, medicine, anodyne, antiphlogistic drug, immunosuppressor, or combinations thereof group.

The exemplary cells factor can include but not limited to angiogenin, vascular endothelial growth factor (VEGF, include but not limited to VEGF-165), interleukin-, fibroblast growth factor (for example, but be not limited to, FGF-1 and FGF-2), pHGF (HGF), transforming growth factor-beta (TGF-β), endothelin is (as ET-1, ET-2, and ET-3), rhIGF-1 (IGF-1), the vascularization element is (as Ang-1, Ang-2, Ang-3/4), the plain sample albumen of vascularization is (as ANGPTL-1, ANGPTL-2, ANGPTL-3, and ANGPTL-4), Thr6 PDGF BB (PDGF, include but not limited to PDGF-AA, PDGF-BB and PDGF-AB), Urogastron (EGF), endothelial cell growth factor (ECGF) (ECGF comprises ECGS), platelet-derived endothelial cell growth factor (ECGF) (PD-ECGF), placenta growth factor (PLGF) etc.Cytokine (comprising recombinant cytokine) and chemokine usually can be available from many sources, for example, and R﹠amp; D Systems (Minneapolis, Minn.); Endogen (Woburn, Wash.); And Sigma (St.Louis, Mo.).The technician will understand, be used for adding the chemokine of particular organization graft or cell suspending liquid and destination organization or the organ of wanting vascularization, reproduce, increase or rebuilding will be partly depended in the selection of cytokine.

In certain embodiments, tissue grafts or cell suspending liquid further comprise at least a genetically engineered cell.In certain embodiments, comprise the tissue grafts of at least a genetically engineered cell or at least a gene product that cell suspending liquid is expressed composition ground or the expression of inducibility ground is encoded by at least a genetically engineered cell, this is owing to caused by the change of the heredity at least a genetically engineered cell of technological guide known in the art.The description of exemplary genetic engineering technique can be referring to (except that other place): Ausubel et al., Current Protocols in Molecular Biology (comprising augmenting in March, 2002), John Wiley﹠amp; Sons, New York, N.Y., 1989; Sambrooket al., Molecular Cloning:A Laboratory Manual, 2.sup.nd Ed., ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; Sambrook and Russell, Molecular Cloning:A Laboratory Manual, 3.sup.rd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; Beaucage et al., Current Protocols in Nucleic AcidChemistry, John Wiley﹠amp; Sons, New York, N.Y., 2000 (comprising augmenting) in March, 2002; Short Protocols in Molecular Biology, 4.sup.th Ed., Ausbel, Brent, and Moore, eds., John Wiley﹠amp; Sons, New York, N.Y., 1999; Davis et al., Basic Methods in Molecular Biology, McGraw HillProfessional Publishing, 1995; Molecular Biology Protocols (referring to the highveld.com website), and agreement online (protocol-online.net).The exemplary gene product that is used for genetic modification genetically engineered cell of the present invention comprises plasminogen activator, solubility CD4, Factor IX, factors IX, Feng-von willebrand's factor, urokinase, r-hirudin, Interferon, rabbit (comprises alpha-interferon, beta-interferon and gamma-interferon), tumour necrosis factor, interleukin-, hemopoieticgrowth factor, antibody, glucocerebrosidase, adenosine deaminase, Phenylalanine hydroxylase, human growth hormone, Regular Insulin, erythropoietin, VEGF, the vascularization element, pHGF, PLGF etc.

In an embodiment of the present invention, tissue or organ are selected from the group of being made up of heart tissue, lung tissue, cardiac muscular tissue, striated muscle tissue, hepatic tissue, pancreas tissue, cartilage, bone, pericardium, peritonaeum, kidney, unstriated muscle, skin, mucosal tissue, small intestine, large intestine and fatty tissue.

The step that cell suspending liquid is expelled in curee's tissue or the organ can include but not limited to utilize at least one syringe, pin, intubate, conduit, pipe or micro-needle.When using in this article, term " injection ", " injection " or its modification are meant any way of discharging or extruding material, usually by managing or comprise the structure of hole or outer aperture.Aforementioned tube or structure can be flexible, inflexible, maybe can comprise at least one flexible portion and at least one rigid element.Exemplary injection device comprises the syringe that has or do not have pin, intubate, conduit, flexible duct etc.Can also carry specific cell suspending liquid by the device (as micro-needle) that uses the saturating tissue of thorn.Inject on the contrary with traditional usefulness standard specifications hypodermic needle, by producing the microcosmic hole, micro-needle (radius-of-curvature with about 1 μ m is defined usually) or microneedle arrays are stung transdermal or endothelial layer.These holes are in fact as the conduit that is used for materials conveyance and can strengthen cell suspending liquid of the present invention and adhere to or be transported to blood vessel, tissue or organ.Therefore, the technician will understand, any comprise hole or outer aperture (by described hole or at least a cell suspending liquid of outer aperture can be expressed on tissue or the organ or among) structure or any can piercing tissue or the structure on organ (comprising engineered tissue) surface all within the scope of the invention.In certain embodiments, after injection, the composition of above-mentioned injection is at polymerization in vitro.

In a kind of specific embodiment, tissue grafts of the present invention or cell suspending liquid comprise the cell that is selected from the group of being made up of skin, skeletal muscle, cardiac muscle, auricle, lung, mesentery or fatty tissue.Fatty tissue can be selected from the group of being made up of fat, perirenal fat, pericardial fat, subcutaneous lipids, chest fat or epididymal adipose tissues before nethike embrane fat, the peritonaeum.

The curee's who is used for increasing needs the tissue or the method for organ also are provided, comprise and to implant in the organ or tissue by the tissue grafts of device preparation of the present invention, maybe will inject tissue or organ by the cell suspending liquid of device preparation of the present invention.When using in this article, " increase " is meant volume and/or the density that increases tissue or organ.

Also provide and be used for by injecting regenerate curee's the method for tissue or organ of tissue or organ with at least a tissue grafts implanting tissue of device described herein preparation or organ or by at least a cell suspending liquid with device preparation of the present invention.When using in this article, " regeneration " is meant and substitutes forfeiture, ill or impaired tissue under different situations by forming new tissue.

The technician will understand that curee of the present invention can be any animal, comprise Amphibians, birds, fish, Mammals and marsupial, but be preferably Mammals (for example, people; Domestic animal is as cat, dog, monkey, mouse and rat; Or commercially available animal, as milk cow, horse or pig).In addition, curee of the present invention can have any age, comprises fetus, embryo, children and adult.In a preferred embodiment of the invention, the curee is the people.In one embodiment, the curee is a horse, and method of the present invention is used for regenerating tissues around the neutralization of the hoof of animal.In another embodiment, the curee is the people, and the method for tissue regeneration be used for the prevention or the treatment, for example, sacroiliitis and the eye disease, it includes but not limited to glaucoma and macular degeneration.

In addition, be used for rebuilding the method for tissue or organ the curee of needs, comprise will at least a tissue grafts implanting tissue or organ by device described herein preparation in, maybe will inject and organize or organ by at least a cell suspending liquid of these device preparations.When using in this article, " reconstruction " is meant reconstruction, reconstitutes, changes shape and/or repair tissue or organ.Therefore in one embodiment of the invention, for example, the curee has liparitosis, gives the suitable cell suspending liquid of curee's subcutaneous injection so that the partial reconstruction fatty tissue, thereby improves curee's beauty treatment outward appearance.In one embodiment, the curee is the postoperative curee.

Also provide and be used for by injecting tissue or curee's the tissue or the method for organ primary infection and secondary infection are treated or prevented to organ with at least a tissue grafts implanting tissue of device described herein preparation or organ or by at least a cell suspending liquid with these device preparations.

Also provide the cell suspending liquid and the tissue grafts that utilize by device preparation of the present invention to form in pre-preventing tissue or the organ scar tissue and/or treatment or prevention curee's the tissue or the method for the inflammation in the organ.

Also provide and be used for preventing curee's the tissue of needs or the method for organ adhesion formation by at least a cell suspending liquid of device of the present invention preparation or tissue grafts being injected tissue or organ.

In one embodiment, a kind of method that is used for the treatment of or prevents curee's acute myocardial infarction is provided, this method is implemented in the following manner: will inject heart by at least a cell suspending liquid of any device preparation described herein, and wherein increase the vascular system of leading to heart tissue.In another embodiment, provide the myocarditic method that is used for the treatment of the curee, comprised at least a cell suspending liquid by any device preparation of the present invention is injected curee's pericardial fluid.

Also provide by at least a cell suspending liquid and be injected into the method that wound is used for the treatment of curee's wound device preparation of the present invention.In one embodiment, the curee is the postoperative curee.

The present invention also provides by at least a cell suspending liquid with device of the present invention preparation and has injected curee's tissue or be used for the treatment of or prevent the method for curee's histanoxia by the tissue of at least a tissue grafts of device preparation of the present invention being implanted the curee.

The present invention also provides the method for the generation that is used for artificial organ or organ, and the angiopoietic method that is used for artificial organ.In one embodiment, at least a cell suspending liquid of artificial organ injection by any device preparation of the present invention.In specific embodiment, vascularization occurs in external.In another embodiment, vascularization takes place in vivo.

In addition, provide the cell suspending liquid and the tissue grafts that utilize device of the present invention to prepare to screen the method that the curee is had candidate's medicament of useful treatment effect.In one embodiment, tissue grafts or cell suspending liquid contact with candidate's medicament, analyze at least a useful effect of at least a cell of tissue grafts or cell suspending liquid then.

The present invention also provides the method for the deleterious effect that is used to screen the medicament that the curee is concerned about.In one embodiment, tissue grafts or the cell suspending liquid that comprises curee's tissue grafts or cell suspending liquid contacts with the compound of being concerned about; Analyze at least a deleterious effect of at least a cell of tissue grafts or cell suspending liquid then.In one embodiment, the medicament of being concerned about is a medicine.In another embodiment, the medicament of being concerned about is the potential anaphylactogen.

The invention provides the clinical procedure of persistent pressure shop level ground method and automatization: from the desired portion of separate tissue patient cell, and filtration, washing, heating, dipping, proteolysis release, separation, resuspending, then with level ground, cell pressurization shop to permeable graft.Utilization is no more than the experiment of normal experiment, and those skilled in the art will understand that maybe can determine the embodiment of the invention described herein many are equal to replacement.The replacement that is equal to is like this contained by following claim.

All publications, patent and the patent application mentioned in this specification sheets all are incorporated into this specification sheets with way of reference, are incorporated into this paper as each independent publication, patent or patent application particularly and individually with way of reference.

More than description and example only are used for the preferred embodiment of realizing purpose of the present invention, characteristics and advantage is described, rather than are used for limiting the present invention.

Claims (183)

1. device that is used to prepare tissue grafts comprises:

The medium reservoir;

The separate tissue chemistry reservoir that is communicated with the cell inlet;

Biotron is used to hold implant matrix and has inlet and first outlet;

The medium flowing loop, it connects described biotron and described medium reservoir;

Pump, it is configured to cause flowing by described medium flowing loop;

The cell mixture conduit, it is connected described separate tissue chemistry reservoir with described medium flowing loop;

Cell separator, itself and described cell mixture conduit and described medium flowing circuit communication; And

At least one valve, it is configured to guide to described medium flowing loop with flowing from described cell separator.

2. device according to claim 1 further comprises the cell steeping cell that is communicated with described cell mixture conduit.

3. device according to claim 1 further comprises well heater.

4. device according to claim 1 further comprises waste container.

5. device according to claim 4 further comprises the strainer between described cell inlet and described separate tissue chemistry reservoir.

6. device according to claim 5 further comprises at least one valve, and described valve is configured to allow enter described reservoir and allow second section to enter described waste container from the first part of the cell of described strainer.

7. device according to claim 1 further comprises media lines, and described media lines allows flowing between described cell inlet and the described medium reservoir.

8. device according to claim 1 further comprises the strainer between described cell separator and described biotron.

9. device according to claim 1, wherein, described biotron comprises the perforated tubular graft support and second outlet, described second outlet is configured to allow the saturating chamber by described support to flow.

10. device according to claim 1, wherein, described biotron comprises porous the plane sheets graft support and second outlet basically, described second outlet is configured to allow the saturating wall by described support to flow.

11. device according to claim 9, wherein, described biotron comprises:

Outer sleeve, it has proximal end and distal end portion;

Inner sleeve, it is at least partially disposed in the described outer sleeve and has groove between proximal end and distal end portion and the described end, and described groove is set in the described outer sleeve to limit the internal space between described inner sleeve and outer sleeve;

Distal interior conduit and nearside inner conduit, wherein said distal interior conduit ground, distally in the described distal end portion of described inner sleeve extends, and the nearside ground extension in the described proximal end of described inner sleeve of described nearside inner conduit;

Kapillary inner port in the described internal space, described port are suitable for the gillies' graft support is contained between described distal interior conduit and the described nearside inner conduit, and described graft support limits in the kapillary and the extracapillary space; And wherein said first outlet allows to flow by the saturating wall of described graft.

12. device according to claim 10 further comprises the nearside pipeline, it is connected in described nearside inner conduit, and spatial flows in the described at least kapillary that flows to described graft support to provide.

13. device according to claim 10 further comprises the distally pipeline that connects described distal interior conduit.

14. device according to claim 10 further comprises the mobile device that is used to block by described distally pipeline.

15. device according to claim 10, wherein, described distal catheter comprises the chamber inner port.

16. device according to claim 1, wherein, described device is a handheld apparatus.

17. a method for preparing tissue grafts comprises: device according to claim 1 is provided; The medium that will comprise adherent cell is incorporated in the described biotron; And pass mobile being enough to of the saturating wall of lasting low pressure that matrix applies described medium the time cycle of described cytoadherence to described matrix.

18. method according to claim 15, wherein, described matrix is the tubular bracket that is used for vascular tissue transplantation's thing.

19. method according to claim 15, wherein, described matrix is plane sheets support basically.

20. method according to claim 15, wherein, described pressure is that about 10mmHg is to about 55mmHg.

21. method according to claim 15, wherein, described pressure is that about 35mmHg is to about 50mmHg.

22. method according to claim 15, wherein, described pressure is about 50mmHg.

23. method according to claim 15, wherein, the described time cycle is about 5 minutes to about 1 hour.

24. method according to claim 15, wherein, described adherent cell is the cell that discharges from fatty tissue greater than the effect of the separate tissue chemical substance of adipocyte by the density that has.

25. method according to claim 16, wherein, described cell is an endotheliocyte.

26. method according to claim 15, wherein, described cell is a capillary endothelium.

27. method according to claim 15, wherein, described capillary endothelium stems from fatty tissue.

28. method according to claim 23 further comprises and obtain fatty tissue from the patient.

29. method according to claim 23, wherein, described cell comprises the mixture of capillary endothelium and adult stem cell.

30. method according to claim 23, wherein, described capillary endothelium is a stem cell.

31. method according to claim 25, wherein, described cell is from body.

32. method according to claim 25, wherein, described matrix comprises a kind of material in the group that is selected from following material composition: elastin, ePTFE, collagen, polyurethane(s), polypropylene, polyethylene, polymeric amide, nylon, elastin, polyethylene terephthalate, polycarbonate, polystyrene, poly(lactic acid), polyglycolic acid, PLA/PGA mixture, dextran, polyoxyethylene glycol, stainless steel, titanium/nickelalloy and silicone.

33. a device that is used to prepare tissue grafts comprises:

The flow path shell, it comprises one or more fluid reservoirs, at least one inlet and at least one outlet;

The cell separator shell, it has at least one inlet and at least one outlet;

Be used to hold the graft chamber shell of implant matrix, described graft chamber shell has at least one inlet and at least one outlet; At least one pump, described pump quilt

Be configured to cause flowing by flow path;

At least one valve, described valve are configured to guide to described graft chamber shell with flowing from described cell separator shell;

Wherein said flow path shell, cell separator shell and graft chamber shell are communicated with forming continuous flow path, and wherein said flow path shell, cell separator shell and graft chamber shell provide the modularized test kit shell of power to link to each other with giving described device.

34. device according to claim 33, wherein, described flow path shell, cell separator shell and graft chamber shell are disposable.

35. device according to claim 33, wherein, described cell separator shell comprises whizzer.

36. device according to claim 33 further comprises the cell steeping cell that is communicated with described flow path shell.

37. device according to claim 33, wherein, the preload of described flow path shell has medium.

38. device according to claim 33, wherein, described medium is selected from the group of being made up of M199, M199E, PBS, salt solution and unparalleled cationic DPBS.

39. device according to claim 33, wherein, described medium is M199E.

40. device according to claim 33, wherein, described medium is a buffer saline.

41. device according to claim 33, wherein, described flow path shell comprises at least one pump.

42. device according to claim 33 further comprises well heater.

43. device according to claim 33 further comprises waste container.

44. device according to claim 33 further comprises at least one strainer.

45. device according to claim 33, wherein, described strainer is got rid of greater than about 100 microns particle.

46. device according to claim 33, wherein, described strainer is got rid of the particle greater than 30 microns.

47. device according to claim 33, wherein, at least one filter bits is between described cell separator shell and described graft shell.

48. device according to claim 33, wherein, described test kit shell comprises at least one sensor device, is used for detecting the existence of described flow path shell, described cell separator shell and described graft chamber.

49. device according to claim 33, wherein, described test kit shell comprises at least one sensor device, is used for monitoring and controlled temperature, pressure and flow velocity, and wherein said sensor device is communicated with warning howler.

50. device according to claim 33, wherein, described test kit shell comprises the electrical patterns indicating meter.

51. device according to claim 33, wherein, described test kit shell comprises the bar code scanning device.

52. device according to claim 33, wherein, described test kit shell comprises the cell harvesting module with entrance and exit.

53. device according to claim 33 further comprises the device that is used for counting cells.

54. device according to claim 33, wherein, described device is a handheld apparatus.

55. a method for preparing tissue grafts comprises: device according to claim 28 is provided; The medium that will comprise adherent cell is introduced described graft chamber; And pass mobile being enough to of the saturating wall of lasting low pressure that matrix applies described medium the time cycle of described cytoadherence to described matrix.

56. according to the described method of claim 55, wherein, described matrix is the tubular bracket that is used for vascular tissue transplantation's thing.

57. according to the described method of claim 55, wherein, described matrix is porous plane sheets graft support basically.

58. according to the described method of claim 55, wherein, described pressure is that about 10mmHg is to about 55mmHg.

59. according to the described method of claim 55, wherein, described pressure is that about 35mmHg is to about 50mmHg.

60. according to the described method of claim 55, wherein, described pressure is about 50mmHg.

61. according to the described method of claim 55, wherein, the described time cycle is about 5 minutes to about 1 hour.

62. according to the described method of claim 55, wherein, described adherent cell is the cell that discharges from fatty tissue greater than the effect of the separate tissue chemical substance of adipocyte by the density that has.

63. according to the described method of claim 55, wherein, described adherent cell is an endotheliocyte.

64. according to the described method of claim 55, wherein, described cell is a capillary endothelium.

65. according to the described method of claim 55, wherein, described capillary endothelium stems from fatty tissue.

66., further comprise and from the patient, obtain fatty tissue according to the described method of claim 65.

67. according to the described method of claim 65, wherein, described cell comprises the mixture of capillary endothelium and adult stem cell.

68. according to the described method of claim 65, wherein, described cell is from body.

69. according to the described method of claim 55, wherein, described matrix comprises a kind of material in the group that is selected from following material composition: elastin, ePTFE, collagen, polyurethane(s), polypropylene, polyethylene, polymeric amide, nylon, elastin, polyethylene terephthalate, polycarbonate, polystyrene, poly(lactic acid), polyglycolic acid, PLA/PGA mixture, dextran, polyoxyethylene glycol, stainless steel, titanium/nickelalloy and silicone.

70. a device that is used to prepare tissue grafts comprises:

The flow path shell, it comprises one or more fluid reservoirs, cell separator and at least one inlet and at least one outlet;

Be used to hold the graft chamber shell of implant matrix, described graft chamber shell has at least one inlet and at least one outlet; At least one pump, described pump are configured to cause flowing by flow path;

At least one valve, described valve are configured to guide to described graft chamber shell with flowing from described cell separator shell;

Wherein said flow path shell, cell separator shell and graft chamber shell are communicated with forming continuous flow path, and wherein said flow path shell, cell separator shell and graft chamber shell provide the modular reagent box shell of power to link to each other with giving described device.

71. according to the described device of claim 70, wherein, described flow path shell and graft chamber shell are disposable.

72. according to the described device of claim 70, wherein, described cell separator comprises whizzer.

73., further comprise the cell steeping cell that is communicated with described flow path shell according to the described device of claim 70.

74. according to the described device of claim 70, wherein, the preload of described flow path shell has medium.

75. according to the described device of claim 70, wherein, described medium is selected from the group of being made up of M199, M199E, PBS, salt solution and unparalleled cationic DPBS.

76. according to the described device of claim 70, wherein, described medium is M199E.

77. according to the described device of claim 70, wherein, described medium is a buffer saline.

78. according to the described device of claim 70, wherein, described flow path shell comprises at least one pump.

79., further comprise well heater according to the described device of claim 70.

80., further comprise waste container according to the described device of claim 70.

81., further comprise at least one strainer according to the described device of claim 70.

82. according to the described device of claim 70, wherein, described strainer is got rid of greater than about 100 microns particle.

83. according to the described device of claim 70, wherein, described strainer is got rid of the particle greater than 30 microns.

84. according to the described device of claim 70, wherein, at least one filter bits is between described cellular segregation and described graft shell.

85. according to the described device of claim 70, wherein, described test kit shell comprises at least one sensor device, is used for detecting the existence of described flow path shell and described graft chamber.

86. according to the described device of claim 70, wherein, described test kit shell comprises at least one sensor device, is used for monitoring and controlled temperature, pressure and flow velocity, wherein said sensor device links to each other with warning howler.

87. according to the described device of claim 70, wherein, described test kit shell comprises the electrical patterns indicating meter.

88. according to the described device of claim 70, wherein, described test kit shell comprises the bar code scanning device.

89. according to the described device of claim 70, wherein, described test kit shell comprises the cell harvesting module with entrance and exit.

90., further comprise the device that is used for counting cells according to the described device of claim 70.

91. according to the described device of claim 70, wherein, described device is a handheld apparatus.

92. a method for preparing tissue grafts comprises: provide according to the described device of claim 70; The medium that will comprise adherent cell is introduced described graft chamber; And pass the saturating wall of lasting low pressure that matrix applies described medium flow the time enough cycle with described cytoadherence to described matrix.

93. according to the described method of claim 92, wherein, described matrix is the tubular bracket that is used for vascular tissue transplantation's thing.

94. according to the described method of claim 92, wherein, described matrix is porous plane sheets graft support basically.

95. according to the described method of claim 92, wherein, described pressure is that about 10mmHg is to about 55mmHg.

96. according to the described method of claim 92, wherein, described pressure is that about 35mmHg is to about 50mmHg.

97. according to the described method of claim 92, wherein, described pressure is about 50mmHg.

98. according to the described method of claim 92, wherein, the described time cycle is about 5 minutes to about 1 hour.

99. according to the described method of claim 92, wherein, described adherent cell is the cell that discharges from fatty tissue greater than the effect of the separate tissue chemical substance of adipocyte by the density that has.

100. according to the described method of claim 92, wherein, described adherent cell is an endotheliocyte.

101. according to the described method of claim 92, wherein, described cell is a capillary endothelium.

102. according to the described method of claim 92, wherein, described capillary endothelium stems from fatty tissue.

103., further comprise and from the patient, obtain fatty tissue according to the described method of claim 103.

104. according to the described method of claim 103, wherein, described cell comprises the mixture of capillary endothelium and adult stem cell.

105. according to the described method of claim 103, wherein, described cell is from body.

106. according to the described method of claim 92, wherein, described matrix comprises a kind of material in the group that is selected from following material composition: elastin, ePTFE, collagen, polyurethane(s), polypropylene, polyethylene, polymeric amide, nylon, elastin, polyethylene terephthalate, polycarbonate, polystyrene, poly(lactic acid), polyglycolic acid, PLA/PGA mixture, dextran, polyoxyethylene glycol, stainless steel, titanium/nickelalloy and silicone.

107. one kind is used for the curee's of needs tissue or the method that organ carries out revascularization, comprises at least a tissue grafts of claim 1,33 or 70 described devices preparations is implanted in described tissue or the organ.

108. according to the described method of claim 107, wherein, described tissue grafts comprises the cell that is selected from the group of being made up of auricle, lung, mesentery or the fatty tissue of skin, skeletal muscle, cardiac muscle, heart.

109. according to the described method of claim 107, wherein, described fatty tissue is selected from the group of being made up of fat, perirenal fat, pericardial fat, subcutaneous lipids, chest fat or epididymal adipose tissues before nethike embrane fat, the peritonaeum.

110. according to the described method of claim 107, wherein, described tissue grafts comprises at least a relevant cell.

111. according to the described method of claim 107, wherein, described relevant cell is selected from by at least a neurone, the myocardial cell, the chondrocyte, pancreatic acinar cell, the pancreatic endocrine cell that comprises the Langerhans pancreas islet, liver cell, renal epithelial cell, the parathyroid gland cell, Leydig cell, sustenticular cell, gonocyte, ovocyte, blastocyst, Kupffer cell, lymphocyte, inoblast, the myocyte, sarcoplast, satellite cell, adipocyte, preceding adipocyte, osteocyte, scleroblast, osteoclast, the chondrocyte, the courage epithelial cell, Purkinje cell, and the group of pacemaker cell composition.

112. according to the described method of claim 107, wherein, described tissue grafts further comprises a kind of preparation, described preparation be selected from by cytokine, chemokine, microbiotic, medicine, anodyne, antiphlogistic drug, immunosuppressor, or combinations thereof group.

113. according to the described method of claim 107, wherein, described tissue or organ are selected from the group of being made up of heart tissue, lung tissue, cardiac muscular tissue, striated muscle tissue, hepatic tissue, pancreas tissue, cartilage, bone, pericardium, peritonaeum, kidney, unstriated muscle, skin, mucosal tissue, small intestine and large intestine.

114. one kind is used for the curee's of needs tissue or the method that organ carries out revascularization, comprises that the cell suspending liquid with claim 1,33 or 72 described devices preparations injects described tissue or organ.

115. according to the described method of claim 114, wherein, described implantation step comprises and utilizes at least a syringe, pin, intubate, conduit, pipeline or micro-needle.

116. according to the described method of claim 114, wherein, described cell suspending liquid comprises the cell that is selected from the group of being made up of auricle, lung, mesentery or the fatty tissue of skin, skeletal muscle, cardiac muscle, heart.

117. according to the described method of claim 114, wherein, described fatty tissue is selected from the group of being made up of fat, perirenal fat, pericardial fat, subcutaneous lipids, chest fat or epididymal adipose tissues before nethike embrane fat, the peritonaeum.

118. according to the described method of claim 114, wherein, described cell suspending liquid comprises at least a relevant cell.

119. according to the described method of claim 114, wherein, described relevant cell is selected from by at least a neurone, the myocardial cell, the chondrocyte, pancreatic acinar cell, the pancreatic endocrine cell that comprises the Langerhans pancreas islet, liver cell, renal epithelial cell, the parathyroid gland cell, Leydig cell, sustenticular cell, gonocyte, ovocyte, blastocyst, Kupffer cell, lymphocyte, inoblast, the myocyte, sarcoplast, satellite cell, adipocyte, preceding adipocyte, osteocyte, scleroblast, osteoclast, the chondrocyte, the courage epithelial cell, Purkinje cell, and the group of pacemaker cell composition.

120. according to the described method of claim 114, wherein, described tissue grafts further comprises a kind of preparation, described preparation be selected from by cytokine, chemokine, microbiotic, medicine, anodyne, antiphlogistic drug, immunosuppressor, or combinations thereof group.

121. according to the described method of claim 114, wherein, described tissue or organ are selected from the group of being made up of heart tissue, lung tissue, cardiac muscular tissue, striated muscle tissue, hepatic tissue, pancreas tissue, cartilage, bone, pericardium, peritonaeum, kidney, unstriated muscle, skin, mucosal tissue, small intestine and large intestine.

122. a method that is used at curee's augmenting tissue of needs comprises at least a tissue grafts of claim 1,33 or 70 described device preparations is implanted in described tissue or the organ.

123. according to the described method of claim 122, wherein, described tissue grafts comprises the cell that is selected from the group of being made up of auricle, lung, mesentery or the fatty tissue of skin, skeletal muscle, cardiac muscle, heart.

124. according to the described method of claim 122, wherein, described fatty tissue is selected from the group of being made up of fat, perirenal fat, pericardial fat, subcutaneous lipids, chest fat or epididymal adipose tissues before nethike embrane fat, the peritonaeum.

125. according to the described method of claim 122, wherein, described tissue grafts comprises at least a relevant cell.

126. according to the described method of claim 122, wherein, described relevant cell is selected from by at least a neurone, the myocardial cell, the chondrocyte, pancreatic acinar cell, the pancreatic endocrine cell that comprises the Langerhans pancreas islet, liver cell, renal epithelial cell, the parathyroid gland cell, Leydig cell, sustenticular cell, gonocyte, ovocyte, blastocyst, Kupffer cell, lymphocyte, inoblast, the myocyte, sarcoplast, satellite cell, adipocyte, preceding adipocyte, osteocyte, scleroblast, osteoclast, the chondrocyte, the courage epithelial cell, Purkinje cell, and the group of pacemaker cell composition.

127. according to the described method of claim 122, wherein, described tissue grafts further comprises a kind of preparation, described preparation be selected from by cytokine, chemokine, microbiotic, medicine, anodyne, antiphlogistic drug, immunosuppressor, or combinations thereof group.

128. according to the described method of claim 122, wherein, described tissue or organ are selected from the group of being made up of heart tissue, lung tissue, cardiac muscular tissue, striated muscle tissue, hepatic tissue, pancreas tissue, cartilage, bone, pericardium, peritonaeum, kidney, unstriated muscle, skin, mucosal tissue, small intestine and large intestine.

129. a curee who is used to increase needs the tissue or the method for organ comprise that the cell suspending liquid with claim 1,33 or 70 described devices preparations injects described tissue or organ.

130. according to the described method of claim 129, wherein, described implantation step comprises and utilizes at least a syringe, pin, intubate, conduit, pipeline or micro-needle.

131. according to the described method of claim 129, wherein, described cell suspending liquid comprises the cell that is selected from the group of being made up of auricle, lung, mesentery or the fatty tissue of skin, skeletal muscle, cardiac muscle, heart.

132. according to the described method of claim 129, wherein, described fatty tissue is selected from the group of being made up of fat, perirenal fat, pericardial fat, subcutaneous lipids, chest fat or epididymal adipose tissues before nethike embrane fat, the peritonaeum.

133. according to the described method of claim 129, wherein, described cell suspending liquid comprises at least a relevant cell.

134. according to the described method of claim 129, wherein, described relevant cell is selected from by at least a neurone, the myocardial cell, the chondrocyte, pancreatic acinar cell, the pancreatic endocrine cell that comprises the Langerhans pancreas islet, liver cell, renal epithelial cell, the parathyroid gland cell, Leydig cell, sustenticular cell, gonocyte, ovocyte, blastocyst, Kupffer cell, lymphocyte, inoblast, the myocyte, sarcoplast, satellite cell, adipocyte, preceding adipocyte, osteocyte, scleroblast, osteoclast, the chondrocyte, the courage epithelial cell, Purkinje cell, and the group of pacemaker cell composition.

135. according to the described method of claim 129, wherein, described tissue grafts further comprises a kind of preparation, described preparation be selected from by cytokine, chemokine, microbiotic, medicine, anodyne, antiphlogistic drug, immunosuppressor, or combinations thereof group.

136. according to the described method of claim 129, wherein, described tissue or organ are selected from the group of being made up of heart tissue, lung tissue, cardiac muscular tissue, striated muscle tissue, hepatic tissue, pancreas tissue, cartilage, bone, pericardium, peritonaeum, kidney, unstriated muscle, skin, mucosal tissue, small intestine and large intestine.

137. one kind is used in the curee's regenerating tissues of needs or the method for organ, comprises at least a tissue grafts of claim 1,33 or 70 described device preparations is implanted in described tissue or the organ.

138. the described method of claim 137, wherein, described curee is a Mammals.

139. according to the described method of claim 138, wherein, described curee is a horse.

140. according to the described method of claim 138, wherein, described curee is the people.

141. according to the described method of claim 137, wherein, described curee suffers from sacroiliitis.

142. according to the described method of claim 137, wherein, described curee suffers from eye disease.

143. according to the described method of claim 137, wherein, described curee suffers from glaucoma.

144. according to the described method of claim 137, wherein, described curee suffers from macular degeneration.

145. one kind is used in the curee's regenerating tissues of needs or the method for organ, comprises that the cell suspending liquid with claim 1,33 or 70 described device preparations injects described tissue or organ.

146. according to the described method of claim 145, wherein, described curee is a Mammals.

147. according to the described method of claim 146, wherein, described curee is a horse.

148. according to the described method of claim 146, wherein, described curee is the people.

149. according to the described method of claim 145, wherein, described curee suffers from sacroiliitis.

150. according to the described method of claim 145, wherein, described curee suffers from eye disease.

151. according to the described method of claim 145, wherein, described curee suffers from glaucoma.

152. according to the described method of claim 145, wherein, described curee suffers from macular degeneration.

153. a method that is used for rebuilding the curee of needs tissue or organ comprises at least a tissue grafts of claim 1,33 or 70 described device preparations is implanted in described tissue or the organ.

154. according to the described method of claim 153, wherein, described curee has liparitosis.

155. according to the described method of claim 153, wherein, described curee is the postoperative curee.

156. a method that is used for rebuilding the curee of needs tissue or organ comprises that at least a cell suspending liquid with claim 1,33 or 70 described device preparations injects described tissue or organ.

157. according to the described method of claim 153, wherein, described curee has liparitosis.

158. according to the described method of claim 153, wherein, described curee is the postoperative curee.

159. one kind is used for comprising at least a tissue grafts of claim 1,33 or 70 described devices preparations being implanted in described tissue or the organ in the curee's of needs the tissue or the method for organ treatment or preventing infection.

160. one kind is used for comprising that in the curee's of needs the tissue or the method for organ treatment or preventing infection at least a cell suspending liquid with claim 1,33 or 70 described devices preparations injects described tissue or organ.

161. the method for treatment or preventing inflammation in the curee's of needs a tissue or an organ comprises at least a tissue grafts of claim 1,33 or 70 described devices preparations is implanted in described tissue or the organ.

162. the method for treatment or preventing inflammation in the curee's of needs a tissue or an organ comprises that at least a cell suspending liquid with claim 1,33 or 70 described devices preparations injects described tissue or organ.

163. tissue or the synulotic method of organ prevention that is used for the curee of needs comprises that at least a cell suspending liquid with claim 1,33 or 70 described device preparations injects described tissue or organ.

164. the method that tissue or the organ Film with Preventing Adhesion that is used for the curee of needs forms comprises that at least a cell suspending liquid with claim 1,33 or 70 described devices preparations injects described tissue or organ.

165. a method that is used in curee's treatment or prophylaxis of acute myocardial infarction comprises that at least a cell suspending liquid with claim 1,33 or 70 described device preparations injects heart, thereby increases the vascular system of leading to heart tissue.

166. one kind is used in the myocarditic method of curee's treatment, comprises that at least a cell suspending liquid with claim 1,33 or 70 described device preparations injects described curee's pericardial fluid.

167. a method that is used at curee's treatment wound comprises with at least a cell suspending liquid of claim 1,33 or 70 described device preparations and injects described wound.

168. a method that is used in curee's treatment or prevention histanoxia comprises that at least a cell suspending liquid with claim 1,33 or 70 described device preparations injects described curee's tissue.

169. an angiopoietic method that is used for artificial organ comprises with at least a cell suspending liquid of claim 1,32 or 67 described device preparations and injects described artificial organ.

170. according to the described method of claim 168, wherein, described vascularization occurs in external.

171. according to the described method of claim 168, wherein, described vascularization takes place in vivo.

172. one kind is used for screening the method that the curee has candidate's medicament of useful treatment effect, comprise: with described candidate's medicament contact tissue graft, wherein said tissue grafts comprises the curee's of claim 1,33 or 70 described device preparations cell; And at least a useful effect of analyzing at least a cell of described tissue grafts.

173. one kind is used for screening the method that the curee has candidate's medicament of useful treatment effect, comprise: with described candidate's medicament exposing cell suspension, wherein said cell suspending liquid comprises the curee's of claim 1,33 or 70 described device preparations cell; And at least a useful effect of analyzing at least a cell of described cell suspending liquid.

174. method that is used for screening the deleterious effect of the compound that the curee is concerned about, comprise: use the compound contact tissue graft of being concerned about, wherein said tissue grafts comprises the curee's of claim 1,33 or 70 described device preparations cell; And at least a deleterious effect of analyzing at least a cell of described tissue grafts.

175. according to the described method of claim 174, wherein, the described compound of being concerned about is a medicine.

176. according to the described method of claim 174, wherein, the described compound of being concerned about is the potential anaphylactogen.

177. method that is used for screening the deleterious effect of the compound that the curee is concerned about, comprise: use the compound exposing cell suspension of being concerned about, wherein said cell suspending liquid comprises the curee's of claim 1,33 or 70 described device preparations cell; And at least a deleterious effect of analyzing described cell suspending liquid.

178. according to the described method of claim 177, wherein, the described compound of being concerned about is a medicine.

179. according to the described method of claim 177, wherein, the described compound of being concerned about is the potential anaphylactogen.

180. a tissue treatment apparatus comprises: have the isolation catheter of the longitudinal axis, comprise at least one inlet, be used to be provided to pending tube material; At least one outlet is used for removing material from described isolation catheter; The flow path that between described inlet and described outlet, extends; Inner room with the internal communication of described isolation catheter; Wherein said isolation catheter is connected in centrifuge rotor, and wherein said centrifuge rotor is configured to be rotated by motor.

181. according to the described device of claim 180, wherein, described device is disposable.

182., further comprise heating unit according to the described device of claim 180.

183., further comprise pump according to the described device of claim 180.

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