EP1649000A2 - Seeding pancreatic cells on porous matrices - Google Patents

Abstract

A method was developed for seeding cells, particularly for seeding cells having at least one marker characteristic of a pancreatic cell, onto a biocompatible scaffold. The method utilizes centrifugal forces and a specially designed ePTFE plate to uniformly guide cell seeding into the scaffold with no loss in viability.

Description

Seeding Pancreatic Cells On Porous Matrices

FIELD OF THE INVENTION The present invention relates to a method for seeding cells, particularly cells having one or more markers characteristic of pancreatic cells, onto a biocompatible scaffold before transplantation. The present method employs centrifugation forces to uniformly guide cell seeding into the scaffold with a high seeding efficiency and little or no loss in viability.

BACKGROUND OF THE INVENTION Islet transplantation typically involves direct injection of a cadeveric islet suspension into the portal vein of a patient and subsequent localization of islets in the liver matrix. Although the procedure has been successful, injecting the islets directly into the blood stream is associated with significant complications, such as thrombosis, which reduces islet survival, Tissue engineering can provide a solution to this problem by providing a three dimensional porous polymeric matrix that acts as a substrate for islet attachment. It has been previously demonstrated that cells seeded in a properly designed 3-D scaffold could recreate the in-vivo microenvironment, thereby facilitating cell-cell interactions and expression of differentiated functions. To construct such complex structures, the efficiency of the cell seeding process is critical to the overall performance of the tissue-engineered construct, especially in case of constructing 3D islet scaffold considering the low proliferative capacity islet cells and their sensitivity to surrounding environment. Prior to the present invention, islet seeding onto polymeric scaffolds has involved simple depositing of islets onto the scaffold by relying on passive diffusion of islets into the scaffold which was not very successful (Vacanti et al., "Selective cell transplantation using bioabsorbable artificial polymers as matrices," J. Pedia tr. Surg. 23(1 Pt 2) : 3-9, 1988) . Several other approaches have been developed to enhance the efficiency of cell seeding, but none of them focused on pancreatic islets. For example, spinner flasks have been used in seeding of chondrocytes onto polyglycolic acid scaffolds (Vunjak-Novakovic et al., "Dynamic cell seeding of polymer scaffolds for cartilage tissue engineering," Biotechnol . Prog. 14 (2 ): 193-202, 1998). The procedure involved suspending the scaffolds via needles in a cell suspension and mixing with a magnetic stir bar at 50 rpm. The process required a long time to complete, ranging from several hours to one day. Another approach is the use of centrifugation forces in cell seeding which yields minimum stress to the seeded cells and enhances seeding efficiency. A cell seeding method was developed by Yang et al. (J. Biomed. Mater. Res . 55(3) : 379- 86, 2001) , referred to as Centrifugational Cell

Immobilization (CCI) . Hepatocytes were seeded onto hydrophilic porous poly (vinyl formal) cubes. Both the cubes and hepatocytes were suspended in media in a centrifugation tube and were exposed to alternating centrifugation and resuspension steps. The procedure yielded 40% seeding efficiency and required a large number of hepatocytes [(2-8) x 10⁷ cells]. Dar et al. {Biotechnol . Bioeng. 80(3): 305-12, 2002) utilized a more controlled approach in cell seeding via centrifugation forces. Cardiomyocytes were seeded onto a hydrophilic alginate scaffold by placing the scaffold into a well of a 96-well plate and pipetting lOμl of cell suspension onto it. The plate was then placed onto a plate holder-type rotor and centrifuged for 6 minutes at 1000 x g, 4°C. A seeding efficiency of 80-90% was reported in an alginate scaffold, which decreased to 60% when higher seeding densities were used per scaffold. Thus, there remains a need to develop a simple and reproducible method to seed pancreatic cells onto porous scaffolds with high seeding efficiency and little or no loss in cell viability.

SUMMARY OF THE INVENTION The present invention provides a method for seeding cells onto a biocompatible scaffold. The method employs centrifugal forces to uniformly guide cell seeding into the scaffold with high seeding efficiency and no significant loss in cell viability. The present seeding method applies to any cell type, and is especially useful for seeding cells having at lest one marker, preferably two or more markers, characteristic of pancreatic cells. The present method involves placing scaffolds into a plate or any other device that fits into a centrifuge chamber. In a preferred embodiment of the present invention, scaffolds are placed into wells of an expanded polytetrafluoro-ethylene (ePTFE) plate. The ePTFE plates are designed to allow easy fit into a holder within a centrifuge chamber, and to contain wells of a diameter made to tight fit a scaffold of a desired size and of a height to ensure containment of the cell suspension to prevent any loss during the centrifugation process. The cells are introduced to scaffolds, which are typically already placed in a plate or device suitable for centrifugation. The plate is covered and slightly shaken sideways to ensure uniform distribution of cells or islets onto the top surface of the scaffold. The plate is then placed in a centrifugation chamber and spun down at a speed sufficient to help the islets spread evenly and penetrate into the 3D matrix of the scaffold. The present invention also provides a kit for seeding cells onto a biocompatible scaffold. The kit contains biocompatible scaffolds, at least two plates each designed to allow easy fit into a holder within a centrifuge chamber, and to contain wells of a diameter made to tight fit the scaffolds and of a height to ensure containment of the cell suspension to prevent any loss during the centrifugation process .

BRIEF DESCRIPTION OF THE FIGURES Figures 1A-1B are SEM pictures of one embodiment of a 3-D VICRYL nonwoven scaffold described herein. 1A. Top view; IB. Cross section. Figure 2 is a drawing of the Teflon plate including a detailed description of its suggested dimensions. Figures 3A-3B depict murine islets of Langerhan in a Live/Dead stain seeded on a scaffold. 3A. Live islets are stained with Calcein AM; 3B. Dead islets are stained with Ethidium homodimer-1. Figures 4A-4B illustrate murine islet distribution in a composite scaffold composed of 90/10 PGA/PLA fibers reinforced with a 0.5% 65/35 PGA/PCL porous foam component. Seeding was performed at 800 rpm (4A) , or 2000 rpm (4B) . Figure 5 is an image showing results of murine islet seeding on a foam scaffold with low porosity as described herein after.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for seeding cells onto a biocompatible scaffold by employing centrifugation forces to uniformly guide cell seeding into the scaffold. The seeding method of the present invention achieves a high seeding efficiency with no significant loss in cell viability. The term "biocompatible", as used herein, means that the material does not elicit a substantial detrimental response in the host. Biocompatible scaffolds appropriate for use in the method of the present invention include both synthetic polymer based scaffold or non-synthetic biopolymers. By "non-synthetic biopolymers" is meant to include, but not limited to biopolymers such as hyaluronic acid, collagen, recombinant collagen, cellulose, elastin, alginates, chondroitin sulfate, chitosan, chitin, keratin, silk, small intestine submucosa (SIS) , and blends thereof. Biocompatible synthetic scaffolds suitable for use in the present invention are typically 3-dimentional matrices that can take any geometrical shape, preferably a cylindrical disk with a height of less than 1cm and a diameter of less than 20cm. Such biocompatible synthetic scaffolds can be hydrophilic or hydrophobic scaffolds, and can be made from polymeric materials such as, but not limited to, aliphatic polyesters, polyalkylene oxalates, polyamides, polycarbonates, polyorthoesters, polyoxaesters, polyamidoesters, polyanhydrides and polyphosphazenes . Currently, aliphatic polyesters are among the preferred biodegradable polymers for use in making the composite scaffold according to the present invention. Aliphatic polyesters can be homopolymers or copolymers (random, block, segmented, tapered blocks, graft, triblock, etc.) having a linear, branched or star structure. Suitable monomers for making aliphatic homopolymers and copolymers may be selected from the group consisting of, but are not limited to, lactic acid, lactide (including L-, D-, meso and L,D mixtures), glycolic acid, glycolide, ε-caprolactone, p-dioxanone, trimethylene carbonate, polyoxaesters, δ-valerolactone, β- butyrolactone, ε-decalactone, 2, 5-diketomorpholine, pivalolactone, α, α-diethylpropiolactone, ethylene carbonate, ethylene oxalate, 3-methyl-l, 4-dioxane-2, 5-dione, 3,3- diethyl-1, 4-dioxan-2, 5-dione, γ-butyrolactone, 1, 4-dioxepan- 2-one, 1, 5-dioxepan-2-one, 6, 6-dimethyl-dioxepan-2-one and 6, 8-dioxabicycloctane-7-one . Preferably, the biodegradable polymers are selected from polylacetic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), polydioxanone (PDO), trimethylene carbonate (TMC) , polyvinyl alcohol (PVA) , polyoxaesters, copolymers or blends thereof. Scaffolds suitable for use in the present invention can be a highly fibrous or nonwoven scaffold, as illustrated in Figure 1, or a composite scaffold (or scaffold composite) which is typically composed of a nonwoven component and a foam component . Scaffolds for use in the present method can be prepared by a variety of methods known to those skilled in the art. For example, a nonwoven scaffold can be prepared using wet- lay or dry-lay fabrication techniques well known in the art. A scaffold composite is usually prepared by lyophilization of a dry lay needle-punched nonwoven mat with a polymer solution which typically has a weight ratio of 0.5/99.5 polymer in 1,4-dioxane solvent. A laboratory scale lyophilizer, or freeze dryer, (Model Duradry, FTS Kinetics, Stone Ridge, NY) , cane be used to form the composite scaffold. For example, 25ml of the polymer solution is slowly poured into a 4-inch by 4-inch aluminum mold and then the nonwoven mat is carefully placed into the mold. The mold assembly then is placed on the shelf of the lyophilizer and the freeze dry sequence is begun. The sequence usually is 1) -17°C for 60 minutes, 2) -5°C for 60 minutes under vacuum 100 mT, 3) 5°C for 60 minutes under vacuum 20 mT, 4) 20°C for 60 minutes under vacuum 20 mT . After the cycle is completed, the mold assembly is taken out of the freeze drier and allowed to degas in a vacuum hood for 2 to 3 hours after which the composite scaffold is stored under nitrogen. The scaffold suitable for use in the present invention is characterized by having interconnecting pores. The pore size and porosity of the scaffold should be such that facilitates the uniform distribution of the cells, the transport of nutrients and/or the growth of cells into the scaffold. The term "porosity" refers to the ratio of the volume of the interconnected pores within a scaffold to the total volume of the scaffold. The porosity of a scaffold can be determined using mercury porosimetery. Scaffold porosity may vary depending on the application and can be controlled by a variety of means such as the density of the fibers in the nonwoven component, the concentration or amount of the polymer solution used in forming the scaffold. Preferably, the scaffolds suitable for use in the present method have a porosity of 70-95%, preferably a porosity of 90%. The scaffolds suitable for use in the present invention have a pore size in the range of 10-500 μm, and preferably in the range of 50-400 μm. A particularly preferred scaffold for use in the method of the present invention is a nonwoven scaffold made from a 90/10 copolymer of PGA/PLA, sold under the tradename VICRYL (Ethicon, Inc., Somerville, NJ) . A nonwoven VICRYL based scaffold is highly fibrous with a porosity range of 70-95%. Preferably, the nonwoven VICRYL based scaffold has a porosity of 90%. Another particularly preferred scaffold for use in the method of the present invention is a nonwoven scaffold made of a 95/5 copolymer of PLA/PGA, sold under the trade name PANACRYL (Ethicon, Inc., Somerville, NJ) . A nonwoven PANACRYL based scaffold is highly fibrous with a porosity range of 70-95%. Preferably, the nonwoven PANACRYL based scaffold has a porosity of 90%. Another particularly preferred scaffold for use in the method of the present invention is a nonwoven scaffold made of a 100% homopolymer of polydioxanone, sold under the trade name PDS II (Ethicon, Inc., Somerville, NJ) . A nonwoven PDS II based scaffold is highly fibrous with a porosity range of 70-95%. Preferably, the nonwoven PDS II based scaffold has a porosity of 90%. Those skilled in the art appreciate that nonwoven scaffolds may also be prepared by mixing different ratios of the PDS II, PANACRYL, and VICRYL fibers. Another particularly preferred scaffold for use in the method of the present invention is a composite scaffold comprised of a nonwoven component and a porous foam component surrounding the fibers of the nonwoven component. The preferred foam component is prepared from a 65/35 PGA/PCL, 60/40 PLA/PCL, or blends thereof. A composite scaffold for use in the present invention should have a porosity range of 70-95%. Preferably, the composite scaffold has a porosity of 90%. Another preferred scaffold is a highly porous foam scaffold. The preferred foam scaffold is prepared from a 65/35 PGA/PCL copolymer, 60/40 PLA/PCL copolymer, or blends thereof. A foam scaffold for use in the present invention should have a porosity range of 70-95%. Preferably, the foam scaffold has a porosity of 90%. In another embodiment, a scaffold as described hereinabove may be coated with a biopolymer, growth factor, or a pharmaceutical prior to addition of cells. The seeding method of the present invention can be applied to any cells. The term "cells", as used herein, refers to isolated cells, cells lines (including cells engineered in vitro) , any preparation of living tissue, including primary tissue explants and preparations thereof. Although the seeding method of the present invention is not limited to seeding any particular cell type, the present method is especially useful for seeding cells having at least one marker, preferably two or more markers, characteristic of a pancreatic cell, either isolated from a mammalian pancreas or engineered in vi tro . The term "mammalian" is meant to include human and other primate species, porcine, canine, murine, and the like . By "pancreatic cell" is meant to include cells of both endocrine and exocrine pancreatic tissues. The endocrine pancreas is composed of hormone-producing cells arranged in clusters or islets of Langerhans . Of the four main types of cells that form the islets ("islet cells") , the alpha cells produce glucagons, the beta cells produce insulin, the delta cells produce somatostatin, and the PP cells produce pancreatic polypeptide (PP) . The endocrine pancreas includes the pancreatic acini and the pancreatic duct. Pancreatic acinar cells synthesize a range of digestive enzymes. Ductal cells secret bicarbonate ions and water in response to the hormone secreted from the gastrointestinal tract. Thus, "pancreatic cells" as used herein include alpha cells, beta cells, delta cells, PP cells, acinar cells, ductal cells or other cells found in a mammalian pancreas. Markers characteristic of a pancreatic cell include the expression of cell surface proteins or the encoding genes, the expression of intracellular proteins or the encoding genes, cell morphological characteristics, and the production of secretory products such as glucagons, insulin and somatostatin. Those skilled in the art will recognize that known immunofluorescent, immunochemical, polymerase chain reaction, in si tu hybridization, Northern blot analysis, chemical or radiochemical methods can readily ascertain the presence of absence of a islet cell specific characteristic. In a particularly preferred embodiment, the seeding method of the present invention is applied to seed pancreatic islets. The term "islets" as used herein includes both islets isolated from a mammalian pancreas as masses or structures formed by alpha cells, beta cells, delta cells and PP cells, and islets formed in vi tro from isolated or engineered islet cells or cells that bear at least one marker, preferably two or more markers, characteristic of an islet cell. In another embodiment, the seeding method of the present invention is applied to seed cells engineered in vitro to have at least one marker characteristic of a pancreatic islet cell, preferably, a pancreatic beta cell. Markers characteristic of beta cells include, but are not limited to, the expression of transcription factors such as PDX-1 (pancreatic and duodenal homeobox gene-1) , NGN-3 (neurogenin-3) , Hlxb9, Nkx6, Isll, Pax6, Neurod, Hnfla, Hnf6, among others. Such cells can be produced in vitro by, e.g., differentiating adipose stromal cells. Typically, before the cells are introduced to a biocompatible scaffold, the scaffold has been sterilized and placed in a plate, container or device that fits onto a rotor in a centrifugation chamber. In a preferred embodiment, a sterilized scaffold has been placed in a well of a sterile TEFLON plate, which has dimensions similar to a standard 96-well cell culture plate (127 mm X 85 mm) to allow easy placement of the TEFLON plate onto a plate holder within a centrifugation chamber, e.g., a Allegra 6R Centrifuge chamber. The TEFLON plate can be designed to tight fit one scaffold per well. For example, for a 5mm scaffold, a TEFLON plate can be made to have wells with a diameter of 4.8+0.05mm, as shown in Figure 2, which gives 54 wells per plate. The depth of the wells is about 0.78mm, which is long enough to accommodate the scaffold and the added cell suspension volume without any risk of cell loss during centrifugation. TEFLON plates, which are designed to allow easy fit into a holder within a centrifuge chamber, and to contain wells of a diameter made to tight fit a desired scaffold and of a height to ensure containment of the islet suspension to prevent any loss during the centrifugation process, are also referred herein as "ePTFE plates". For each design two plates are manufactured to guarantee accurate balance during centrifugation. Covers of standard 96-well cell culture plates are used to cover the TEFLON plates during the centrifugation process to maintain sterility. The cells can be introduced into a scaffold in any manner convenient and appropriate. In one embodiment, isolated islets are counted and mixed with a known volume of media, and the resulting islet suspension is pippetted onto a scaffold in 60-100μl of suspension per well. Preferably, an islet suspension is pippetted at about 60μl into each 8 mm diameter scaffold (i.e., each well) . Those skilled in the art will appreciate that the volume of media added depends on the size of the scaffold and the number of islets needed per scaffold for transplantation. The Teflon plate is then covered and slightly shaken side ways to ensure uniform spread of the islets onto the surface of the scaffold. The plate is placed on a plate holder in a centrifugation chamber and a counter plate placed on the opposite side. In another embodiment, pancreatic cells are suspended in a biopolymer, such as, hyaluronic acid, collagen, recombinant collagen, cellulose, elastin, alginates, chondroitin sulfate, chitosan, chitin, keratin, silk, small intestine submucosa (SIS), and blends thereof. The cells suspended in the biopolymer can be subsequently added to a synthetic scaffold and centrifuged according to the present invention. Alternatively, the cells suspended in the biopolymer may be centrifuged, in the absence of a synthetic scaffold. The speed and length of time of centrifugation depend on a number of factors, including the type of the centrifuge rotor, the type of scaffold, the size of cells, and the density of cells in the scaffold. Generally speaking, higher centrifugation forces can be applied to cells of larger sizes. Islets of Langerhans have an average diameter of 50 - 350μm, which is considerably larger as compared to most individual cells. Insufficient centrifugation forces may result in an insufficient penetration of cells through the scaffold, which might lead to a loss of cells remaining on the surface during transplantation. It may also lead to over-crowdedness of cells in one area of the scaffold, which in turn may lead to aggregation or competition for nutrition and oxygen resulting in a loss of cells. On the other hand, high centrifugation speed might force cells all the way through the scaffold to the bottom of the well resulting in loss in cell seeding efficiency. Typically, to achieve a highly uniform distribution of islets across a scaffold without significant loss of islet viability, an islet-containing scaffold can be centrifuged at 100 to 700 g for about 2-5 minutes, preferably, at about 200 g for about 3 minutes. In a preferred embodiment, an islet-containing scaffold, placed within a TEFLON plate which is placed in a plate holder in a chamber of an Allegra 6R Centrifuge or other similar or equivalent centrifuges, is centrifuged at a speed of about 800-2000 rpm (equivalent to 100-700 g) for about 2-5 minutes, or preferably 3 minutes. Optimal centrifugation forces and time parameters can be determined by those skilled in the art given the particular type of scaffold, cells and centrifuge. Cell viability within the scaffolds after centrifugation can be determined by using any appropriate assay, e.g., a Live/Dead assay kit (Molecular probes, Eugene, OR) , where Calcein AM and Ethidium homodimer-1 are used for staining live and dead cells, respectively. Figures 3A-3B illustrate the staining of live (florescent green) and dead (red) islets. Seeding efficiency can be determined by estimating the percentage of live cells or islets on the scaffold and determining the number of cells or islets left behind on the well surface after removal of scaffold following centrifugation. The number of cells or islets left behind on the well surface can be determined by washing the well surface with a known volume of media and performing an cell or islet count under the florescence microscope. By employing the method of the present invention, highly uniform cell seeding, especially islet seeding, onto a porous polymer scaffold is achieved with a high seeding efficiency without significant loss of cell or islet viability. Seeding efficiency is considered to be high if it is more than 90%, or preferably more than 95% or nearly 100%. Loss of viability of more than 70% is considered to be significant. Preferably, the loss of viability is less than 10%, more preferably, less than 5%. In another aspect, the present invention also provides a kit for seeding cells onto a biocompatible polymer based scaffold. The kit contains biocompatible polymer based scaffolds, at least two plates each designed to allow easy fit into a holder within a centrifuge chamber, and to contain wells of a diameter made to tight fit the scaffolds and of a height to ensure containment of the cell suspension to prevent any loss during the centrifugation process. The following examples illustrate some of the scaffolds that were seeded with islets using the method disclosed in this invention. Those skilled in the art will realize that these specific examples do not limit the scope of this invention to any specific materials.

EXAMPLE 1 Islet Seeding Onto A Foam Reinforced VICRYL Nonwoven Scaffold A biodegradable sheet approximately 2mm in thickness was prepared from nonwoven VICRYL (90/10 PGA/PLA) , reinforced with 0.5% foam (65/35 PGA/PCL) matrix via freeze- drying techniques known to those skilled in the art. Scaffolds were then punched from the sheet using a biopsy punch with a diameter of 5mm. Three scaffolds were placed each in a separate well in a custom made TEFLON plate. Islets were isolated from Balb/C mice with an average of 220 ± 40 islets per pancreas. Immediately after isolation, islet purity, as determined by dithazone staining, ranged from 80 to 90%. Islet suspension in aliquots of 60μl containing 200 islets were pipetted slowly onto each scaffold. The TEFLON plate was then covered with a 96-well plate cover to ensure sterility and shaken side ways slowly to distribute the islets evenly on the surface of the scaffold. The plate was then placed in the centrifugation chamber with a counter plate for balance at 4°C. A Beckman Coulter Allegra 6 series centrifuge was used in this example with a GH3.8 stainless steel rotor with an average diameter of 326mm and multiwell plate carriers to accommodate the Teflon plates. After 3 minutes at 200g the plate was removed from the chamber. The viability of seeded islets was determined using the Live/Dead assay (Molecular Probes) and the seeding efficiency was calculated by counting the number of islets remaining in the bottom of each well. For this construct, the seeding efficiency was ~95% and the majority of the cells were viable.

EXAMPLE 2 Appropriate Centrifugation Speed And Time Scaffolds made from several different materials with different porosities were tested to obtain an appropriate range of centrifugation speed and time. Initially time was kept constant at 3 minutes, temperature at 4°C, and three different speeds (300, 800, 2000 rpm) with a GH3.8 rotor Beckman Coulter Allegra 6 series centrifuge were used. It was observed that at 300rpm the islets were mainly concentrated on the top portion of the scaffold and their distribution was not uniform, as indicated by highly concentrated areas of the florescence intensity on the top of the scaffold. The run performed at 800 rpm yielded a much better distribution, as shown in Figure 4A. Islets with high intensity were closer to the surface and those with lower intensity were deeper in the scaffold. Jn order to determine a maximum speed at which the islets would penetrate all the way through the scaffold, the experiment was repeated again at 2000 rpm while keeping other parameters constant. A uniform distribution was maintained at 2000 rpm, as indicated in Figure 4B. Therefore, an appropriate range of the centrifugation speed is 800-2000 rpm (100-700 g) . Furthermore, cell seeding efficiency was -95% in the 800-2000 RPM range. EXAMPLE 3 Islet Seeding Onto Biodegradable Foam Scaffolds With Lower Porosity This example illustrates the importance of the scaffold porosity in the success of islet seeding. A low porosity sheet was prepared from a 5% PGA/PCL (65/35) in dioxane solution via lyophilization. The porosity of this scaffold was determined by mercury porosimetery to be -75%.

Scaffolds of 5mm were punched from the sheet and placed in the wells of the Teflon plate. Stained islets were added to the scaffolds and centrifuged, as described in Example 1. Following centrifugation, the constructs were examined under the florescence microscope. As indicated in Figure 5, due to the low porosity of the foam, the islets were not seeded uniformly throughout the scaffolds and the cell seeding efficiency was -50%.

Claims

WHAT IS CLAIMED IS :

1. A method for seeding cells onto a biocompatible scaffold, comprising adding the cells to said scaffold, and subjecting the scaffold to centrifugation.

2. The method of claim 1, wherein said scaffold is a nonwoven scaffold or a composite scaffold comprising a nonwoven component and a foam component .

3. The method of claim 2, wherein said nonwoven scaffold or said nonwoven component of said composite scaffold is formed from a polymer selected from polylacetic acid (PLA) , polyglycolic acid (PGA) , polycaprolactone (PCL) , polydioxanone (PDO) , trimethylene carbonate (TMC) , polyvinyl alcohol (PVA) , or polyoxaester, or a copolymer or blend thereof.

4. The method of claim 3, wherein said nonwoven scaffold or said nonwoven component of said composite scaffold is formed from a 90/10 copolymer of PGA/PLA.

5. The method of claim 4, wherein said nonwoven scaffold is a VICRYL based scaffold.

6. The method of claim 3, wherein said nonwoven scaffold or said nonwoven component of said composite scaffold is formed from a 95/5 copolymer of PLA/PGA.

7. The method of claim 6, wherein said nonwoven scaffold is a PANACRYL based scaffold.

8. The method of claim 3, wherein said nonwoven scaffold or said nonwoven component of said composite is made of a 100% homopolymer of polydioxanone.

9. The method of claim 8, wherein said nonwoven scaffold is a PDS II based scaffold.

10. The method of claim 2, wherein said foam component is prepared from a 65/35 PGA/PCL copolymer, a 60/40 PLA/PCL copolymer, or a blend thereof.

11. The method of claim 1, wherein said scaffold has a porosity of 70-95%.

12. The method of claim 1, wherein said scaffold is a biopolymer .

13. The method of claim 12, wherein said biopolymer is selected from hyaluronic acid, collagen, recombinant collagen, cellulose, elastin, alginates, chondroitin sulfate, chitosan, chitin, keratin, silk, small intestine submucosa (SIS), or blends thereof.

14. The method of claim 1, wherein said cells have at least one marker characteristic of a pancreatic cell.

15. The method of claim 14, wherein said pancreatic cell is selected from an alpha cell, a beta cell, a delta cell, a PP cell, an ancinar cell or a ductal cell.

16. The method of claim 14, wherein said cells are islets isolated from a mammal.

17. The method of claim 16, wherein said mammal is selected from a primate, porcine, canine, or murine.

18. The method of claim 14, wherein said cells are engineered in vi tro .

19. The method of claim 1, wherein said scaffold is placed in a plate suitable for centrifugation prior to the addition of the cells.

20. The method of claim 19, wherein said plate is designed to allow easy fit into a holder within a centrifuge chamber, and have wells of a diameter made to fit said scaffold and of a height suitable for containing a liquid suspension of said cells.

21. The method of claim 20, wherein said plate has wells with a diameter of about 4.8 mm and a height of about 0.78 mm, and wherein said plate fits into a holder of a standard 96-well culture plate within a centrifuge chamber.

22. The method of claim 21, wherein said plate is an expanded polytetrafluoro-ethylene (ePTFE) plate.

23. The method of claim 1, wherein the centrifugation is performed at about 100-700 g.

24. The method of claim 23, wherein the centrifugation is performed at about 200 g.

25. The method of claim 1, wherein the centrifugation is performed for about 2-5 minutes.

26. The method of claim 25, wherein the centrifugation is performed for about 3 minutes.

27. The method of claim 1, wherein the centrifugation is performed in a Beck an Coulter Allegra 6 series centrifuge with a GH3.8 rotor at a speed of 800-2000 rpm.

28. A kit for seeding cells onto a biocompatible scaffold, comprising said biocompatible scaffold, at least two identical plates, wherein said plates are designed to allow easy fit into a plate holder within a centrifuge chamber, and to have wells of a diameter to fit said scaffold and of a height suitable for containing a liquid suspension of said cells.

29. The kit of claim 28 wherein said scaffold is a nonwoven scaffold or a composite scaffold comprising a nonwoven component and a foam component .

30. The kit of claim 29 wherein said nonwoven scaffold or said nonwoven component of said composite scaffold is formed from a polymer selected from polylacetic acid (PLA) , polyglycolic acid (PGA), polycaprolactone (PCL) , polydioxanone (PDO), trimethylene carbonate (TMC) , polyvinyl alcohol (PVA) , or polyoxaester, or a copolymer or blend thereof.

31. The kit of claim 29 wherein said nonwoven scaffold or said nonwoven component of said composite scaffold is formed from a 90/10 copolymer of PGA/PLA.

32. The kit of claim 31wherein said nonwoven scaffold is a VICRYL scaffold.

33. The kit of claim 29 wherein said nonwoven scaffold or said nonwoven component of said composite scaffold is formed from a 95/5 copolymer of PLA/PGA.

34. The kit of claim 33 wherein said nonwoven scaffold is a PANACRYL scaffold.

35. The kit of claim 29 wherein said nonwoven scaffold or said nonwoven component of said composite is made of a 100% homopolymer of polydioxanone.

36. The kit of claim 35 wherein said nonwoven scaffold is a PDS II scaffold.

37. The kit of claim 29 wherein said foam component is prepared from a 65/35 PGA/PCL copolymer, a 60/40 PLA/PCL copolymer, or a blend thereof.

38. The kit of claim 29 wherein said scaffold has a porosity of 70-95%.

39. The kit of claim 28, wherein said scaffold is a biopolymer.

40. The kit of claim 39, wherein said biopolymer is selected from yaluronic acid, collagen, recombinant collagen, cellulose, elastin, alginates, chondroitin sulfate, chitosan, chitin, keratin, silk, small intestine submucosa (SIS), or blends thereof.

41. The kit of claim 28, wherein said plates have wells with a diameter of about 4.8 mm and a height of about 0.78 mm, and wherein said plates fit into a holder of a standard 96-well culture plate within a centrifuge chamber.

42. The kit of claim 41, wherein said plates are expanded tubular polytetrafluoro-ethylene (ePTFE) plates.

3. The kit of claim 37, wherein said centrifuge is a Beckman Coulter Allegra 6 series centrifuge or a similar centrifuge.