EP1902128A2 - 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

APPARATUS AND METHODS FOR PREPARING TISSUE GRAFTS

[0001] This application claims priority to United States Patent

Application Serial No. 60/697,954, filed July 12, 2005, and United States Patent Application Serial No. 11/314,281, filed December 22, 2005, both of which are incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

[0002] This invention was made with Government support under

# W81XWH-04-1-0503 and # W81XWH-05-1-0620 awarded by the United States Army Medical Research and Materiel Command.

FIELD OF THE INVENTION

[0003] The present invention relates to tissue engineering systems involving mammalian cells and cell products for the manufacture of tissue implants and grafts, including, but not limited to, prosthetic vascular and arteriovenous grafts. The present invention relates, in particular, to methods and an automated apparatus to adhere fat-derived mesenchymal cells or other cells to permeable scaffold materials by sustained pressure cell sodding, which includes applying a sustained pressure gradient across a permeable material using media containing cells to be deposited on the material. The methods and apparatus of the invention promote accelerated adhesion and maturation of cells upon the scaffold material. Using sustained pressure sodding and operating room compatible automation, preferred embodiments of the present invention allow one to harvest a patient's own cells from a sample of their adipose tissue, wash, separate, and deposit these cells to prepare a suitable graft for clinical use. Such embodiments involve cell extraction followed by sustained pressure sodding in an automated, sterile, closed system.

BACKGROUND OF THE INVENTION

[0004] Tissue engineering is developing toward clinical applications for the repair and restoration of damaged or diseased tissues and organs. In particular, the development of vascular grafts is a major goal in the field of cardiac and peripheral vascular surgery. Cardiovascular disease is the leading cause of mortality and morbidity in the first world. The standard of care, the autograft, is not without serious morbidity. Patients with systemic disease, leaving no appropriate autograft material or having already undergone autografts, numbering 100,000 a year in the United States alone, have few autograft options.

[0005] Researchers have thus been studying synthetic grafts for over 30 years. A major challenge is providing graft materials that are biocompatible, i.e., nonthrombogenic, nonimmunogenic, mechanically resistant, and have acceptable wound healing and physiological responses (e.g., vasoconstriction/relaxation responses, solute transportation ability, etc.). Furthermore, tissue graft materials should be easy to handle, store and ship, and be commercially feasible.

[0006] Vessels have two principal failure modes: mechanical and biological, caused by thrombosis within the vessel and subsequent occlusion and/or cellular ingrowth. Synthetic vessels having material properties capable of withstanding arterial pressure are commonplace, making the search for nonthrombogenic materials the prime research interest. Endothelial cells obtained from the patient have been shown to decrease the thrombogenicity of implanted vessels (Williams et al., 1994, J. Vase. Surg., 19:594-604; Arts et al., 2001 Lab

Invest 81:1461-1465).

[0007] Endothelial cells are of critical importance in establishing a non-thrombogenic cell lining within synthetic grafts. Thus, it is desirable to achieve rapid cellular adhesion in or on a permeable matrix, scaffold, or other permeable cell substrate material in a matter of minutes or hours with an instrument that lends itself to the operating room environment, maintains a sterile barrier, is easy to use, and produces consistent graft results.

[0008] Currently, there are four main approaches for meeting these requirements, but with limited success: (i) the use of decellularized tissue materials; (ii) the use of a self-assembly mechanism, wherein cells are cultured on tissue culture plastic in a medium that induces extracellular matrix (ECM) synthesis; (iii) the use of synthetic biodegradable polymers, onto which cells are subsequently seeded and cultured in a simulated physiological environment; and (iv) the use of biopolymers, such as a reconstituted type I collagen gel, which is formed and compacted with tissue cells by the application of mechanical forces to simulate a physiological environment (see, e.g., Robert T. Tranquillo, 2002, Ann. KY. Acad. ScL, 961:251-254).

[0009] Pressure gradients involving transient high pressures have been used to deposit cells onto a permeable scaffold by a sieving action, i.e., providing a bulk flow and using a substrate or scaffold material having pores smaller than the cell population, thus capturing cells in the matrix (e.g., US Patent 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 cells have been shown to subsequently adhere to the scaffold material, but with only limited clinical applicability due to failure to fully meet the requisites for successful grafts discussed above, i.e., biocompatibility, mechanical strength, and necessary physiological properties.

[0010] Beginning in the late 1970s, endothelial cell seeding was employed experimentally to improve the patency of small diameter, polymeric vascular grafts to counteract adverse reactions. Since that time, advances have been made toward this goal, with the majority of the focus on engineering a biological or a bio-hybrid graft.

[0011] Endothelial cells are more complex than was originally believed in that they do not merely create a single cell lining on the lumenal surface of blood vessels. Endothelial cells also release molecules that modulate coagulation, platelet aggregation, leukocyte adhesion, and vascular tone. In the absence of these cells, e.g., in the case of the lumen of an implanted synthetic polymeric vascular graft, the host reaction progresses to eventual failure. Loss of patency within the first thirty days post-implantation is due to acute thrombosis. This early stage failure is a consequence of the inherent thrombogenicity of the biomaterial's blood-contacting surface, which is non-endothelialized. To date, the only known completely non-thrombogenic material is an endothelium; any other material that comes into contact with the bloodstream is predisposed to platelet deposition and subsequent thrombosis. The long-term failure mode of small diameter polymeric vascular grafts is anastomotic hyperplasia leading to a loss of patency. The precise mechanisms behind initiation of anastomotic hyperplasia are still being defined; however, endothelial cell and smooth muscle cell dysfunctions and improper communications are likely involved. [0012] Early workers in the field of small diameter graft development sought to promote graft endothelialization and, thereby, increase patency by transplanting a varying degree of autologous endothelial cells onto vascular grafts prior to implantation. This process has become known as endothelial cell seeding (partial coverage relying on continued cell proliferation) or cell sodding (full coverage). The underlying hypothesis is fairly simple; that is, by promoting the establishment of the patient's own endothelial cells on the blood contacting surface of a vascular prosthesis, a "normal" endothelial cell lining and associated basement membrane, together known as the neo-intima, will form on the graft and counteract the rheologic, physiologic, and biomaterial forces working synergistically to promote graft failure. After 30 years of research in this area, including promising animal data, this simple hypothesis has not yet yielded a clinical device.

[0013] The failure modes with endothelial-seeded grafts have been identical to untreated polymeric grafts, namely thrombosis and intimal hyperplasia. The failure modes, at least partially, are linked to the lack of a functional endothelial layer, neo-intima, on the lumenal surface of the graft and/or abnormal endothelial and smooth muscle cell direct and indirect communication. These failures in early human trials came despite successful demonstrations of seeded grafts developing into a cell lining development. These data show that neo-intimal formation on polymeric vascular graft lumenal surfaces in animal models occurs by endothelial cell proliferation from perianastomotic arteries, the microvessels of graft interstices, or circulating progenitor endothelial cells not strictly from the seeded cells.

[0014] A potential source for endothelial cell seeding is microvascular endothelial cells (MVEC). Williams et al. pioneered both freshly isolated and cultured human, canine, rabbit, rat, bovine and pig endothelial cells, specifically MVEC, in their laboratory to study cellular function. The source for human MVEC was aspirated tissue from cosmetic liposuction. Two separate protocols for human fat MVEC isolation were used depending on the end use of the cell population. The protocols differed in isolation complexity from a simple, operating room-compatible procedure for immediate sodding of human or animal grafts to a more elaborate procedure if the MVEC will be subsequently cultured.

[0015] The isolation of human MVEC has been enhanced by the use of liposuction to obtain samples of human fat. The process of aspirating fat through a liposuction cannula dissociates subcutaneous fat into small pieces which boosts the efficacy of the digestion process. The fat may be digested with collagenase (4 mg/cc) for 20 minutes, at 37°C which releases >10⁶ cells per gram of fat. These MVEC can be separated from the fat by gradient centrifugation. The MVEC will form a pellet and can subsequently be resuspended in culture medium after discarding the supernatant. These cells have undergone routine characterization to determine the cellular makeup of the primary isolates. A majority of the cells isolated via this procedure are endothelial cells due to their expression of von Willebrand antigen, lack of expression of mesothelial cell specific cytokeratins, synthesis of angiotensin converting enzyme, prostacyclin and prostaglandin E2, synthesis of basement membrane collagens and the morphologic expression of micropinocytic vesicles. [0016] A human clinical trial was undertaken to evaluate endothelial cell transplantation in patients requiring peripheral bypass. During the trial, large quantities of endothelial cells were placed directly on the lumenal surface of an ePTFE graft. To improve cell deposition, all grafts were pre-wetted in culture medium containing autologous serum. Cells were suspended in the same medium at a density of 2 x 10^s cells/cm² graft lumenal area. This solution was held at a cross-wall, or transmural, pressure gradient of 5 psi to force cells onto the surface, a process termed pressure sodding. After institutional approval, 11 patients were enrolled and received the experimental graft. During surgical prep, the patients underwent liposuction to remove approximately 50 grams of abdominal wall fat. The fat was processed using the aforementioned procedure and the resulting cell population was pressure sodded on the intended graft and immediately implanted. After more than 4 years of follow-up, these grafts have maintained a patency rate similar to that of saphenous vein grafts.

[0017] Pressure gradients involving transient (<1 min.) relatively high pressures (250mmHg) have previously been used to deposit cells onto a permeable scaffold by a sieving action, i.e., providing a bulk flow and using a substrate or scaffold material having pores smaller than the cell population, thus capturing cells in the matrix (e.g., US Patent 5,628,781; Williams et al., 1992, J Biomed Mat Res 26:103-117; Williams et al., J Biomed Mat Res 28:203-212.) However, despite the aforementioned advances, clinical coronary applicability has been limited to date because the vessels do not maintain sufficiently cohesive non-thrombogenic surfaces; research has focused on additional maturation time in vitro.

[0018] Endothelial cells are of critical importance in establishing a non-thrombogenic cell lining. In addition, a need still exists for an efficient and reliable method for producing endothelial cell linings on a synthetic graft in an operating room setting, and the current invention provides a solution. It is desirable to achieve rapid cell adhesion in or on a permeable matrix, scaffold or other permeable cell substrate material in a matter of minutes or hours with an instrument that lends itself to the operating room environment, maintains a sterile barrier, is easy to use, produces consistent graft results, and is inexpensive. The present invention enables the isolation of large quantities of endothelial cells from fat tissue and the rapid cell sodding of synthetic grafts, and enables automation and adhesion of cells in a turn-key, operating room-ready instrument for the rapid sodding of the graft. This invention will likely have other applications in addition to the lining of grafts for implantation.

SUMMARY OF THE INVENTION

[0019] The present invention provides devices and methods and to achieve rapid cellular adhesion in or on a matrix, scaffold, or other cell substrate material during tissue engineering involving an automated method and apparatus to harvest a patient's own cells from a sample of their adipose tissue, and to wash, separate, and deposit these cells on a suitable graft for clinical use. [0020] The present invention is based, in part, on the inventors' discovery that a sustained low pressure gradient formed across a graft scaffold or substrate material, for as short a period as about 5 min up to about 24 hours, preferably in a cell perfusion system, provides significantly more rapid adhesion and subsequent maturation of cells on a substrate material than the application of previously known methods. Employing sustained low magnitude pressure for sodding cells onto permeable materials also provides greater control over cellular activation and differentiation pathways, a lack of control of which has impeded previous tissue engineering efforts. The present invention also provides an automated, sterile and safe method and devices to form cells on a suitable graft for clinical use in a short period of time, as well as methods and devices for collecting a sample of cells suitable for therapeutic use. 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.

[0021] The present invention also is directed to cell sodding biochambers, which provide for controlled sustained transmural flow, thereby creating a sustained differential pressure gradient across a porous material for preparing tissue implants and grafts via adherence, growth, and differentiation of the cells.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] FIG. 1 illustrates a schematic of one embodiment of the automated apparatus of the present invention. [0023] FIG. 2 illustrates a perspective view of one embodiment of the apparatus of the present invention.

[0024] FIG. 3 shows a vessel biochamber in accordance with the present invention and having a proximal stopcock for the introduction of cells, and a Y-connector allowing for the application of transmural pressure, followed by luminal flow. [0025] FIGS. 4A-4D show views of another biochamber in accordance with the present invention. This embodiment employs a tubular configuration. Ancillary equipment, such as a stopcock for introduction of cells, Y-connector for application of transmural and translumenal flow, and associated automated perfusion equipment, are not shown. Such equipment may be any suitable equipment, including, but not limited to, as shown in FIG. 1.

[0026] FIG. 5 provides a perspective view of one embodiment of the apparatus including a cell separation unit, the sodding unit and the graft chamber. [0027] FIG. 6 depicts the cell separation module durable and disposable components.

[0028] FIG. 7 provides a perspective view of the cell separation module with disposables loaded onto the cell separation durable.

[0029] FIG. 8 shows the graft sodding module durable and disposable components.

[0030] FIG. 9 shows the graft sodding module durables connected to the cell separation unit with disposable components loaded for use.

[0031] FIG. 10 shows the major components within the graft sodding durable and disposable components. [0032] FIG. 11 shows the cell collection module durable and disposable components.

[0033] FIG. 12 shows the cell collection module durables connected to the cell separation module with disposable components loaded for use. [0034] FIG. 13 shows the mounted barcode scanner.

[0035] FIG. 14 shows a cross-sectional view of one embodiment of the centrifuge bowl.

[0036] FIG. 15 illustrates the overall system flowpath.

DETAILED DESCRIPTION OF THE INVENTION [0037] The present invention provides devices and methods of preparing various tissue implants or grafts by applying pressure, preferably sustained low magnitude pressure, for adhering or "sodding" cells onto any suitable graft scaffolds or other permeable substrate materials. In a specific embodiment, the tissue is a tubular tissue, such as a vascular tissue. However, the invention is also applicable to any type of tissue grafts involving the adhesion of cells to scaffolds or other substrate materials, including, but not limited to, skin, cartilage, bone, bone marrow, tendon, ligament, gastrointestinal tract, genitourinary tracts, liver, pancreas, kidney, adrenal gland, mucosal epithelium, and nerve grafts. The method is particularly well suited to tubular tissues, including, but not limited to, those of the cardiovascular system and the urinary system.

[0038] The term "sustained low magnitude pressure" as used herein means pressure having a head of about 10 mmHg, about 15 mmHg, about 20 mmHg, about 25 mmHg and about 30 mmHg and about 55 mmHg, for about 5 min, about 20 min, about 30 min, about 40 min, about 50 min, 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 enhance the adhesion, growth and/or differentiation of the cells. One of ordinary skill in the art can select appropriate conditions for applying specific low magnitude sustained pressures according to the types of cells, tissue grafts, substrate materials, and given the teachings herein.

[0039] The cells to be adhered may include, for example, fibroblasts, smooth muscle cells, pericytes, macrophages, monocytes, plasma cells, mast cells, adipocytes, tissue-specific parenchymal cells, endothelial cells, urothelial cells, and various other cell types encountered in tissue engineering applications, including undifferentiated adult stem cells from various tissue sources. In a preferred embodiment, the adherent cells are endothelial cells, more preferably human microvascular endothelial cells obtained from autologous microvascular rich adipose tissue as referred to in U.S. Patent Nos. 4,820,626 (by Williams et al., issued April 11, 1989), 5,230,693 (by Williams et al., issued July 27, 1993), and 5,628,781 (by Williams et al., issued May 13, 1997), all of which are hereby incorporated by reference in their entireties. The adherent cells may be autologous, allogeneic, or xenogeneic, but preferably are autologous in origin. [0040] The graft substrate ("scaffold") materials used in the present invention may be any preferably permeable material of various sizes and geometries. The material may be natural or synthetic materials, including, but not limited to, polyethyleneterathalate, polyurethane, or expanded poly- tetrafluoroethylene (ePTFE). In another embodiment, the graft scaffold may be a biopolymer, such as collagen. The material may be preclotted and/or elastin, or allograft vessels, such as cryopreserved vein, decellularized vein or artery. In yet another embodiment, the scaffold may be a composite material such as an elastin scaffold with a polymeric coating, for example electrospun on the surface to improve mechanical properties. The material may be pre-clotted or pre-treated with a protein (e.g., albumin) or plasma, which in certain embodiments can serve to further enhance the adherence, spreading, and growth of tissue cells on the substrate material. The graft substrate or scaffolds may be constructed by any suitable method, including, but not limited to, those referred to in Liu, T.V. et al., 2004, Adv. Drug. Deliv. Rev. 56(11):1635-47; Nygren, P.A. et al., 2004, J.

Immunol. Methods 290(l-2):3-28; Hutmacher, D.W. et al., 2004, Trends Biotechnol. 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):103-19.

[0041] The term "transmural pressure or flow" as used herein refers to pressure or flow from one side to the other side of a graft scaffold, across the wall of the graft scaffold. Where the graft scaffold is a tubular graft scaffold, the term refers to pressure or flow from the lumen or intracapillary (IC) space of the graft to the outside or extracapillary (EC) space of the graft.

[0042] The term "translumenal pressure or flow" as used herein refers to pressure or flow through the lumen of a tubular graft. The terms

"translumenal flow" and "translumenal perfusion" may be used interchangeably. While translumenal perfusion is not required for cellular adhesion in the present invention, it may be used, for example, after the transmural flow to provide a training or cleansing effect. In this case, flow rates up to and including physiologic flow rates (~ 160 ml/min) are preferred, although flow rates as low as 5 ml/min typically are sufficient to provide cellular adhesion capable of withstanding subsequent physiologic flow. [0043] The term "proximal" as used herein refers to a point of reference on the side of media inflow in relation to the center of the biochamber vessel {see Figure 4).

[0044] The term "distal" as used herein refers to a point of reference on the side of media outflow in relation to the center of the biochamber vessel {see Figure 4).

[0045] The term "intracapillary (IC)" refers to the lumen or the internal space of a tubular graft scaffold and may be interchangeably referred to as "translumenal" or "intralumenal." [0046] The term "extracapillary (EC)" refers to the outside space of a tubular graft scaffold and may be interchangeably referred to as "extravascular" or "extralumenal."

[0047] Reference is now made to FIG. 1. FIG. 1 illustrates one embodiment of a system 120 that includes an optionally disposable bag 100 that can be loaded into a suitable hard apparatus for use in or near an operating room or other sterile environment for the harvesting of cells to be deposited onto a suitable graft. The disposable bag 100 includes a tissue dissociating chemical reservoir 20, a medium reservoir 10 and a waste reservoir 30. In use, the reservoir 20 and medium reservoir may be pre-loaded. Preferably, the tissue dissociating chemicalreservoir 20 includes a collagenase.

[0048] The system 120 further includes a macerator and heating apparatus 50, a pump 60, a buoyancy separator 40 and a biochamber 70. The pump 60 may be any suitable pump or combination or pumps, including, but not limited to, gear pumps, peristaltic pumps, diaphragm pumps, centrifugal pumps, and passive pressure heads created by a column of fluid. In one embodiment, the biochamber 70 is a vessel biochamber, such as discussed below and in copending U.S. Patent Application Serial No. 11/314,281 filed December 22, 2005, which is herein incorporated by reference.

[0049] The preferred system 120 allows for the separation of cells from the patient's tissue and deposition of a desired fraction of those cells onto a graft material. This may be accomplished within an operating room setting in an automated fashion within a clinically feasible timeframe. A clinically feasible timeframe is generally considered to be from about 30 minutes to about 24 hours, depending upon a variety of factors, such as, for example, the types of cells, the amount of starting material, the amount of grafted cells needed, the time required for a maturation of the cell layer into a tissue, and the like. These factors are readily understood by a person having ordinary skill in the relevant art.

[0050] The preferred system provides sustained pressure sodding and automation of the clinical procedures of separating a desired fraction of the patient's cells from tissue and one or more of filtering, washing, heating, macerating, proteolytically releasing, separating, resuspending, and pressure sodding the cells onto a permeable graft.

[0051] More specifically, still with reference to the illustrative embodiment of FIG. 1, the tissue dissociating chemical reservoir 20 and the medium reservoir 10 are preferably pre-loaded. The cells to be grafted are input into the system 120 through inlet 90. The cells are preferably from a patient's adipose tissue, which have been collected via relatively non-invasive means such as, for example, the Tulip™ system. The cells are first washed and filtered in filter 18 to remove small particles from the cells like red blood cells. The small particles are filtered to waste reservoir 30 through valve 14. The remaining parts of the cells are mixed with the medium from the medium reservoir 10 by closing valves 34 and 14 and allowing the medium to flow from medium reservoir 10 through conduit 76 and 38. The medium is mixed with the cells by being pumped by pump 60 through conduit 28 and 24 and through open valves 12 and 16 and being mixed with protease in the tissue dissociating chemical reservoir 20. By this operation, the washed cells, medium and protease are mixed. [0052] The mixed product is macerated and heated in macerator and heater 50 by flowing through conduit 26. Maceration of the tissue may be accomplished by any means that increases the surface area of the tissue, allowing higher protease activity on the tissue with less cellular damage. The macerator and heater 50 may be combined or separate and may include any suitable devices, such as, for example, centrifugal impellors, blenders, spiral mixers, or the like. [0053] The tissue slurry in the macerator is preferably maintained at a temperature of from about 35 to about 40 degrees centigrade, preferably about 37 degrees centigrade, so as to maintain high cell viability and to allow maximum activity of the protease. Heating can be accomplished in a number of ways, including, for example, resistive heating elements, peltier blocks, and others, and can be transferred to the tissue slurry through any suitable conductive media, including a water bath, and through any suitable heat-conductive reservoir materials.

[0054] Preferably while the slurry is being macerated, or at any other desired or appropriate time, the user may input the patient's serum to the system. The serum is input into the illustrative system 120 through input 80. The serum flows through conduit 78 into the medium reservoir 10.

[0055] The macerated slurry is then passed through conduit 42 to the cell separator 40. The separator 40 can be, for example, a buoyancy separator, centrifuge or any other suitable apparatus. The slurry may be maintained in the separator 40 by closing valves 36 and 52. While the slurry is being separated, the serum and medium in medium reservoir 10 flows through a flow loop. Valves 12, 14, 16, 32, 36, 52, 62 and 68 are shut and valves 34, 46, 56, 66 are opened to create the flow loop. The pump 60 pumps the serum/medium mixture through the flow loop, which includes conduits 76, 38,

44, 54, 58 and 72. The ratio of the serum: medium mixture in the flow loop is preferably about 1:6. The serum/medium mixture preferably flows through the flow loop to provide translumenal flow through the vessel while the slurry is separated in the separator 40. After a predetermined time, valve 56 is closed and valve 62 is opened to provide transmural flow to the biochamber 70 to put a coating of the patient's serum proteins on the scaffold material to improve cell adhesion. The separation may take from about 2 minutes to about 30 minutes, and preferably from about 5 to about 10 minutes. The predetermined time for translumenally flow is preferably from about 1 minute to about 10 minutes, more preferably about 3 to 5 minutes.

[0056] The cells are then separated in the separator 40 by fraction.

Cells having the desired fraction are processed through filter 48. The proteases associated with these cells have been inactivated by the addition of media/serum to the cells. Once the cells have been separated from the slurry, valve 52 is opened to allow the desired fraction to proceed to the biochamber 70 for grafting the cells on the surface of the graft material, which typically has pore sizes that are smaller than the size of the cells. The flow may initially be sent to the waste reservoir 30 through a waste line (not shown) or through conduit 74 for a short period of time to allow the growth factors to be removed. This may be done for about 2 minutes to about 30 minutes. Thereafter, the flow is re-routed into the continuous flow loop that includes the biochamber 70, the medium reservoir 10 and the pump 60 through conduits 54, 58, 72, 76, 38, and 44. During flow through this flow loop, valves 12, 14, 16, 32, 36, 52, 56, and 68 are closed and valves 46, 66, 62, and 34 are opened. The material is circulated through the flow loop, preferably to provide sustained low magnitude pressure sodding, until the cells have been grafted onto the graft material. This may be from about 5 minutes to about 24 hours, depending on conditions. The system 120 may include a visual indicator such as a light or an aural indicator or alarm to indicate that the cells have been grafted onto the grafting material.

[0057] A sustained low-pressure gradient of at least 10 mmHg and not more than 100 mmHg may be used over a period from 5 minutes to 24 hours to deposit the cells upon the surface or in the graft material, depending upon the nominal pore size of the graft. The pressure gradient can be accomplished with any combination of positive and/or negative pressures such that the net gradient causes flow through the graft material.

[0058] The separation and sodding media may be a commercially available media including DMEM, F 12, AlphaMEM, University of Wisconsin

Solution, etc., or any combination thereof, without or without additional factors, which may include heparin or other factors that accommodate the desired cell type.

[0059] As set forth above, the biochamber 70 holds the graft material and can allow for flow of the media through a permeable graft. The preferred biochamber 70 has a positive seal such as a double o-ring seal and is easy to load and separable from the rest of the disposable container 100 for use in the operating room. The preferred biochamber 70 is also easy to unload for delivery of the graft to the operating room and requires no tools to load or unload. It may be a trough in a tube design that has no net force acting on the biochamber 70 by virtue of canceling all pressures from fluid flow in the system. Such a design allows for easy placement of, for example, a tubular graft.

[0060] In the case of a tubular graft, cells are deposited upon the lumenal surface of the graft and the biochamber holds the graft to allow for uniform cell deposition by virtue of uniform permeability along the long axis of the graft. Such a tubular graft may also by preloaded along its long axis to change the permeability of the graft, including opening up the pores of the graft material. In the case of a substantially planar graft, cells are deposited on one surface of the graft.

[0061] Reference is now made to FIG. 2, which illustrates a view of the system 120 showing the removable biochamber 70 and the system casing 110. The system casing 110 has a cell separator access means 112 for accessing the separator 40. The system casing 110 also has an access means 114 for inserting and removing the disposable bag 100.

[0062] The illustrative systems of FIGS. 1 and 2 will typically include a microprocessor and associated software to control the system and automate one or more steps based on user input. The software may allow full or partial automation of, for example, controlling flow through tubular conduits by controlling pumps and valves, controlling temperature, and controlling cell separator and macerator devices. Preferably the system is fully automated, but capable of being reconfigured based on one or more input parameters. The systems may further include various sensors to detect or measure system parameters, such as pressures that would indicate a blockage, and signal same to the microprocessor or user. In one embodiment, the system is a hand-held system.

[0063] While the automated methods of the invention may be carried out in any suitable apparatus, the inventors have found that particular biochamber designs are especially well-suited to achieve optimal results in terms of consistent and uniform cell adherence and user convenience. [0064] A vessel biochamber in accordance with one embodiment of the present invention is shown in Figure 3. The biochamber includes two halves comprising a top vessel 201 and a bottom vessel 202, each having a proximal end and a distal end. The vessels are mutually disposed to define an interior space 203 by virtue of a double O-ring seal 216. Intracapillary (IC) double barb connectors 204 are positioned within the space defined by the vessels. The connectors 204 are adapted to hold a graft scaffold 205 at each of the proximal and distal ends of the biochamber. A proximal tubing 206 connects to the IC connector to provide an IC flow in relation to the graft scaffold 205. Distal tubing 207 connects the distal IC and distal EC flow spaces via a Y- connector 208. The IC tubing 206 and 213 are sealed upon entry to the biochamber by virtue of a pair of O-rings 214 on each side. The biochamber may include an additional extravascular port 215.

[0065] The permeable scaffold material may be mounted via the connectors 204 to the IC proximal 206 and distal tubing 213. In a specific embodiment, the biochamber includes a stopcock 209 attached to the proximal tubing 206 via a T-connector 210, to allow for injection of cells into the biochamber. In yet another specific embodiment, the biochamber further includes at least one clamp or valve 211 that can close either the distal EC tubing 212 or the distal IC tubing 213 to create, or shift between, transmural or translumenal pressure gradients, as explained below. In a preferred embodiment, each of the distal IC tubing 213 and the distal EC tubing 212 has its own valve or slide clamp.

[0066] Preferably, the top and the bottom vessels of the biochamber are made of optically clear materials (e.g., polystyrene or polycarbonate) so that intra- and extra-luminal flow can be visually monitored. The biochamber is preferably made from materials which are autoclavable, gamma, or gas-sterilizable. Furthermore, the biochamber may contain a multiple silicone O-ring system, providing double seal contact for vessel attachment, so that the vessel length and angular position may be adjusted after a specimen is mounted between the two barbs within the biochamber. In addition, metal thread inserts may be used to eliminate the need for threading manufactured components, and also eliminate the potential failure of plastic threads.

[0067] The configuration of the distal tubing 207, 212, 213, which couples the distal IC and distal EC flow spaces, allows the user to switch between a transmural pressure gradient and a translumenal pressure gradient using the slide clamps 211, or in automated fashion within a bioreactor. This switch would typically take place to provide translumenal pressure after cell adhesion. In a specific embodiment, cells are introduced via stopcock 209 or a septum connected via a T-connector 210 to the proximal tubing 206. The distal IC slide clamp 211 is then closed to allow only outflow from the EC space, thereby establishing a transmural pressure gradient from the proximal IC to distal EC space, and a small flux of media through the permeable scaffold while depositing adhering cells on the lumenal surface and/or within the wall of the graft. The pressure gradient may be established either by generation of a positive pressure at the proximal IC side, a negative pressure at the distal EC side, or a combination of positive pressure at the proximal IC and negative pressure at the distal EC spaces. If desired, after cell adhesion to the lumenal surface, the distal EC slide clamp may be closed and the distal IC slide clamp opened to allow flow through the lumen of the vessel to ensure cellular adhesion in the presence of a shear stress, which simulates a physiological environment.

[0068] The controlled, sustained differential pressure gradient across the permeable scaffold material may be created by any suitable configuration, including, but not limited to, gear pumps, peristaltic pumps, diaphragm pumps, centrifugal pumps,ι and passive pressure heads created by a column of fluid, so long as the pressure is sufficiently sustained and at a magnitude sufficient to achieve the advantages of the invention. In a particularly preferred embodiment, the pressure is applied transmurally to a vascular graft scaffold using media containing endothelial cells at a pressure head of about 50 mmHg and for a duration of about 5 minutes. [0069] Another embodiment of a preferred biochamber apparatus for cell sodding is the tubular biochamber shown in FIGs. 4A-4D. A barb-to- barb connector (not shown) is fitted into the interior ends of two pieces of tubing 303, preferably hard silicone tubing, that rests in a "trough" 302 of an inner sleeve 301. A vascular graft is attached to both barbs, thereby defining the interior intra-vascular flow (IVF) space or lumenal flow space. This space lies within the trough 302 created by the inner sleeve 301 and is sealed from the exterior by virtue of an interference fit between the hard silicone tubing 303 and the inner sleeve ID. IVF flow can then be initiated by connecting the two pieces of silicone tubing 303 to any flow system through the intra-lumenal ports 305. The biochamber is then sealed by sliding the inner sleeve 303 into the outer sleeve such that both pairs of O-rings 307 are within the outer sleeve. Extra- vascular flow (EVF) is accomplished by a pair of threaded barbs (not shown) attached to the extra-lumenal ports 308. The EVF is sealed from the exterior by virtue of the double O-ring 307 at each end of the inner sleeve. Transmural flow is accomplished by flowing into the lumenal space proximally by way of the silicone tubing and clamping the lumenal space distally while opening the EVF to the pump return via tubing connections to either or both of the extra-lumenal ports 308. Not shown are threaded barb connectors on the outer sleeve at the extra-lumenal port, and barbs and a graft attached to the two hard silicone tubes in the interior trough of the inner sleeve.

[0070] The preferred biochambers of the present invention have: 1) two independent flow spaces for lumenal and extravascular flow, 2) silicone

O-ring clamps to provide double seal contact, 3) minimal number of threaded fasteners, minimizing handling time and production cost, 4) optically clear materials for visualization of specimen and intra- and extra-lumenal flows, 5) manufacturable from autoclavable and gas sterilizable materials, 6) embedded multiple silicone O-ring system for vessel attachment to allow vessel length and angular position to be adjusted after specimen mounting, and 7) metal thread inserts to eliminate the need for threading manufactured components, and eliminate failure of plastic threads. In addition, an automated rocking bed or other rotating apparatus may be provided to facilitate even distribution of the cells within the lumen of the vessel during pressure sodding. The rocking bed may fit within and be powered by, an automated perfusion system, and the biochamber may fit within the rocking bed. However, a rocking bed is not required for complete 360 degree coverage of the graft.

[0071] The tubular configuration of FIG. 4 has all of the benefits listed above, and in addition, includes the following benefits: tool-less open/close, minimal volume, convenient length and rotational adjustment of graft, lengthwise scale-up, and pressures balanced such that leakage does not occur by virtue of the O-ring sealing mechanism.

[0072] The methods and biochamber apparatus of the present invention may be employed in combination with various media perfusion systems. The advantages of the invention may be optimized for certain tissue engineering applications by use of an automated cell culture apparatus, preferably as described in U.S. Patent Application Ser. Nos. 09/967,995 and 10/109,712.

[0073] The above-referenced patent applications provide automated perfusion culture platforms to provide controlled media flow, shear stress, nutrient delivery, waste removal, and improved mass transfer. These address many of the shortcomings of traditional culture systems by providing a sterile barrier to contamination while maintaining more uniform and controlled physiologic environments for cells and tissues, providing the user with sample access and data, and providing affordable reproducibility and reliability with data tracking and logging.

[0074] Such an automated perfusion system may include a durable cartridge containing a pump, valve array, flow meter, and user interface. It preferably has an embedded microcontroller and pre-programmed flow regimes with programmable flow states. Multiple perfusion loop cartridges may be housed within a single docking station rack, designed to be housed within a laboratory incubator. The disposable perfusion flowpath integrates with the cartridge and may have integrated media reservoirs, tubing for gas exchange, and a valve matrix controlling media flow. A biochamber, in accordance with the present invention, may be mated with the flowpath. Periodic flow reversal can be employed to decrease differences in media composition from inlet to outlet. An automated sampling system also may be provided to allow the user to obtain a sample of media for analysis. Flow rates may vary from, for example, 1 up to 120 ml/min or more; flow may be monitored by an optical drop meter.

[0075] Because at least a portion of the flow for the current invention is typically transmural, the flow rate is dependent upon the permeability of the graft material, and decreases as the cells are applied to the lumenal surface. Transmural flow rates before the introduction of cells can be from 5-50 ml/min depending on the graft material and generally decrease to 1-10 ml/min after the introduction of cells. Preferred endothelial cell numbers include 120,000-2,000,000 cells/cm² of luminal surface area, more preferably about 250,000 cells/cm².

. [0076] In another particular embodiment of the present invention, the device system is modular, such that the tissue digestion and separation portion of the device can be used with interchangeable modules to either apply cells to a vascular graft or collect cells in a syringe. Fig. 5 shows the device assembled in a modular system. Because the cell separation portion of the device is housed in a distinct, separate unit, this embodiment also provides flexibility for pairing the cell separation unit other with other systems. Preferably, the device is divided into the three distinct modules: a cell separation module, a graft sodding module, and a cell collection module. Cell Separation Module

[0077] In one embodiment, the cell separation module is a standalone piece of equipment that contains all necessary electronics and components to cut, heat, digest, and separate adipose tissue. In a preferred embodiment, the cell separation component comprises a centrifuge. An outlet from the cell separation module supplies a single cell suspension of isolated cells, to be connected to either the graft sodding or cell collection modules. Fig. 6 shows the cell separation module durable and disposable components. Fig. 7 shows the cell separation module with disposables loaded onto the cell separation durable. [0078] In one embodiment, the cell separation module durable unit houses all of the electronics necessary for operation of the device, including the computer boards, software, power supply, and an user interface. In a preferred embodiment, the user interface includes an LCD screen with buttons that guides the user through the set-up and operation of the device. The cell separation module durable can also house the necessary pinch valves, motors, sensors and other durables required for cutting, heating, digesting, and centrifuging the subject tissue. In a preferred embodiment, the subject tissue is adipose tissue. Pinch valves protrude from the enclosure on a top flat surface to allow valves to engage the disposable fluid pathway. In a preferred embodiment, electronics are located a maximum distance from any fluid pathways.

[0079] In another embodiment, the device includes a mountable hook to hang media and waste bags. Preferably, the bag hook is mounted to either the graft sodding durable or the cell collection durable to maximize the distance between the media bags and electronics housed in the cell separation durable. This separation reduces risk of electronics damage from fluid spills.

[0080] In a particular embodiment of the invention, all elements of the cell separation module flowpath are disposable. In one embodiment, these disposable components can be assembled on a rigid tray that loads onto the cell separation module durable. The user loads the disposable tray by placing the tray onto the flat surface of the durable by aligning the pinch valves with the valve cutouts in the disposable tray. The user then slides the tray forward to engage tubing loops in the pinch valves and lock the disposable tray in place. All disposable components are located in the tray to align with and engage the necessary durable components in the cell separation durable by this loading operation. The tray design minimizes the user's burden for set-up and disposal by eliminating the need for many tubing connections and individual loading of many disposable components. After loading the tray, the user can load the disposable centrifuge bowl into a recess provided in the durable component and attach inlet and outlet tubing from the disposable tray to the centrifuge, media bag, waste bag, and sodding or collection unit. Graft Sodding Module

[0081] The graft sodding module refers to the durable and disposable components that are necessary to apply the cells provided by the cell separation unit onto a porous graft scaffold using a pressure sodding technique. The graft sodding module durable and disposable components are shown in Fig. 8. Fig. 9 further shows the graft sodding module durables connected to the cell separation unit with disposable components loaded for use.

[0082] In an embodiment of the invention, the sodding module contains two durable components: the sodding unit durable and the graft chamber durable. These durable components physically mate with the cell separation durable to provide a power and communication connection. In another embodiment of the invention, the sodding module durables are controlled by the electronics in the cell separation module durable. The graft chamber durable provides secure mounting for the disposable graft biochamber and houses components necessary for heating of the chamber. The sodding durable contains the hardware (e.g., pinch valves, sensors) that is specifically required to manipulate flow through the graft chamber as needed for the pressure sodding application. In one embodiment, the sodding durable has a top flat surface with protruding durable equipment where the sodding disposable can be loaded. Fig. 10 shows the major components within the graft sodding durable and disposable components.

[0083] In a further embodiment, sodding disposable components include the disposable graft biochamber and a sodding disposable tray. The scaffold or other substrate material is typically preloaded in the disposable graft chamber, which provides a sealed environment for delivery of liquids to the graft while prohibiting all other gaseous, liquid, and solid matter exchange with surroundings. In one embodiment, three ports on the graft chamber connect with tubing from the sodding disposable tray to provide inlet, transmural outlet, and lumenal outlet from the graft chamber. In an embodiment, the graft chamber rests inside the chamber durable which has a closing door to enclose the chamber during the sodding operation.

[0084] In an embodiment, the sodding disposable rigid tray includes all disposable components and connecting materials required for the sodding operation. The tray loads onto the flat surface of the sodding durable by aligning the pinch valves with the valve cutouts in the disposable tray and sliding forward to engage tubing in the pinch valves. The user connects the cell separation disposable, sodding disposable, and graft chamber disposable to form the complete flowpath for sodding. Collection Module

[0085] The collection module refers to the durable and disposable components that are necessary to collect cells from the cell separation unit in a syringe for use in cell therapies. The cell collection module durable and disposable components are shown in Fig. 11. Fig. 12 shows the cell collection module durables connected to the cell separation module with disposable components loaded for use. In an embodiment, the collection module durable physically mates with the cell separation unit to provide a power and communication connection. The collection durable houses a linear actuator that interfaces with a syringe to automatically collect the cell product produced in the cell separation unit.

[0086] In another embodiment, the disposable component in the collection unit is the syringe to collect the cell product. The syringe is held in place by a clip on the collection unit durable. The top of the syringe is loaded into the durable such that the syringe plunger can be drawn by the motion of the actuator. The user connects the outlet tube from the cell separation module to the tip of the syringe. System Operation

[0087] In one embodiment, the user installs the durable components required for the current application (i.e. graft sodding durables or cell collection durables) before switching on the device. When the device is switched on, it boots, detects that the durable modules are engaged properly, performs initial diagnostics, and goes into a standby mode. The user then presses a button near the display to initialize device set-up. The OR Kit enters a mode to allow installation of the disposables. The user is prompted to scan each disposable component using a bar code scanner mounted on the cell separation durable. When the user scans the disposable, the OR Kit will verify that the correct durables are in place, then guide the user through each step to load the disposable and make necessary tubing connections. The device will sense that the disposable components are properly loaded and ensure that all required disposables are installed for the current application. In a preferred embodiment, the barcode scanner is located on device such that scanning of the disposables does not interfere with loading of the disposables. The mounted barcode scanner is shown in Fig. 13. [0088] After completing device set-up, the user presses an "OK" button to proceed. The device performs an air purge operation in which media and serum are pumped through the flow paths, pushing air to a waste collection point which has a vent port that allows air to escape to the atmosphere. The graft chamber is bypassed so that the graft is never exposed to air. [0089] The user is then prompted to inject adipose into a port on the centrifuge disposable. The adipose tissue is macerated as it enters the centrifuge by passing through stationary blades. The user presses "OK" to proceed. The user is then prompted to introduce the protease solution through the same inlet. In a preferred embodiment, the protease solution is a collagenase/PBS solution. The user presses "OK" to proceed. The user interface display indicates that the cell separation process is initiated.

[0090] In a preferred embodiment, from this point on, no user interaction is required until the entire OR Kit process is complete. The user interface display provides continuous updates on the process, indicating the specific operation being performed, the estimated time to complete the operation, and the estimated time to complete the entire process. In one embodiment, other important process parameters (temperatures, pump speed, etc.) can also be made available to the user via the display.

[0091] In an embodiment, the graft scaffold is packed in alcohol or other appropriate sterile substance within the disposable graft chamber. Graft preparation is concurrent with the cell separation steps provided below. The following steps are involved in preparing the graft for sodding. (1) Alcohol Purge ~ alcohol is purged from the graft chamber by flowing media through the graft chamber and directing the liquid outlet to waste; (2) Scaffold pretreatment - media is recirculated through the graft chamber until the cell suspension is available for graft sodding. The media can include, without limitation, Ml 99, M199E, PBS, Saline, or Di-Cation Free DPBS. In a preferred embodiment, the media is a 6:1 mixture of M199E and serum from the patient.

[0092] The cell separation process is identical for sodding and cell collection operation modes. In one embodiment, the cell separation steps include: (1) adipose tissue digestion - the centrifuge is temperature controlled at about 37⁰C and provides a low speed mixing action (mixing is maintained for an appropriate amount of time to ensure adequate digestion); (2) centrifugation - the centrifuge spins at high RPM, separating the adipose tissue into its constituent materials; and (3) endothelial cell isolation and resuspension ~ the separated contents are pumped into a thin, transparent tube where an optical sensor detects the location and volume of the endothelial cells. Unwanted materials are directed to a waste reservoir, and a specific volume of endothelial cells is returned to the centrifuge. A 6:1 mixture of M199E and serum is pumped into the centrifuge. The centrifuge suspends the separated cells in the mixture by a low speed mixing action. The cell suspension is then pumped from the centrifuge through a 30- micron filter and directed to the graft sodding unit or the cell collection unit for collection into a syringe. Fig. 14 shows a cross-sectional view of one embodiment of the centrifuge bowl.

[0093] In the process of graft sodding, liquid passes between the separation module and graft module via the sodding module. Preferably, the graft is temperature controlled to about 37°C. In an embodiment, the graft sodding steps include cell sodding and "feed and bleed" flow. In the cell sodding step, the endothelial suspension is introduced into the recirculating flowpath, allowing the cell suspension to flow into the graft at one end and out through the graft walls. Initially, the liquid mixture that leaves the graft chamber is directed to waste until the entire volume of cell suspension has entered the recirculating path. The cell suspension then recirculates until graft sodding is complete. The microporous ePTFE permits the passage of the media/serum mixture, but the cells are embedded into the ePTFE. During this process, transmural pressure is monitored by a pressure sensor in the sodding module. In the "feed and bleed" flow step, graft flow is switched to luminal when a specific transmural pressure is reached, indicating complete sodding. During this process, flow is alternately directed to waste and the pump(s) for recirculation. During periods when the flow is directed to waste, makeup media and serum are pumped from the reservoirs. The "feed and bleed" process is maintained for an appropriate amount of time. [0094] In one embodiment of the cell collection process of the present invention, the cell suspension is pumped from the separation module to a syringe in the collection module. A linear actuator pulls the syringe plunger, drawing cell suspension into the syringe.

[0095] When the operation is complete, visual and/or auditory indications are provided to the user. The user is prompted to remove the product

(the sodded graft or full syringe). The user selects "OK" on the user interface when the product has been removed. The device then enters a mode for the removal of the disposables. In this mode, the disposables can be removed from the unit for disposal. The user selects "OK", and the device enters standby mode awaiting either another process run or a system shutdown. Fig. 15 illustrates the system flowpath.

[0096] The inventors have demonstrated that a sustained pressure head, applied to a liquid medium with suspended cells across a permeable scaffold material, offers the advantage of rapid cell adhesion, without large pressure gradients as used in transient pressure sodding techniques. One skilled in the art could readily practice the invention with a myriad of cell types, scaffold materials and geometries with any number of device designs. Those skilled in the art will recognize, or be able to ascertain, many equivalents to the specific embodiments of the invention described herein using no more than routine experimentation. Such equivalents are intended to be encompassed by the claims. Therapeutic Uses

[0097] The tissue grafts and cell suspensions prepared by the above-described devices can be employed in a myriad of therapeutic uses. For example, in one embodiment of the invention methods are provided for revascularizing a tissue or organ of a subject in need thereof, by implanting into the tissue or organ at least one tissue graft or cell suspension that is prepared by any of the above-described devices.

[0098] In an embodiment, the tissue graft or cell suspension comprises cells selected from the group consisting of skin, skeletal muscle, cardiac muscle, atrial appendage of the heart, lung, mesentery, or adipose tissue.

The adipose tissue may be from omental fat, preperitoneal fat, perirenal fat, pericardial fat, subcutaneous fat, breast fat, or epididymal fat.

[0099] In certain embodiments, the tissue graft or cell suspension further comprises appropriate stromal cells, stem cells, Relevant Cells, or combinations thereof. As used herein, the term "stem cells" is used in a broad sense and includes traditional stem cells, progenitor cells, preprogenitor cells, reserve cells, and the like. Exemplary stem cells include embryonic stem cells, adult stem cells, pluripotent stem cells, neural stem cells, liver stem cells, muscle stem cells, muscle precursor stem cells, endothelial progenitor cells, bone marrow stem cells, chondrogenic stem cells, lymphoid stem cells, mesenchymal stem cells, hematopoietic stem cells, central nervous system stem cells, peripheral nervous system stem cells, and the like. Descriptions of stem cells, including method for isolating and culturing them, may be found in, among other places, Embryonic Stem Cells, Methods and Protocols, Turksen, ed., Humana Press, 2002; Weisman et al., Annu. Rev. Cell. Dev. Biol. 17:387 403; Pittinger et al., Science, 284:143 47, 1999; Animal Cell Culture, Masters, ed., Oxford University Press, 2000; Jackson et al., PNAS 96 (Shepherd BR et al. Rapid perfusion and network remodeling in a microvascular construct after implantation. Arterioscler Thromh Vase Biol 24: 898-904, 2004):14482 86, 1999; Zuk et al., Tissue Engineering, 7:211 228, 2001 ("Zuk et al."); Atala et al., particularly Chapters 33 41; and U.S. Pat. Nos. 5,559,022, 5,672,346 and 5,827,735. Descriptions of stromal cells, including methods for isolating them, may be found in, among other places, Prockop, Science, 276:71 74, 1997; Theise et al., Hepatology, 31:235 40, 2000; Current Protocols in Cell Biology, Bonifacino et al., eds., John Wiley & Sons, 2000 (including updates through

March, 2002); and U.S. Pat. No. 4,963,489. The skilled artisan will understand that the stem cells and/or stromal cells selected for inclusion in a tissue graft or cell suspension are typically appropriate for the intended use of that construct.

[00100] In a particular embodiment, the tissue graft or cell suspension comprises endothelial cells which are capable of differentiating into, without limitation, a neuron, myocardiocyte, chondrocyte, pancreatic ancinar cell, pancreatic endocrine cells including islet of Langerhans, hepatocyte, renal epithelial cell, parathyroid cell, Leydig cell, Sertoli cell, gonocyte, oocyte, blastocyst, Kupffer cell, lymphocyte, fibroblast, myocyte, myoblast, satellite cell, adipocyte, preadipocyte, osteocyte, osteoblast, osteoclast, chondrocyte, biliary epithelial cell, Purkinje cell, and pacemaker cell.

[00101] In another particular embodiment, the tissue graft or cell suspension comprises at least one stem cell, progenitor cell or Relevant Cell, which may be without limitation a neuron, myocardiocyte, chondrocyte, pancreatic ancinar cell, pancreatic endocrine cells including islet of Langerhans, hepatocyte, renal epithelial cell, parathyroid cell, Leydig cell, Sertoli cell, gonocyte, oocyte, blastocyst, Kupffer cell, lymphocyte, fibroblast, myocyte, myoblast, satellite cell, adipocyte, preadipocyte, osteocyte, osteoblast, osteoclast, chondrocyte, biliary epithelial cell, Purkinje cell, and pacemaker cell.

[00102] The term "Relevant Cell(s)" as used herein refers to cells that are appropriate for incorporation into a tissue graft or cell suspension prepared by the devices of the present invention, based on the intended use of that tissue graft or cell suspension. By way of example, Relevant Cells that are appropriate for the repair, restructuring, or repopulation of damaged liver may include, without limitation, hepatocytes, biliary epithelial cells, Kupffer cells, fibroblasts, and the like. Exemplary Relevant Cells for incorporation into tissue graft or cell suspensions include neurons, myocardiocytes, myocytes, chondrocytes, pancreatic acinar cells, islets of Langerhans, osteocytes, hepatocytes, Kupffer cells, fibroblasts, myocytes, myoblasts, satellite cells, endothelial cells, adipocytes, preadipocytes, biliary epithelial cells, and the like. These types of cells may be isolated and cultured by conventional techniques known in the art. Exemplary techniques can be found in, among other places, Atala et al., particularly Chapters 9 32; Freshney, Culture of Animal Cells A Manual of Basic Techniques, 4th ed., Wiley Liss, John Wiley & Sons, 2000; Basic Cell Culture: A Practical Approach, Davis, ed., Oxford University Press, 2002; Animal Cell Culture: A Practical Approach, Masters, ed., 2000; and U.S. Pat. Nos. 5,516,681 and 5,559,022. [00103] The skilled artisan will appreciate that such stromal cells, stem cells, and/or Relevant Cells may be incorporated into the tissue graft or cell suspension during or after preparation. For example, but not limited to, combining the cell suspension, stem cells, Relevant Cells, and/or stromal cells in a liquid three-dimensional culture, such as collagen, fibrin, or the like, or seeding or sodding stem cells, Relevant Cells, and/or stromal cells in or on the tissue graft may be achieved. Exemplary combinations of appropriate stem cells, stromal cells, and Relevant Cells for incorporation into tissue grafts or cell suspensions include: islets of Langerhans and/or pancreatic acinar cells in a tissue graft or cell suspension for revascularizing a damaged pancreas; hepatocytes, hepatic progenitor cells, Kupffer cells, endothelial cells, endodermal stem cells, liver fibroblasts, and/or liver reserve cells in a tissue graft or cell suspension for revascularizing a damaged liver. For example, but not limited to, appropriate stem cells or stromal cells for a tissue graft or cell suspension for vascularizing, repairing, and reconstructing a damaged or disease liver might comprise liver reserve cells, liver progenitor cells, such as, but not limited to, liver fibroblasts, embryonic stem cells, liver stem cells, cardiomyocytes, Purkinje cells, pacemaker cells, myoblasts, mesenchymal stem cells, satellite cells, and/or bone marrow stem cells for revascularizing a damaged or ischemic heart (see, e.g., Atkins et al., J. of Heart and Lung Transplantation, December 1999, at pages 1173 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), and the like.

[00104] In one embodiment, the tissue graft or cell suspension further comprises an agent selected from the group consisting of cytokines, chemokines, antibiotics, drugs, analgesic agents, anti-inflammatory agents, immunosuppressive agents, or combinations thereof. [00105] Exemplary cytokines may include, without limitation, angiogenin, vascular endothelial growth factor (VEGF, including, but not limited to VEGF- 165), interleukins, fibroblast growth factors, for example, but not limited to, FGF-I and FGF-2, hepatocyte growth factor, (HGF), transforming growth factor beta (TGF-.beta.), endothelins (such as ET-I, ET-2, and ET-3), insulin-like growth factor (IGF-I), angiopoietins (such as Ang-1, Ang-2, Ang- 3/4), angiopoietin-like proteins (such as ANGPTLl, ANGPTL-2, ANGPTL-3, and ANGPTL-4), platelet-derived growth factor (PDGF), including, but not limited to, PDGF-AA, PDGF-BB and PDGF-AB, epidermal growth factor (EGF), endothelial cell growth factor (ECGF), including ECGS, platelet-derived endothelial cell growth factor (PD-ECGF), placenta growth factor (PLGF), and the like. Cytokines, including recombinant cytokines, and chemokines are typically commercially available from numerous sources, for example, R & D Systems (Minneapolis, Minn.); Endogen (Woburn, Wash.); and Sigma (St. Louis, Mo.). The skilled artisan will understand that the choice of chemokines and cytokines for incorporation into particular tissue graft or cell suspensions will depend, in part, on the target tissue or organ to be vascularized, revascularized, augmented or reconstructed.

[00106] In certain embodiments, tissue graft or cell suspensions further comprise at least one genetically engineered cell. In certain embodiments, tissue graft or cell suspensions comprising at least one genetically engineered cell will constitutively express or inducibly express at least one gene product encoded by at least one genetically engineered cell due to the genetic alterations within at least one genetically engineered cell induced by techniques known in the art. Descriptions of exemplary genetic engineering techniques can be found in, among other places, Ausubel et al., Current Protocols in Molecular Biology (including supplements through March 2002), John Wiley & Sons, New York, N. Y., 1989; Sambrook et al., Molecular Cloning: A Laboratory Manual, 2.sup.nd Ed., Cold Spring 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 Acid Chemistry, John Wiley & Sons, New York, N.Y., 2000 (including supplements through March 2002); Short Protocols in Molecular Biology, 4.sup.th Ed., Ausbel, Brent, and Moore, eds., John Wiley & Sons, New York, N. Y., 1999; Davis et al., Basic Methods in Molecular Biology, McGraw Hill Professional Publishing, 1995; Molecular Biology Protocols (see the highveld.com website), and Protocol Online (protocol- online.net). Exemplary gene products for genetically modifying the genetically engineered cells of the invention include plasminogen activator, soluble CD4, Factor VIII, Factor IX, von Willebrand Factor, urokinase, hirudin, interferons, including alpha-, beta- and gamma-interferon, tumor necrosis factor, interleukins, hematopoietic growth factor, antibodies, glucocerebrosidase, adenosine deaminase, phenylalanine hydroxylase, human growth hormone, insulin, erythropoietin, VEGF, angiopoietin, hepatocyte growth factor, PLGF, and the like.

[00107] In an embodiment of the invention, the tissue or organ is selected from the group consisting of heart tissue, lung tissue, cardiac muscle tissue, striated muscle tissue, liver tissue, pancreatic tissue, cartilage, bone, pericardium, peritoneum, kidney, smooth muscle, skin, mucosal tissue, small intestine, and large intestine and adipose tissue.

[00108] The step of injecting a cell suspension into a subject tissue or organ may include, without limitation, using at least one syringe, needle, cannula, catheter, tube, or microneedle. The terms "injecting", "injection", or variations thereof as used herein shall refer to any means of ejecting or extruding a substance, typically through a tube or structure comprising a bore or external opening. Such tube or structure can be flexible, inflexible, or can comprise at least one flexible portion and at least one inflexible portion. Exemplary injection means include a syringe with or without a needle, a cannula, a catheter, flexible tubing, and the like. Delivery of the particular cell suspension might also be accomplished through the use of devices that permeablize tissue, such as microneedles. In contrast to traditional injections with standard-gauge hypodermic needles, microneedle (typically defined by a radius of curvature

.about.1 um) or microneedle arrays permeabilize the skin or endothelial cell layer by producing microscopic holes. These holes, in effect, act as conduits for materials delivery and may enhance the attachment or delivery of a cell suspension of the present invention to a vessel, tissue, or organ. Thus, the skilled artisan will understand that any structure comprising a bore or external opening through which at least one cell suspension can be extruded on or into a tissue or organ, or any structure that can permeabilize the surface of a tissue or and organ, including an engineered tissue, is within the intended scope of the invention. In certain embodiments, such injected construct polymerizes in vitro, following injection.

[00109] In a particular embodiment, the tissue graft or cell suspension of the present invention comprises cells selected from the group consisting of skin, skeletal muscle, cardiac muscle, atrial appendage of the heart, lung, mesentery, or adipose tissue. The adipose tissue may be selected from the group consisting of omental fat, properitoneal fat, perirenal fat, pericardial fat, subcutaneous fat, breast fat, or epididymal fat. [00110] Also provided are methods for augmenting a tissue or organ of a subject in need thereof, comprising implanting into the organ or tissue a tissue graft prepared by the devices of the present invention or injecting into the tissue or organ a cell suspension prepared by the devices of the present invention. As used herein, "augmenting" refers to increasing the volume and/or density of the tissue or organ.

[00111] Methods are also provided for regenerating a tissue or organ in a subject by implanting into the tissue or organ at least one tissue graft prepared by the devices described herein or by injecting into the tissue or organ at least one cell suspension prepared by the devices of the invention. As used herein, "regenerating" refers to replacing lost, diseased or otherwise damaged tissue by the formation of new tissue.

[00112] A skilled artisan will appreciate that the subject of the present invention may be any animal, including amphibians, birds, fish, mammals, and marsupials, but is preferably a mammal (e.g., a human; a domestic animal, such as a cat, dog, monkey, mouse, and rat; or a commercial animal, such as a cow, horse or pig). Additionally, the subject of the present invention may be of any age, including a fetus, an embryo, a child, and an adult. In a preferred embodiment of the present invention, the subject is human. In one embodiment, the subject is a horse and methods of the subject invention are used to regenerate tissues in and around the hooves of the animal. In further embodiments, the subject is a human, and the methods of tissue regeneration are used to prevent or treat, for example arthritis and diseases of the eye, including but not limited to, glaucoma and macular degeneration.

[00113] Additionally, methods for reconstructing a tissue or organ in a subject in need thereof comprising implanting into the tissue or organ at least one tissue graft prepared by the devices described herein or by injecting into the tissue or organ at least one cell suspension prepared by these devices. As used herein, "reconstructing" refers to rebuilding, reconstituting, reshaping and/or restoring a tissue or organ. In one embodiment of the invention, for example the subject has cellulite, and the subject is administered a subcutaneuous injection of an appropriate cell suspension in order to locally reconstruct the adipose tissue, thus improving the cosmetic appearance of the subject. In one embodiment, the subject is a post-surgical subject.

[00114] Also provided are methods for treating or preventing primary and secondary infections in a tissue or organ of a subject by implanting into the tissue or organ at least one tissue graft prepared by the devices described herein or by injecting into the tissue or organ at least one cell suspension prepared by these devices.

[00115] Methods for using the cell suspensions and tissue grafts prepared by the devices of the present invention to prevent the formation of scar tissue in a tissue or organ, and/or to treat or prevent inflammation in a tissue or organ of a subject are also provided.

[00116] Also provided are methods for preventing adhesion formation in a tissue or organ of a subject in need thereof by injecting into the tissue or organ at least one cell suspension or tissue graft prepared by the devices of the invention. [00117] In one embodiment, a method is provided for treating or preventing acute myocardial infarction in a subject by injecting into the heart at least one cell suspension prepared by any of the devices described herein, wherein vasculature to the heart tissue is increased. In another embodiment, methods for treating myocarditis in a subject are provided comprising injecting into the pericardial fluid of the subject at least one cell suspension prepared by any of the devices of present invention. [00118] Methods for treating a wound in a subject by injecting the wound with at least one cell suspension prepared by the device of the present invention are also provided. In one embodiment, the subject is a post-surgical subject.

[00119] The present invention also provides methods for treating or preventing tissue hypoxia in a subject by injecting into the tissue of the subject at least one cell suspension prepared by the devices of the invention, or implanting into the tissue of the subject at least one tissue graft prepared by the devices of the invention.

[00120] Methods for generation of an artificial tissue or organ, as well as methods for vascularization of an artificial tissue are also provided by the present invention. In one embodiment, the artificial tissue is injected with at least one cell suspension prepared by any of the devices of the invention. In a particular embodiment, the vascularization occurs in vitro. In another embodiment, the vascularization occurs in vivo. [00121] Additionally provided are methods for using the cell suspensions and tissue grafts prepared by the devices of the invention for screening a candidate agent for a beneficial therapeutic effect in a subject. In one embodiment, the tissue graft or cell suspension is contacted with the candidate agent; and at least one cell of the tissue graft or cell suspension is then assayed for at least one beneficial effect.

[00122] The present invention also provides methods for screening for adverse effects of a agent of interest in a subject. In an embodiment, a tissue graft or cell suspension containing the subject's is contacted with the compound of interest; and at least one cell of the tissue graft or cell suspension is then assayed for at least one adverse effect. In one embodiment, the agent of interest is a drug. In another embodiment, the agent of interest is a potential allergen. [00123] The current invention provides sustained pressure sodding and automation of the clinical procedures of separating a desired fraction of the patient's cells from tissue and filtering, washing, heating, macerating, proteolytically releasing, separating, resuspending, and pressure sodding the cells onto a permeable graft. Those skilled in the art will recognize, or be able to ascertain, many equivalents to the embodiments of the inventions described herein using no more than routine experimentation. Such equivalents are intended to be encompassed by the following claims.

[00124] All publications, patents and patent applications mentioned in this specification are herein incorporated by reference into the specification to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference.

[00125] The above description and example are only illustrative of preferred embodiments which achieve the objects, features, and advantages of the present invention, and it is not intended that the present invention be limited thereto.

Claims

CLAIMS What is claimed is:

1. An apparatus for preparing a tissue graft, comprising a media reservoir; a tissue dissociating chemical reservoir in communication with a cell inlet; a biochamber for holding a graft substrate and having an inlet and a first outlet; a media flow loop connecting the biochamber and the media reservoir; a pump configured to cause flow through the media flow loop; a cell mixture conduit connecting the tissue dissociating chemical reservoir with the media flow loop; a cell separator in communication with the cell mixture conduit and the media flow loop; and at least one valve configured to direct flow from the cell separator to the media flow loop.

2. The apparatus of claim 1 further comprising a cell macerator in communication with the cell mixture conduit.

3. The apparatus of claim 1 further comprising a heater.

4. The apparatus of claim 1 further comprising a waste reservoir.

5. The apparatus of claim 4 further comprising a filter between the cell inlet and the tissue dissociating chemical reservoir.

6. The apparatus of claim 5 further comprising at least one valve configured to permit a first fraction of cells from the filter to enter the reservoir and a second fraction to enter the waste reservoir.

7. The apparatus of claim 1 further comprising a media conduit permitting flow between the cell inlet and the media reservoir.

8. The apparatus of claim 1 further comprising a filter between the cell separator and the biochamber.

9. The apparatus of claim 1 wherein the biochamber comprises a porous tubular graft scaffold and a second outlet configured to permit translumenal flow through the scaffold.

10. The apparatus of claim 1, wherein the biochamber comprises a porous substantially planar sheet graft scaffold and a second outlet configured to permit transmural flow through the scaffold.

11. The apparatus of claim 9, wherein the biochamber comprises: an outer sleeve having a proximal end and a distal end; an inner sleeve at least partially disposed within the outer sleeve and having proximal and distal ends and a trough between said ends, the trough being disposed within the outer sleeve to define an interior space between the inner and outer sleeves; a distal interior conduit extending distally from within the distal end of the inner sleeve, and a proximal interior conduit extending proximally from within the proximal end of the inner sleeve; intracapillary posts within the interior space, the posts being adapted to hold a tubular graft scaffold between the distal, interior conduit and the proximal interior conduit, the graft scaffold defining an intracapillary and an extracapillary space; and . wherein the first outlet permits transmural flow through the graft.

12. The apparatus of claim 10, further comprising proximal tubing connected to the proximal interior conduit to provide flow to at least the intracapillary space of the graft scaffold.

13. The apparatus of claim 10, further comprising distal tubing connecting the distal interior conduit.

14. The apparatus of claim 10, further comprising a means for blocking flow through the distal tubing.

15. The apparatus of claim 10, wherein the distal conduit comprises an intralumenal port.

16. The apparatus of claim 1, wherein the apparatus is a handheld apparatus.

17. A method of preparing a tissue graft, comprising providing an apparatus in accordance with claim 1; introducing media containing adherent cells into the biochamber; and applying a sustained low pressure transmural flow of the media across the substrate for a time period sufficient to adhere the cells to the substrate.

18. The method of claim 15, wherein the substrate is a tubular scaffold for a vascular tissue graft.

19. The method of claim 15, wherein the substrate is a substantially planar sheet scaffold.

20. The method of claim 15, wherein the pressure is from about 10 tnmHg to about 55 mmHg.

21. The method of claim 15, wherein the pressure is from about 35 mmHg to about 50 mmHg.

22. The method of claim 15, wherein the pressure is about 50 mmHg.

23. The method of claim 15, wherein the time period is from about 5 minutes to about 1 hour.

24. The method of claim 15, wherein the adherent cells are cells released from fat tissue by the action of tissue dissociation chemicals having a density greater than adipocytes.

25. The method of claim 16, wherein the cells are endothelial cells.

26. The method of claim 15, wherein the cells are microvascular endothelial cells.

27. The method of claim 15, wherein the microvascular endothelial cells are derived from adipose tissue.

28. The method of claim 23, further comprising harvesting the adipose tissue from a patient.

29. The method of claim 23, wherein the cells comprise a mixture of. microvascular endothelial cells and adult stem cells.

30. The method of claim 23, wherein the microvascular endothelial cells are stem cells.

31. The method of claim 25, wherein the cells are autologous.

32. The method of claim 25, wherein the substrate comprises a material selected from the group consisting of elastin, ePTFE, collagen, polyurethane, polypropylene, polyethylene, polyamides, nylon, elastin, polyethylene terephthalate, polycarbonate, polystyrene, polylactic acid, polyglycolic acid, a PLA/PGA mixture, dextran, polyethylene glycol, stainless steel, titanium/nickel alloys, and silicone.

33. An apparatus for preparing a tissue graft, comprising: a flowpath cartridge comprising one or more fluid reservoirs, at least one inlet and at least one outlet; a cell separator cartridge having at least one inlet and at least one outlet; a graft chamber cartridge for holding a graft substrate, the graft chamber cartridge having at least one inlet and at least one outlet; at least one pump configured to cause flow through a flowpath; at least one valve configured to direct flow from the cell separator cartridge to the graft chamber cartridge; wherein said flowpath cartridge, cell separator cartridge and graft chamber cartridge communicate to form a continuous flowpath, and wherein said flowpath cartridge, cell separator cartridge, and graft chamber cartridge communicate with a modular kit enclosure capable of providing power to the apparatus.

34. The apparatus of claim 33, wherein the flowpath cartridge, cell separator cartridge and graft chamber cartridge are disposable.

35. The apparatus of claim 33, wherein the cell separator cartridge comprises a centrifuge.

36. The apparatus of claim 33, further comprising a cell macerator in communication with the flowpath cartridge.

37. The apparatus of claim 33, wherein the flowpath cartridge is preloaded with media.

38. The apparatus of claim 33, wherein the media is selected from the group consisting of M199, M199E, PBS, Saline, and Di-Cation Free DPBS.

39. The apparatus of claim 33, wherein the media is Ml 99E.

40. The apparatus of claim 33, wherein the media is buffered physiologic saline.

41. The apparatus of claim 33, wherein the flowpath cartridge comprises at least one pump.

42. The apparatus of claim 33, further comprising a heater.

43. The apparatus of claim 33, further comprising a waste reservoir.

44. The apparatus of claim 33, further comprising at least one filter.

45. The apparatus of claim 33, wherein the filter excludes particles greater than about 100 microns.

46. The apparatus of claim 33, wherein the filter excludes particles greater than 30 microns.

47. The apparatus of claim 33, wherein at least one filter is located between the cell separator cartridge and the graft cartridge.

48. The apparatus of claim 33, wherein the kit enclosure comprises at least one sensor means for detecting the presence of the flowpath cartridge, the cell separator cartridge and the graft chamber.

49. The apparatus of claim 33, wherein the kit enclosure comprises at least one sensor means for monitoring and controlling temperature, pressure and flow rate, wherein the sensor means is in communication with an alarm.

50. The apparatus of claim 33, wherein the kit enclosure comprises an electronic graphical display.

51. The apparatus of claim 33, wherein the kit enclosure comprises a bar code scanner.

52. The apparatus of claim 33, wherein the kit enclosure comprises a cell collection module having an inlet and an outlet.

53. The apparatus of claim 33, further comprising a means for counting cells.

54. The apparatus of claim 33, wherein the apparatus is a handheld apparatus.

55. A method of preparing a tissue graft, comprising: providing an apparatus in accordance with claim 28; introducing media containing adherent cells into the graft chamber; and applying a sustained low pressure transmural flow of the media across the substrate for a time period sufficient to adhere the cells to the substrate.

56. The method of claim 55, wherein the substrate is a tubular scaffold for a vascular tissue graft.

57. The method of claim 55, wherein the substrate is a porous substantially planar sheet graft scaffold.

58. The method of claim 55, wherein the pressure is from about 10 mmHg to about 55 mmHg.

59. The method of claim 55, wherein the pressure is from about 35 mmHg to about 50 mmHg.

60. The method of claim 55, wherein the pressure is about 50 mmHg.

61. The method of claim 55, wherein the time period is from about 5 minutes to about 1 hour.

62. The method of claim 55, wherein the adherent cells are cells released from fat tissue by the action of tissue dissociation chemicals having a density greater than adipocytes.

63. The method of claim 55, wherein the adherent cells are endothelial cells.

64. The method of claim 55, wherein the cells are microvascular endothelial cells.

65. The method of claim 55, wherein the microvascular endothelial cells are derived from adipose tissue.

66. The method of claim 65, further comprising harvesting the adipose tissue from a patient.

67. The method of claim 65, wherein the cells comprise a mixture of microvascular endothelial cells and adult stem cells.

68. The method of claim 65, wherein the cells are autologous.

69. The method of claim 55, wherein the substrate comprises a material selected from the group consisting of elastin, ePTFE, collagen, polyurethane, polypropylene, polyethylene, polyamides, nylon, elastin, polyethylene terephthalate, polycarbonate, polystyrene, polylactic acid, polyglycolic acid, a PLA/PGA mixture, dextran, polyethylene glycol, stainless steel, titanium/nickel alloys, and silicone.

70. An apparatus for preparing a tissue graft, comprising: a flowpath cartridge comprising one or more fluid reservoirs, a cell separator, and at least one inlet and at least one outlet; a graft chamber cartridge for holding a graft substrate, the graft chamber cartridge having at least one inlet and at least one outlet; at least one pump configured to cause flow through a flowpath; at least one valve configured to direct flow from the cell separator cartridge to the graft chamber cartridge; wherein said flowpath cartridge, cell separator cartridge and graft chamber cartridge communicate to form a continuous flowpath, and wherein said flowpath cartridge, cell separator cartridge, and graft chamber cartridge communicate with a modular kit enclosure capable of providing power to the apparatus.

71. The apparatus of claim 70, wherein the flowpath cartridge and graft chamber cartridge are disposable.

72. The apparatus of claim 70, wherein the cell separator comprises a centrifuge.

73. The apparatus of claim 70, further comprising a cell macerator in communication with the flowpath cartridge.

74. The apparatus of claim 70, wherein the flowpath cartridge is preloaded with media.

75. The apparatus of claim 70, wherein the media is selected from the group consisting of M199, M199E, PBS, Saline, and Di-Cation Free DPBS.

76. The apparatus of claim 70, wherein the media is Ml 99E.

77. The apparatus of claim 70, wherein the media is buffered physiologic saline

78. The apparatus of claim 70, wherein the flowpath cartridge comprises at least one pump.

79. The apparatus of claim 70, further comprising a heater.

80. The apparatus of claim 70, further comprising a waste reservoir.

81. The apparatus of claim 70, further comprising at least one filter.

82. The apparatus of claim 70, wherein the filter excludes particles greater than about 100 microns.

83. The apparatus of claim 70, wherein the filter excludes particles greater than 30 microns.

84. The apparatus of claim 70, wherein at least one filter is located between the ^• cell separator and the graft cartridge.

85. The apparatus of claim 70, wherein the kit enclosure comprises at least one sensor means for detecting the presence of the flowpath cartridge and the graft chamber.

86. The apparatus of claim 70, wherein the kit enclosure comprises at least one sensor means for monitoring and controlling temperature, pressure and flow rate, wherein the sensor means is in communication with an alarm.

87. The apparatus of claim 70, wherein the kit enclosure comprises an electronic graphical display.

88. The apparatus of claim 70, wherein the kit enclosure comprises a bar code scanner.

89. The apparatus of claim 70, wherein the kit enclosure comprises a cell collection module having an inlet and an outlet.

90. The apparatus of claim 70, further comprising a means for counting cells.

91. The apparatus of claim 70, wherein the apparatus is a handheld apparatus.

92. A method of preparing a tissue graft, comprising: providing an apparatus in accordance with claim 70; introducing media containing adherent cells into the graft chamber; and applying a sustained low pressure transmural flow of the media across the substrate for a time period sufficient to adhere the cells to the substrate.

93. The method of claim 92, wherein the substrate is a tubular scaffold for a vascular tissue graft.

94. The method of claim 92, wherein the substrate is a porous substantially planar sheet graft scaffold.

95. The method of claim 92, wherein the pressure is from about 10 mmHg to about 55 mmHg.

96. The method of claim 92, wherein the pressure is from about 35 mmHg to about 50 mmHg.

97. The method of claim 92, wherein the pressure is about 50 mmHg.

98. The method of claim 92, wherein the time period is from about 5 minutes to about 1 hour.

99. The method of claim 92, wherein the adherent cells are cells released from fat tissue by the action of tissue dissociation chemicals having a density greater than adipocytes.

100. The method of claim 92, wherein the adherent cells are endothelial cells.

101. The method of claim 92, wherein the cells are microvascular endothelial cells.

102.The method of claim 92, wherein the microvascular endothelial cells are derived from adipose tissue.

103. The method of claim 103, further comprising harvesting the adipose tissue from a patient.

104.The method of claim 103, wherein the cells comprise a mixture of microvascular endothelial cells and adult stem cells.

105.The method of claim 103, wherein the cells are autologous.

106. The method of claim 92, wherein the substrate comprises a material selected from the group consisting of elastin, ePTFE, collagen, polyurethane, polypropylene, polyethylene, polyamides, nylon, elastin, polyethylene terephthalate, polycarbonate, polystyrene, polylactic acid, polyglycolic acid, a PLA/PGA mixture, dextran, polyethylene glycol, stainless steel, titanium/nickel alloys, and silicone.

107. A method for revascularizing a tissue or organ of a subject in need thereof, comprising implanting into the tissue or organ at least one tissue graft prepared by the apparatus of claims 1, 33 or 70.

108. The method of claim 107, wherein the tissue graft comprises cells selected from the group consisting of skin, skeletal muscle, cardiac muscle, atrial appendage of the heart, lung, mesentery, or adipose tissue.

109. The method of claim 107, wherein the adipose tissue is selected from the group consisting of omental fat, properitoneal fat, perirenal fat, pericardial fat, subcutaneous fat, breast fat, or epididymal fat.

110. The method of claim 107, wherein the tissue graft comprises at least one

Relevant Cell.

111. The method of claim 107, wherein the Relevant Cell is selected from the group consisting of at least one neuron, myocardiocyte, chondrocyte, pancreatic ancinar cell, pancreatic endocrine cells including islet of Langerhans, hepatocyte, renal epithelial cell, parathyroid cell, Leydig cell, Sertoli cell, gonocyte, oocyte, blastocyst, Kupffer cell, lymphocyte, fibroblast, myocyte, myoblast, satellite cell, adipocyte, preadipocyte, osteocyte, osteoblast, osteoclast, chondrocyte, biliary epithelial cell, Purkinje cell, and pacemaker cell.

112. The method of claim 107, wherein the tissue graft further comprises an agent selected from the group consisting of cytokines, chemokines, antibiotics, drugs, analgesic agents, anti-inflammatory agents, immunosuppressive agents, or combinations thereof.

113. The method of claim 107, wherein the tissue or organ is selected from the group consisting of heart tissue, lung tissue, cardiac muscle tissue, striated muscle tissue, liver tissue, pancreatic tissue, cartilage, bone, pericardium, peritoneum, kidney, smooth muscle, skin, mucosal tissue, small intestine, and large intestine.

114. A method for revascularizing a tissue or organ of a subject in need thereof, comprising injecting into the tissue or organ a cell suspension prepared by the apparatus of claims 1, 33 or 72.

115. The method of claim 114, wherein the step of injecting comprises using at least one syringe, needle, cannula, catheter, tube, or microneedle.

116. The method of claim 114, wherein the cell suspension comprises cells selected from the group consisting of skin, skeletal muscle, cardiac muscle, atrial appendage of the heart, lung, mesentery, or adipose tissue.

117. The method of claim 114, wherein the adipose tissue is selected from the group consisting of omental fat, properitoneal fat, perirenal fat, pericardial fat, subcutaneous fat, breast fat, or epididymal fat.

118. The method of claim 114, wherein the cell suspension comprises at least one Relevant Cell.

119. The method of claim 114, wherein the Relevant Cell is selected from the group consisting of at least one neuron, myocardiocyte, chondrocyte, pancreatic ancinar cell, pancreatic endocrine cells including islet of Langerhans, hepatocyte, renal epithelial cell, parathyroid cell, Leydig cell, Sertoli cell, gonocyte, oocyte, blastocyst, Kupffer cell, lymphocyte, fibroblast, myocyte, myoblast, satellite cell, adipocyte, preadipocyte, osteocyte, osteoblast, osteoclast, chondrocyte, biliary epithelial cell, Purkinje cell, and pacemaker cell.

120. The method of claim 114, wherein the tissue graft further comprises an agent selected from the group consisting of cytokines, chemokines, antibiotics, drugs, analgesic agents, anti-inflammatory agents, immunosuppressive agents, or combinations thereof.

121. The method of claim 114, wherein the tissue or organ is selected from the group consisting of heart tissue, lung tissue, cardiac muscle tissue, striated muscle tissue, liver tissue, pancreatic tissue, cartilage, bone, pericardium, peritoneum, kidney, smooth muscle, skin, mucosal tissue, small intestine, and large intestine.

122. A method for augmenting tissue in a subject in need thereof comprising implanting into the tissue or organ at least one tissue graft prepared by the apparatus of claims 1, 33 or 70.

123. The method of claim 122, wherein the tissue graft comprises cells selected from the group consisting of skin, skeletal muscle, cardiac muscle, atrial appendage of the heart, lung, mesentery, or adipose tissue.

124. The method of claim 122, wherein the adipose tissue is selected from the group consisting of omental fat, properitoneal fat, perirenal fat, pericardial fat, subcutaneous fat, breast fat, or epididymal fat.

125. The method of claim 122, wherein the tissue graft comprises at least one Relevant Cell.

126. The method of claim 122, wherein the Relevant Cell is selected from the group consisting of at least one neuron, myocardiocyte, chondrocyte, pancreatic ancinar cell, pancreatic endocrine cells including islet of Langerhans, hepatocyte, renal epithelial cell, parathyroid cell, Leydig cell, Sertoli cell, gonocyte, oocyte, blastocyst, Kupffer cell, lymphocyte, fibroblast, myocyte, myoblast, satellite cell, adipocyte, preadipocyte, osteocyte, osteoblast, osteoclast, chondrocyte, biliary epithelial cell, Purkinje cell, and pacemaker cell.

127. The method of claim 122, wherein the tissue graft further comprises an agent selected from the group consisting of cytokines, chemokines, antibiotics, drugs, analgesic agents, anti-inflammatory agents, immunosuppressive agents, or combinations thereof.

128. The method of claim 122, wherein the tissue or organ is selected from the group consisting of heart tissue, lung tissue, cardiac muscle tissue, striated muscle tissue, liver tissue, pancreatic tissue, cartilage, bone, pericardium, peritoneum, kidney, smooth muscle, skin, mucosal tissue, small intestine, and large intestine.

129. A method for augmenting a tissue or organ of a subject in need thereof, comprising injecting into the tissue or organ a cell suspension prepared by the apparatus of claims 1, 33 or 70.

130. The method of claim 129, wherein the step of injecting comprises using at least one syringe, needle, cannula, catheter, tube, or microneedle.

131. The method of claim 129, wherein the cell suspension comprises cells selected from the group consisting of skin, skeletal muscle, cardiac muscle, atrial appendage of the heart, lung, mesentery, or adipose tissue.

132. The method of claim 129, wherein the adipose tissue is selected from the group consisting of omental fat, preperitoneal fat, perirenal fat, pericardial fat, subcutaneous fat, breast fat, or epididymal fat.

133. The method of claim 129, wherein the cell suspension comprises at least one Relevant Cell.

134. The method of claim 129, wherein the Relevant Cell is selected from the group consisting of at least one neuron, myocardiocyte, chondrocyte, pancreatic ancinar cell, pancreatic endocrine cells including islet of Langerhans, hepatocyte, renal epithelial cell, parathyroid cell, Leydig cell, Sertoli cell, gonocyte, oocyte, blastocyst, Kupffer cell, lymphocyte, fibroblast, myocyte, myoblast, satellite cell, adipocyte, preadipocyte, osteocyte, osteoblast, osteoclast, chondrocyte, biliary epithelial cell, Purkinje cell, and pacemaker cell.

135. The method of claim 129, wherein the tissue graft further comprises an agent selected from the group consisting of cytokines, chemokines, antibiotics, drugs, analgesic agents, anti-inflammatory agents, immunosuppressive agents, or combinations thereof.

136. The method of claim 129, wherein the tissue or organ is selected from the group consisting of heart tissue, lung tissue, cardiac muscle tissue, striated muscle tissue, liver tissue, pancreatic tissue, cartilage, bone, pericardium, peritoneum, kidney, smooth muscle, skin, mucosal tissue, small intestine, and large intestine.

137. A method for regenerating a tissue or organ in a subject in need thereof comprising implanting into the tissue or organ at least one tissue graft prepared by the apparatus of claims 1, 33, or 70.

138. The method of claim 137, wherein the subject is a mammal.

139. The method of claim 138, wherein the subject is a horse.

140. The method of claim 138, wherein the subject is human.

141. The method of claim 137, wherein the subject has arthritis.

142. The method of claim 137, wherein the subject has a disease of the eye.

143. The method of claim 137, wherein the subject has glaucoma.

144. The method of claim 137,wherein the subject has macular degeneration.

145. A method for regenerating a tissue or organ in a subject in need thereof comprising injecting into the tissue or organ a cell suspension prepared by the apparatus of claims 1, 33 or 70.

146. The method of claim 145, wherein the subject is a mammal.

147. The method of claim 146, wherein the subject is a horse.

148. The method of claim 146, wherein the subject is human.

149. The method of claim 145, wherein the subject has arthritis.

150. The method of claim 145, wherein the subject has a disease of the eye.

151. The method of claim 145, wherein the subject has glaucoma.

152. The method of claim 145,wherein the subject has macular degeneration.

153. A method for reconstructing a tissue or organ in a subject in need thereof comprising implanting into the tissue or organ at least one tissue graft prepared by the apparatus of claims 1, 33, or 70.

154. The method of claim 153, wherein the subject has cellulite.

155. The method of claim 153, wherein the subject is a post-surgical subject.

156. A method for reconstructing a tissue or organ in a subject in need thereof comprising injecting into the tissue or organ at least one cell suspension prepared by the apparatus of claims 1, 33 or 70.

157. The method of claim 153, wherein the subject has cellulite.

158. The method of claim 153, wherein the subject is a post-surgical subject.

159. A method for treating or preventing infection in a tissue or organ of a subject in need thereof comprising implanting into the tissue or organ at least one tissue graft prepared by the apparatus of claims 1, 33 or 70.

160. A method for treating or preventing infection in a tissue or organ of a subject in need thereof comprising injecting into the tissue or organ at least one cell suspension prepared by the apparatus of claims 1, 33 or 70.

161. A method of treating or preventing inflammation in a tissue or organ of a subject in need thereof comprising implanting into the tissue or organ at least one tissue graft prepared by the apparatus of claims 1, 33 or 70.

162. A method of treating or preventing inflammation in a tissue or organ of a subject in need thereof comprising injecting into the tissue or organ at least one cell suspension prepared by the apparatus of claims 1, 33 or 70.

163. A method for preventing the formation of scar in a tissue or organ of a subject in need thereof comprising injecting into the tissue or organ at least one cell suspension prepared by the apparatus of claims 1, 33 or 70.

164. A method for preventing adhesion formation in a tissue or organ of a subject in need thereof comprising injecting into the tissue or organ at least one cell suspension prepared by the apparatus of claims 1, 33 or 70.

165. Method for treating or preventing acute myocardial infarction in a subject comprising injecting into the heart at least one cell suspension prepared by the apparatus of claims 1, 33 or 70, thereby increasing vasculature to the heart tissue.

166. Method for treating myocarditis in a subject comprising injecting into the pericardial fluid of the subject at least one cell suspension prepared by the apparatus of claims 1, 33 or 70.

167. A method for treating a wound in a subject comprising injecting the wound with at least one cell suspension prepared by the apparatus of claims 1, 33 or 70.

168. A method for treating or preventing tissue hypoxia in a subject comprising injecting into the tissue of the subject at least one cell suspension prepared by the apparatus of claims 1, 33 or 70.

169. Method for vascularization of an artificial tissue comprising injecting the artificial tissue with at least one cell suspension prepared by the apparatus of clams 1, 32, or 67.

170. The method of claim 168, wherein the vascularization occurs in vitro.

111. The method of claim 168, wherein the vascularization occurs in vivo.

112. A method for screening a candidate agent for a beneficial therapeutic effect in a subject comprising: contacting a tissue graft comprising the subject's cells prepared by the apparatus of claims 1, 33 or 70 with the candidate agent; and assaying at least one cell of the tissue graft for at least one beneficial effect.

173. A method for screening a candidate agent for a beneficial therapeutic effect in a subject comprising: contacting a cell suspension comprising the subject's cells prepared by the apparatus of claims 1, 33 or 70 with the candidate agent; and assaying at least one cell of the cell suspension for at least one beneficial effect.

174. A method for screening for adverse effects to a compound of interest in a subject comprising: contacting a tissue graft comprising the subject's cells prepared by the apparatus of claims 1, 33 or 70 with the compound of interest; and assaying at least one cell of the tissue graft for at least one adverse effect.

175. The method of claim 174, wherein the compound of interest is a drug.

176. The method of claim 174, wherein the compound of interest is a potential allergen.

177. A method for screening for adverse effects to a compound of interest in a subject comprising: contacting a cell suspension comprising the subject's cells prepared by the apparatus of claims 1, 33 or 70 with the compound of interest; and assaying the cell suspension for at least one adverse effect.

178. The method of claim 177, wherein the compound of interest is a drug.

179. The method of claim 177, wherein the compound of interest is a potential allergen.

180. A tissue processing apparatus comprising: a separation vessel having a longitudinal axis, including at least one inlet for providing to the vessel material to be processed; at least one outlet for removing material from the separation vessel; a flow path extending between the inlet and the outlet; an inner chamber in communication with the interior of the separation vessel; wherein the separation vessel is attached to a centrifuge rotor configured to be rotated by a motor.

181. The apparatus of claim 180, wherein the apparatus is disposable.

182. The apparatus of claim 180, further comprising a heating means.

183. The apparatus of claim 180, further comprising a pump.