WO2009006600A2 - Density gradient isolation of pancreatic islet cells - Google Patents

Abstract

Methods of isolating cells or clusters of cells from stromal components following enzymatic treatment and/or physical disruption of an organ or tissue using density gradients and kits for isolating such cells are disclosed. Cells or clusters of cells, in particular, pancreatic islet cells, produced by these methods, and methods of using said cells for transplantation are also disclosed.

Description

DENSITY GRADIENT ISOLATION OF PANCREATIC ISLET CELLS

[0001] The invention was made with U.S. Government support under National Institute of Health (NIH) grant RFA-RR 05-003. The U.S. Government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Field of the Invention

[0002] The application relates to the field of cell biology. In particular, the application relates to solutions, cells, methods, kits, and processes useful for the isolation, culture and transplantation of cells and tissues.

Description of Related Art

[0003] The transplantation of cells, tissues, and organs holds great promise for the treatment of many diseases. For example, pancreatic islet transplantation can reverse insulin-dependent diabetes, therapeutically delivered mesenchymal stem cells can improve heart function after an acute myocardial infarction (Scheuleri et al., 2007, Handb Exp Pharmacol 180:195-218), implantation of bone marrow mononuclear cells (BM-MNC) into ischemic tissue can improve tissue vascularization (Hernandez P et al., 2006, Atherosclerosis, Sep 15, Epub ahead of print; Zenovich AG et al., 2007, Handb Exp Pharmacol, 180: 117-65), and transplantation of hematopoietic stem cells can resolve multiple sclerosis (Portaccio E et al., 2007, Mult Scler, 13(5): 676-8). [0004] With respect to pancreatic islets transplantation, unfortunately, the procedure is hampered by a short supply of islets and a gradual loss of islet function after transplantation. The inconsistency of islet isolation outcomes has been a major limitation to widespread clinical application of islet transplantation. The following U.S. Patents describe known methods of isolating, culturing, and/or transplanting pancreatic islets: 6,506,599, 6,562,620, 6,783,964, and 6,815,203.

[0005] Current methods for isolating mammalian cells for transplantation are not highly efficient. Despite significant advancements in human pancreatic islet isolation techniques, islet yield remains highly variable and unpredictable (Weir et al., 1990, J Clin Invest. 85: 983-7). Purification of cells liberated from tissues after successful enzymatic treatment is a crucial step to ensure a good recovery of a sufficient number of viable pancreatic islet cells for transplantation. Density gradient centrifugation has been attempted as an islet purification method, but the method gave variable reported outcome. Moreover, the specific details of certain of these methods are not readily available {see, for example, Matsumoto et al, 2006, Transplantation 82: 460-5; Huang et al, 2004, Transplantation 77: 143-145). One such method known in the art uses a continuous density gradient of Biocoll (a polymeric substance commercially available from Biochrome, Berlin, DE). However, the conventional density gradient protocol suffers from several disadvantages, for example, the pancreatic islet cells so obtained are not sufficiently pure for transplantation, the permitted sample volume in the gradient is often too small, and the concentration of Biocoll used in the gradient is often toxic to pancreatic islet cells. Thus, there is a need for better and more efficient techniques for isolation of mammalian cells.

SUMMARY OF THE INVENTION

[0006] This invention provides methods and reagents for density gradient isolation and purification of cells liberated by enzymatic treatment. In particular, the invention provides methods for isolating pancreatic islet cells from enzyme-treated pancreata. Specifically, the methods of the invention provides Biocoll density gradient and methods for using such gradients in centrifugation to purify pancreatic islet cells derived from enzyme -treated pancreata away from stromal components. Preferably, the gradient is a narrow gradient prepared using a mixture of Biocoll and University of Wisconsin (UW) solution or equivalent solution for preserving cells and tissue for transplantation. [0007] In one aspect, the invention provides methods of isolating pancreatic islet cells from a mammal, comprising (a) treating pancreatic tissue from the mammal with one or a plurality of enzymes for producing unpurified pancreatic islet cells therefrom; (b) collecting the unpurified pancreatic islet cells from the tissue treated with the enzyme; (c) incubating the unpurified pancreatic islet cells in a tissue preservation solution; and (d) subjecting the unpurified pancreatic islet cells to a continuous density gradient to isolate pancreatic islet cells, wherein the density gradient is formed in a mixture of Biocoll and the tissue preservation solution, and wherein the density gradient formed has a density range of from about 1.06 g/mL to about 1.08 g/mL. In preferred embodiments, the mammal is a human, and the density gradient has a density range from about 1.068 g/mL to about 1.079 g/mL. In certain embodiments, the unpurified pancreatic islet cells are collected by spinning down the cells and resuspending the cells in the tissue preservation solution. Advantageously, in certain embodiments, the tissue preservation solution is UW solution. In certain embodiments, the unpurified pancreatic islet cells are isolated by centrifugation in the density gradient at 3000 rpm for 5 min.

[0008] In certain embodiments, pancreatic tissues are treated with an enzyme, most preferably a protease such as collagenase, to liberate islet cells from associating stromal components. Advantageously, said treatment is performed using cell culture media, such as RPMI or CMRL or other commercially-available or proprietary media. In certain embodiments, pancreatic tissues are treated with an enzyme in the presence of UW solution. [0009] In certain embodiments, included in the pancreas treatment and isolation solutions are hemoglobin-based O₂ carriers (HBOCs) that are useful in delaying or reducing ischemia in the isolated islets, as set forth in more detail in International Application No. PCT/US2007/60987, published as WO 2008/021577, which is incorporated herein by reference in its entirety. In certain advantageous embodiments, the pancreatic tissue from the mammal is incubated in the presence of polymerized hemoglobin at 4⁰C prior to enzymatic treatment and at 37⁰C during enzymatic treatment. In certain embodiments, the polymerized hemoglobin is derived from human blood. In preferred embodiments, the polymerized hemoglobin is pyridoxylated.

[0010] In certain embodiments of this aspect, the methods further comprise the step of spinning down the unpurified pancreatic islet cells after enzymatic treatment, resuspending the cells in a tissue preservation solution, such as UW solution, before incubating the unpurified pancreatic islet cells in the tissue preservation solution. In preferred embodiments, the unpurified pancreatic islets are incubated in UW solution for at least 30 minutes. In certain embodiments, the methods further comprise the step of separating the unpurified pancreatic islet cells by gentle physical dissociation. In certain embodiments, the step of gentle physical dissociation occurs after the enzymatic treatment step, before, during or after the incubation with the tissue preservation solution step, or before the step of subjecting the unpurified pancreatic islet cells to the density gradient. In preferred embodiments, the tissue preservation solution is UW solution.

[0011] In another aspect, the invention provides purified pancreatic islet cells produced according to the methods of the invention. In a further aspect, the invention provides methods for treating a mammal that suffers from a defect, disorder, disease or deficiency of pancreatic islet cells, comprising a step of administering to the mammal pancreatic islet cells purified according to the method of the invention. In certain embodiments, the defect, disorder, disease or deficiency of pancreatic islet cells is diabetes mellitus. In preferred embodiments, the mammal is a human.

[0012] In another aspect, the invention provides methods for treating a mammal that suffers from a defect, disorder, disease or deficiency of pancreatic islet cells, comprising administering to the animal in need of such treatment a pharmaceutical composition or a cellular preparation of a therapeutically effective amount of the purified pancreatic islet cells of the invention. In certain embodiments, the methods of the invention for treating a mammal having a defect or deficiency of pancreatic islet cells comprise administering to the animal in need of such treatment a pharmaceutical composition of a therapeutically effective amount of purified pancreatic islet cells and at least one pharmaceutically-acceptable carrier, excipient or diluent. In preferred embodiments, the mammal is a human. [0013] In yet another aspect, the invention provides purified pancreatic islet cells for use in therapy in treating a mammal having a defect, disorder, disease or deficiency of pancreatic islet cells. In a further aspect, the invention provides methods for using the purified pancreatic islet cells to prepare a pharmaceutical composition or formulation for treating a defect or deficit of pancreatic islet cells in a mammal in need thereof. In preferred embodiments, the mammal is a human.

[0014] In yet another aspect, the invention provides pharmaceutical compositions comprising a therapeutic effective amount of purified pancreatic islet cells of the invention and at least one pharmaceutically acceptable excipient, diluent or carrier. In a further aspect, the invention provides compositions comprising pancreatic islet cells purified according to the methods of the invention and at least one pharmaceutically acceptable excipient, diluent or carrier.

[0015] In another aspect, the invention provides kits for isolating pancreatic islet cells comprising (a) a first Biocoll and a tissue preservation solution gradient mixture having a density of about 1.06 g/mL; and (b) a second Biocoll and a tissue preservation solution gradient mixture having a density of about 1.08 g/mL. In preferred embodiments, the first Biocoll and UW solution gradient mixture has a density of about 1.068 g/mL and the second Biocoll and UW solution gradient mixture has a density of about 1.079 g/mL. In most preferred embodiments, the tissue preservation solution is UW solution. In certain embodiments, the kit further comprises at least one enzyme that is capable of digesting pancreatic tissue and liberating pancreatic islet cells from the associating stromal components. In certain embodiments, the enzyme is a protease, preferably a collagenase. In yet other embodiments, the kit further comprises polymerized hemoglobin. [0016] Specific preferred embodiments of the invention will become evident from the following more detailed description of certain preferred embodiments and the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 shows the viability of islets from both groups expressed in percentages

24 hours after isolation, represented as means±SEM. PoIySFH-P isolations (n=9) Control isolations (n=9) for each group. *p=0.047.

[0018] Figure 2 shows caspase-3 levels measured in islets from PoIySFH-P and control groups 24 hrs after isolation as a marker for apoptosis, n=3 isolations per group. Caspase-3 levels are significantly lower in the PoIySFH-P group than in the control. *p=0.011.

[0019] Figure 3 A shows changes in ratio-metric values (Fura 2/AM) as a measurement of intracellular calcium levels in two representative islets under basal (2 mM) and stimulated

(5, 8, or 14 mM) glucose conditions.

[0020] Figure 3B shows the percentage of intracellular calcium change in response to glucose stimulation (5, 8 or 14 mM glucose concentrations) in islets from PoIySFH-P and control groups, (n=25 islets per group), mean±SEM. *p<0.05.

[0021] Figure 4 A shows changes in ratio-metric values (Fura 2/AM) as a measurement of intracellular calcium levels in two representative islets under basal glucose (2 mM) conditions after the addition of Tolbutamide (100 μM).

[0022] Figure 4B shows the area under the curve (AUC) for intracellular calcium levels under basal glucose concentration (2 mM) in islets from both groups after the addition of

Tolbutamide (100 μM). (n=25 islets per group), represented as mean±SEM. p=0.183.

[0023] Figure 5 shows insulin secretion of islets in response to glucose challenge, expressed as a stimulation index (SI), represented as mean±SEM. PoIySFH-P isolations

(n=5), control isolations (n=5), *p=0.03.

[0024] Figure 6A shows levels of Rhodamine 123 (Rhl23)-fluorescence outside the mitochondrial inner membrane in two representative islets under basal (2 mM) and glucose- stimulated conditions (14 niM). A gradual decrease in fluorescence represents the incorporation of Rh 123 into the membrane as an indirect measurement of membrane potentials.

[0025] Figure 6B shows the percentage change in mitochondrial potentials in islets from

PoIySFH-P and control groups, (n=25 islets per group), represented as mean±SEM. p<0.05.

[0026] Figure 7 shows mitochondrial morphology. Mitochondria were stained with

Rh 123 dye. Two representative images (confocal reconstructions) from individual islets from PoIySFH-P and control groups are shown. Images are maximum intensity projections, lμm slice thickness. Cell nuclei in the islets are identified with the letter "n". Mitochondrial morphology and distribution around the nuclei appear superior in the PoIySFH-P group than in the control. Contrast has been balanced to reveal details of mitochondrial morphology.

Scale bar is 5 μm.

[0027] Figure 8 shows the number of days (lag time) to reach normoglycemia after islet transplantation in mice. PoIySFH-P mice (n=6) and control mice (n=4). *p=0.02.

[0028] Figure 9 shows the results of an Intraperitoneal Glucose/ Arginine Tolerance Test

(IPG/ ATT) in a representative sample of mice that reached normoglycemia after transplantation with islets from PoIySFH-P (n=5) and control (n=3). Values indicate mean blood glucose levels ±SEM. p=0.03.

[0029] Figure 10 is a graph showing viability staining specific for beta and non-beta cells from isolated islet cell populations. Cells were assayed for cell membrane stability

(7aaD), mitochondrial membrane stability (TMRE) in beta cells (gating the Newport Green

(NG) high population) versus non-beta cell (NG low population), n=3 per group. *p< 0.001;

** /K0.001; t/XO.OOl; tt/XO.OOl.

[0030] Figures HA through HD are graphs showing purity and tissue volume distribution in each fraction after purification using conventional methods (SM) (Figure

1 IA) or the inventive methods (UIC-UB; Figure 1 IB). Figures 11C and 1 ID show the purity distribution based on the density of each fraction for both SM and UIC-UB gradient groups, respectively. Power lines in Figures 11C and 1 ID are theoretical lines fitted to the density lines (the least squares fit through the experimental data points). DETAILED DESCRIPTION OF THE INVENTION

[0031] This invention provides methods for isolating cells or collections of cells that have been liberated from stromal components of organs or tissues. The cells or collections of cells can be liberated from stromal components using physical or more preferably chemical methods, including but not limited to enzymatic treatment. In particular embodiments, the invention provides methods for isolating pancreatic islet cells from enzyme-treated pancreata. Preferred enzymes include without limitation proteases. [0032] In one aspect, the invention provides methods for isolating pancreatic islet cells by using modified and improved Biocoll density gradient centrifugation, particularly with regard to the composition and density range of the gradient and the specifics concerning the handling of enzyme-treated tissues as set forth herein. The inventive methods are illustrated by improved separation of islet cells from stromal components of enzyme -treated pancreata as compared to the isolated islets from methods known in the art, wherein the isolated islets show superior yields, purity, and viability. Additional advantages include higher digested tissue volume capacity of the gradient, which reduces the processing time and ischemic insult to the cells as well as resulting in higher yield. In addition, incubation in a tissue preservation solution, preferably UW solution, allows a better distinction between the density of islets and exocrine tissue. Without being limited to a certain mechanism, incubation in a tissue preservation solution prevents cell swelling of the exocrine tissue, thus preserves the distinction in density between pancreatic islet cells and other exocrine tissue. [0033] As used herein, the term "pancreatic islets", "islets", "islet cells", and "pancreatic islet cells" are used interchangeably to refer to endocrine cells of the pancreas located and grouped in the islets of Langerhans.

[0034] As used herein, the term "unpurified pancreatic islet cells" refers to the pancreatic islets cells liberated from pancreatic tissue by enzymatic treatment before purification by density gradient centrifugation. In preferred embodiments, the unpurified pancreatic islet cells are spun down and resuspended in UW solution before loading onto the density gradient. In certain embodiments, the unpurified pancreatic islet cells may further comprise polymerized hemoglobin and/or UW solution.

[0035] The term "purified pancreatic islet cells" as used herein refers to isolated or enriched pancreatic islet cells that are substantially free of contaminating cells or tissues such as stromal components. [0036] A tissue preservation solution refers to a solution used for preserving the viability of organs, tissues, or cells for transplantation, the use of such solution allows prolonged tolerable ischemic time. A number of transplantation preservation solutions are known in the art. See, e.g., P. Michel et al, 2002, J. Heart Lung Transplant. 21(9): 1030-39. More than ten different preservation solutions are already in clinical use, including UW solution, histidine-tryptophan-ketoglutarate (e.g., "HTK" "Bretschneider's" or "CUSTODIOL®") solution, Stanford ("STF") solution, and Eurocollins ("EC") solution. See, e.g., G. M. Collins, 1997, Transplant. Proc. 29:3543-44, D. G. Stein et al., 1991, J. Thorac. Cardiovasc. Surg. 102:657-665, and D.T. Hsu et al., 1993, J. Thorac. Cardiovasc. Surg. 106:651-657. In most preferred embodiments, a tissue preservation solution is UW solution. [0037] In certain aspects, the invention provides a modified density gradients for isolating pancreatic islet cells, methods for preparing and using the gradients, and kits for isolating pancreatic islet cells comprising the solutions for preparing the gradient. In other aspects, the invention provides purified pancreatic islet cells isolated using the inventive methods, and methods for using the purified pancreatic islet cells for transplantation. [0038] Specifically, the inventive methods provide Biocoll gradients having a different density range than methods known in the art, with improved yield, purity, and viability of the isolated islets. In certain embodiments, the gradient is prepared using commercially- available Biocoll and a tissue preservation solution. In an advantageous embodiment, the inventive gradients are prepared using Biocoll and University of Wisconsin (UW) solution, the composition of which is known in the art (for example, see Belzer et al., 1988, Transplantation 45: 673-6).

[0039] The term "density range" as used herein refers to the density differential from the lightest to the heaviest density points in a density gradient. In certain embodiments of the invention, the density gradient has a density range of from about 1.06 g/mL to about 1.08 g/mL, preferably, from about 1.068 g/mL to about 1.079 g/mL. In another embodiment, the density gradient has a density range of from about 1.062 g/mL to about 1.073 g/mL. [0040] As set forth herein, the composition of the gradient used produces a gradient density range that is "shallower" or "narrower" (i.e., does not change as greatly per unit length of the gradient) than previously-attempted gradients using Biocoll. It has been the understanding in the field that a density gradient with a wide or broad density range should be used for islet cells purification to recover cells with different density. It was unexpectedly discovered by the Applicant that a narrower or shallower gradient, in which each density fraction contains a larger volume provides better separation results. In certain embodiments, the density range of the gradient has a density differential, i.e., the difference in density between the lightest and the heaviest density points in the gradient, of about 0.02 g/mL. In preferred embodiments, the density range of the gradient has a density differential of about 0.015 g/mL, more preferably 0.011 g/mL, most preferably not more than 0.011 g/mL. In addition, the gradients of the invention have a lower viscosity. It is unexpectedly discovered that not only did the inventive density gradient with a narrower density range provide better separation of pancreatic islet cells from stromal components, the inventive gradients, because of the overall lower concentrations of Biocoll in the gradients, greatly reduced Biocoll- associated toxicity to the pancreatic islet cells. Moreover, the inventive gradients are advantageously prepared using UW solution, which serves to preserve isolated cells and tissues for transplantation.

[0041] University of Wisconsin (UW) solution, also known as VIASP AN^®, was a tissue or organ preservation solution designed for use in organ transplantation. UW solution is commercially available from, for example, DuPont Pharma, Bad Homburg, Germany and further described in Salehi et al. (Transplantation, 2006, 82: 983-985). The composition of UW solution used in the invention is shown below: Potassium lactobionate: 100 mM, KH₂PO₄: 25 mM, MgSO₄: 5 mM, Raffϊnose: 30 mM, Adenosine: 5 mM, Glutathine: 3 mM, Allopurinol: 1 mM, Hydroxyethyl starch: 50 g/L. The final values are shown below: Na = 25 +/- 5 mM; K = 120 +/- 5 mM; mOsm/L = 320 +/- 10.

[0042] As disclosed herein, the density gradient of the invention having a narrower density range provided markedly better separation of islets from exocrine tissue. The density of an intact islet is approximately 1.070 g/cm , whereas the density of surrounding acinar tissue is approximately 1.10 g/ cm (according to Eckhard et al, 2004, Transplantation Proc. 36: 2849-54). A shallower gradient as provided herein was shown to increase discrimination between the islets and extraneous tissues co-digested from the organ during preparation. It is particularly advantageous to use a preservation solution such as UW solution in the gradient. Moreover, it is advantageous to incubate the unpurifϊed islets derived from the enzyme- treated pancreata in UW solution prior to density gradient centrifugation, the incubation resulting in higher yield of islet cells. This beneficial effect of the inventive gradient and method of using such a gradient was especially significant in cases where the tissue is subjected to prolonged cold ischemia time, which is associated with exposure to proteolytic enzymes and other stressors during isolation. Such insults can result in pancreatic edema and cell swelling (as discussed in Pipeleers et al, 1984, Islet cell purification, in: J. Lamer and S. Pohl, Editors, Methods in Diabetic Research. Laboratory methods. Part A, Wiley, New York (1984), p.185.). Cell swelling has been suggested as the main reason for exocrine contamination of isolated islet cells because acinar tissue can migrate to lower density layers during continuous gradient purification as a result of cell swelling (Eckhard et al., 2004, Transplantation Proc. 36: 2849-54; London et al., 1995, Adult islet purification. In: Ricordi, C. ed. Methods in Cell Transplantation. Austin, TX: RG Landes, 1995, pp. 439- 454). [0043] Generally, the methods disclosed herein are practiced by loading unpurified pancreatic islets (produced^or example by proteolytic digestion as set forth herein), preferably comprising UW solution, onto the gradient. In certain preferred embodiments, the enzyme-treated unpurified pancreatic islet cells are spun down, suspended in 150 ml of UW solution. The unpurified pancreatic islet cells are incubated in the UW solution, preferably for at least 30 min before loading onto the density gradient of the invention. [0044] The combination of Biocoll gradient separation using UW solution has been recently reported by Huang et al. (2004, Transplantation 77: 143-145). However, in this method conventional Biocoll gradient centrifugation was used rather than the modified gradient disclosed herein. Additional distinctions include pre-application of the Biocoll mixtures used to form the gradient, followed by application of unpurified islet cell suspensions and centrifugation. Moreover, the inventive gradients disclosed herein can be used to purify larger amounts of the unpurified islet cells. Packed tissue volume up to 50 mL or more can be purified in one gradient of the invention. The methods of the instant invention are also advantageous because the higher sample volume capacity minimizes the volume of gradient required and shortens the isolation process time. In general only one gradient run will be necessary during one human islet isolation process, as compare to two to four with the conventional method. Consequently, the cold ischemia time is reduced using the inventive methods, which increases islet cell viability.

[0045] Advantageously, in certain embodiments the invention provides methods for isolating cells from tissues wherein the enzymatic treatment solution further contains polymerized hemoglobin (such as PoIySFH-P) to reduce ischemia during isolation of cells, particularly pancreatic islet cells. The descriptions of polymerized hemoglobin are set forth herein and in International Application No. PCT/US2007/60987, which is incorporated herein by reference in its entirety. [0046] Maintaining an appropriate O₂ level is important to prevent ischemic damage and reperfusion injury during organ preservation, pancreatic islet isolation, and cell culture. Indeed, artificial oxygen carriers, such as perfluorocarbons (PFC), have a beneficial effect on islet isolation and transplantation outcomes when used during pancreas preservation with UW solution in the two layer method (TLM). Artificial oxygen carriers are synthetic solutions capable of binding, transporting and unloading O₂. Artificial oxygen carriers have been originally developed as blood substitutes, but none of the PFC based products have been approved for clinical use, and in clinical trials anaphylactic reactions were observed. Moreover, PFCs have the inconvenience of being hydrophobic and difficult to keep in aqueous solution.

[0047] Hemoglobin-based O₂ carriers (HBOCs), such as PoIySFH-P (polymerized stroma-free hemoglobin pyridoxylated, also known as POLYHEME^®), are water soluble. No anaphylactic reactions have been observed in phase I and II trials of POLYHEME^®. U.S. Patent No. 6,498,141, which is hereby incorporated by reference in its entirety, describes the preparation of representative HBOCs. In contrast to PFC, PoIySFH-P gives an O₂ saturation curve similar to that of red blood cells. PoIySFH-P is essentially tetramer-free (thereby eliminating certain biological responses to contaminating tetramer), substantially stroma- free, polymerized, and pyridoxylated hemoglobin derived from human blood. [0048] As used herein, the term "hemoglobin" refers to hemoglobin from mammals (preferably bovine, ovine, or human hemoglobin), synthetic hemoglobin, hemoglobin obtained by transgenic means, hemoglobin obtained from cell lines that naturally produce or have been manipulated to produce hemoglobin in vitro, hemoglobins obtained in mutant form, and chemically modified forms of hemoglobin. In preferred embodiments, the hemoglobin is human hemoglobin. The hemoglobin of the invention comprises hemoglobin species including but not limited to Hemoglobin A, (α₂β₂,), Hemoglobin A2, (α₂δ₂) and fetal hemoglobin (α₂γ₂), as well as mixtures thereof.

[0049] As used herein, the phrase "polymerized hemoglobin" refers to hemoglobin that has been polymerized so that it can serve as a physiologically competent oxygen carrier, wherein the placement of molecular bridges between molecules or tetrameric subunits of the hemoglobin results in the increased size and weight of the resulting polymerized molecule with respect to native or tetrameric hemoglobin. Polymerized hemoglobin can absorb oxygen at the partial pressures of oxygen prevailing at the site of oxygenation of hemoglobin, for example, in the lungs of humans, and release the bound oxygen to the tissues of the same organisms in amounts that are life supporting. Polymerized hemoglobins can be obtained, for example, by treatment with glutaraldehyde or raffϊnose, as discussed in U.S. Patent No. 5,998,361, which is hereby incorporated by reference. Polymerized hemoglobins are also described, for example, in U.S. Patent No. 6,498,141, which is hereby incorporated by reference.

[0050] In certain embodiments, the polymerized hemoglobin derived from human blood is pyridoxylated. Pyridoxylation may be used to modulate the oxygen half-saturation pressure (P-50) of the polymerized hemoglobin to a desirable range. For example, when P- 50 of the solution containing the polymerized hemoglobin is desired to be within the range of normal human blood, the hemoglobin derived from human blood is preferably pyridoxylated, as described in U.S. Patent No. 6,498,141.

[0051] The use of solution containing polymerized hemoglobin to preserve and maintain viability of cells or tissues has been described in PCT application PCT/US2007/60987. Preferred solutions containing polymerized hemoglobin are aqueous and are formulated to contain from about 5-15 g/dL of polymerized hemoglobin, more preferably from about 8-12 g/dL of polymerized hemoglobin, and most preferably from about 9-11 g/dL of polymerized hemoglobin. Particularly preferred solutions contain about 10 g/dL of polymerized hemoglobin. In certain embodiments, the solutions containing polymerized hemoglobin are formulated to have a pH of from about 7-8, more preferably from about 7.5-7.9, most preferably from about 13-1.6.

[0052] Polymerized hemoglobin containing solution described above preferably contains from about 0.5X to 2X of cell culture medium (where IX medium is a concentration equivalent to IX RPMI). More preferred polymerized hemoglobin/cell culture medium solutions contain about IX cell culture medium. In certain embodiments, the polymerized hemoglobin/cell culture medium further contains UW solution.

[0053] "Cell culture medium," as used herein, refers to a medium suitable for the culture, maintenance, proliferation, and/or growth of cells in vitro. Examples of cell culture media that can be used are disclosed in U.S. Patent Nos. 6,670,180 and 6,730,315, which are incorporated by reference. One of skill in the art will recognize that the type of cell culture media useful in a solution of the invention can be selected based on the type of cell, tissue, and or organ for which the solution is to be used. For example, where the cells are pancreatic islets, the cell culture medium can be RPMI, as described herein. Alternative cell culture media, including Eagles Minimal Media, Dulbecco's Modified Eagle's Media, and others known to those with skill in the art, are commercially available (for example, from GIBCO, Long Island, NY and Sigma Chemical Co., St; Louis. MO) and fall within the scope of components of the invention set forth herein.

[0054] Solutions of polymerized hemoglobin and an enzyme, preferably a protease such as, for example, collagenase or liberase, are formulated to contain the above amounts of hemoglobin and from about 0.1 - 10 mg/mL of the enzyme. Preferred solutions are formulated to contain from about 0.5 - 5 mg/mL of enzyme, more preferably from about 0.75 - 1.25 mg/mL of enzyme. Particularly preferred solutions contain about 1 mg/mL of enzyme.

[0055] Solutions of polymerized hemoglobin, cell culture medium, and enzyme are formulated to contain the amounts of these components described above and within the above-recited pH ranges. The solutions and suspensions can be prepared by mixing the components thereof. Oxygenating the solutions and suspensions can be achieved, for example, by bubbling 100% O₂ gas through the solutions and suspensions for a sufficient period of time, or by otherwise contacting the solutions and suspensions with O₂ gas. [0056] "Buffer," as used herein, refers to a system, such as a solution, that acts to minimize the change in concentration of a specific chemical species in solution against addition or depletion of the species, particularly with regard to the hydrogen ion concentration (pH) of the solution. Examples of buffers are well-known to those of skill in the art.

[0057] The term "enzymatic treatment" of pancreatic tissue as used herein comprises enzymatic digestion of the tissue. In preferred embodiments, the enzyme is a protease. [0058] "Protease" (or "proteolytic enzyme"), as used herein, refers to an enzyme that cleaves peptide bonds in a protein. Non-limiting examples of proteases suitable for use in the invention include trypsin, chymotrypsin, pepsin, furin, dispace, thermolysin, elastase, and mixtures thereof such as pancreatin and liberase (a purified enzyme blend of collagenase isoforms I and II from Clostridium histoliticum and thermolysin from Bacillus thermoproteolyticus).

[0059] "Enzymatically produced," as used herein, refers to the action of an enzyme in the presence or absence of polymerized hemoglobin according to the invention, particularly proteolytic enzymes useful in digesting extracellular matrix proteins and other proteins involved in maintaining the integrity of a tissue or organ in vivo. [0060] The term "stromal component" as used herein refers to the connective, nonfunctional supportive framework of a cell, tissue or organ.

[0061] In another aspect, the invention provides a composition, pharmaceutical composition or a cellular preparation comprising a therapeutic effective amount of the pancreatic islet cells prepared according to the inventive density gradient. The cellular preparations and pharmaceutical compositions of the invention may contain formulation materials for modifying, maintaining, or preserving, in a manner that does not hinder the physiological function and viability of the pancreatic islet cells obtained according to the method of the invention, for example, pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobial compounds, antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, betacyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as serum albumin, gelatin, or immunoglobulins), coloring, flavoring and diluting agents, emulsifying agents, hydrophilic polymers (such as polyvinylpyrrolidone), low molecular weight polypeptides, salt-forming counterions (such as sodium), preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, or hydrogen peroxide), solvents (such as glycerin, propylene glycol, or polyethylene glycol), sugar alcohols (such as mannitol or sorbitol), suspending agents, surfactants or wetting agents (such as pluronics; PEG; sorbitan esters; polysorbates such as polysorbate 20 or polysorbate 80; triton; trimethamine; lecithin; cholesterol or tyloxapal), stability enhancing agents (such as sucrose or sorbitol), tonicity enhancing agents (such as alkali metal halides—preferably sodium or potassium chloride—or mannitol sorbitol), delivery vehicles, diluents, excipients and/or pharmaceutical adjuvants. See REMINGTON'S PHARMACEUTICAL SCIENCES (18th Ed., A. R. Gennaro, ed., Mack Publishing Company 1990).

[0062] The primary vehicle or carrier in a pharmaceutical composition may be aqueous in nature. For example, a suitable vehicle or carrier for injection may be physiological saline solution. Optimal pharmaceutical compositions can be determined by a skilled artisan depending upon, for example, the intended route of administration, delivery format, desired dosage and recipient tissue. See, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra. Such compositions may influence the physical state, stability, and effectiveness of the composition.

[0063] The following Examples are illustrative of specific embodiments of the invention, and various uses thereof. They set forth for explanatory purposes only, and are not to be taken as limiting the invention.

EXAMPLE 1 PREPARATION OF POLYMERIZED HEMOGLOBIN SOLUTION

[0064] In vitro culture media containing collagenase with or without the addition of PoIySFH-P polymerized hemoglobin were prepared as follows. A solution containing 10 g/dL PoIySFH-P formulated with RPMI 1640 cell culture medium ("PolySFH-P/RPMI solution") was prepared by Northfϊeld Industries (Evanston, Illinois) for islet isolation. PolySFH-P/RPMI solution was prepared by modifying the procedure described in Example 1 ofU.S. Patent No. 6,498,141. More specifically, Example 1 ofU.S. Patent No. 6,498,141 was followed from the beginning through the step at Tank 8. Starting at Tank 9, the procedure was as follows. Polymerized hemoglobin derived from human blood (PoIySFH- P) was concentrated to about 7 g/dL and the pH of the solution was adjusted to between 7.30 and 7.60 with 0.1 M HCl. This solution was concentrated to 12 g/dL PoIySFH-P. A sufficient amount of 1OX RPMI solution containing 2.5 g/L ascorbic acid and water for injection ("WFI") was added to produce a final PolySFH-P/RPMI solution containing 10 g/dL PoIySFH-P, IX RPMI, and 0.25 g/L ascorbic acid. The pH of the PolySFH-P/RPMI solution was verified to be between 7.30 and 7.60. PolySFH-P/RPMI solution was then sterile filtered and 250 mL were transferred aseptically into 500 mL bags. Bags were filled only half-full to allow for simplified oxygenation of the solution (within the bag) at the time of use. Filled bags were stored at 2-8⁰C.

[0065] 1OX RPMI solution containing 2.5 g/L ascorbic acid was prepared as follows. RPMI 1640 powder without NaHCO₃, phenol red and L-Glutamine, obtained from Cellgro (Mediatech, Herndon, VA), was added to water for injection to obtain a concentration 10 times as concentrated as IX RPMI 1640 (see below). 7.5% NaHCO₃, obtained from Invitrogen (Carlsbad, CA), was added to obtain a concentration of 267 mL/L. 200 mM L- Glutamine, received as a frozen solution from Invitrogen, was thawed and added to obtain a concentration of 102.5 mL/L. In addition, ascorbic acid was added to obtain a final concentration of 2.5 g/L.

EXAMPLE 2

[0066] 2A. Experiments were conducted to determine the rate of oxygenation and conversion of PoIySFH-P polymerized hemoglobin to methemoglobin during oxygenation and holding at 37⁰C . As a control, 10OmL samples of PoIySFH-P polymerized hemoglobin are oxygenated utilizing compressed air (21⁰ZoO₂) or compressed oxygen (99.4⁰ZoO₂) to at least 85% oxyhemoglobin (O₂Hb). The percent oxygen saturation can be measured by cooximetry such as that employed by Instrumentation Laboratories (Lexington, MA) IL-482 or IL-682. The PoIySFH-P polymerized hemoglobin samples are then heated to 37⁰C and held at this temperature for at least 20 minutes. After the 20-minute hold period, samples are tested utilizing cooximetry to determine the amount of methemoglobin (MetHb) conversion. [0067] Cooximetry Results:

TABLE I A- Oxygenation of PoIySFH-P polymerized hemoglobin (using compressed air/21% O₂, 8 standard cubic feet/hour)

TABLE I B- Oxygenation of PoIySFH-P polymerized hemoglobin (using compressed gas/99.4 % O₂)

[0068] Approximately 45 minutes were required to oxygenate 10OmL PoIySFH-P polymerized hemoglobin (using compressed air comprising about 21% O₂) to at least 85% O₂Hb; alternatively, 10OmL PoIySFH-P could be oxygenated to at least 85% O₂Hb in approximately 15 minutes using compressed oxygen (99.4% O₂) . The results shown above established that the process of oxygenating PoIySFH-P polymerized hemoglobin did not lead to significant conversion of hemoglobin to the Met Hb form. However, the method used for oxygenation did affect the percent Met Hb formed once PoIySFH-P polymerized hemoglobin was heated to 37⁰C. The first method, using compressed air, led to a higher conversion to Met Hb (17.6 %Met Hb) as compared to using compressed oxygen (9.3 %Met Hb). The amount of MetHb formed was directly proportional to the amount of time taken to oxygenate PoIySFH-P polymerized hemoglobin or the time kept at 37⁰C, or both. Despite this conversion of a small amount of the oxygenated PoIySFH-P polymerized hemoglobin to the Met Hb form, a significant amount of Hb (79.7%) remained that was capable of carrying oxygen to the islet cells. [0069] 2B. Experiments were also conducted to determine if collagenase or liberase interfered with PoIySFH-P polymerized hemoglobin or caused product degradation. [0070] For these experiments, initial samples were taken and analyzed by Cooximetry and HPLC (Size Exclusion) as reference samples. A 10OmL sample of PoIySFH-P polymerized hemoglobin at 4-8⁰C was oxygenated to at least 85.0% O₂ Hb. Cooximetry samples were then taken at approximately 15 -minute intervals during oxygenation to determine a time course of the extent of oxygenation. Once the oxyhemoglobin level was at least 85.0% O₂Hb, Cooximetry and HPLC samples were analyzed to determine impact to the product and MetHb levels. Once PoIySFH-P polymerized hemoglobin has been oxygenated, one of the enzymes to be tested (collagenase or liberase) was added to PoIySFH-P polymerized hemoglobin (lmg/lmL) at 4-8⁰C and kept at this temperature for 10 minutes. After the 10-minute at 4-8⁰C, Cooximetry and HPLC samples were evaluated for PoIySFH-P polymerized hemoglobin degradation and methemoglobin conversion. The PoIySFH-P polymerized hemoglobin/enzyme solution was heated to 37-39° and this temperature maintained for approximately 20 minutes. Cooximetry and HPLC samples were then tested for PoIySFH-P polymerized hemoglobin degradation and methemoglobin conversion. HPLC analysis was used to determine degradation of the PoIySFH-P polymers by analyzing for differences over time in the integrated areas of the peaks representing each polymeric species.

TABLE II A(I)- Oxygenation of PoIySFH-P polymerized hemoglobin

TABLE II A(2) - PoIySFH-P polymerized hemoglobin + Collagenase Enzyme

TABLE II A(3)- Integrated Area % by HPLC During Oxygenation and Collagenase

Addition

TABLE II B(I)- Oxygenation of PoIySFH-P polymerized hemoglobin

TABLE II B(2) - PoIySFH-P polymerized hemoglobin + Liberase Enzyme

TABLE II B(3)- Integrated Area % by HPLC During Oxygenation and Liberase

Addition

[0071] These consistent integrated areas for each peak demonstrated that collagenase and liberase did not interfere with PoIySFH-P polymerized hemoglobin or cause product degradation as evaluated by HPLC analysis.

[0072] 2C. Experiments were further conducted to establish the effect of RPMI on

PoIySFH-P polymerized hemoglobin to evaluate the suitability of PoIySFH-P polymerized hemoglobin-supplemented RPMI for use in pancreatic islet cell harvesting.

[0073] For the experimental study of PoIySFH-P polymerized hemoglobin with RPMI

1640, a 10OmL sample of PoIySFH-P polymerized hemoglobin at 4-8⁰C was oxygenated to at least 85.0% O₂Hb. A Cooximetry sample was evaluated for the extent of oxygenation. Once the oxyhemoglobin level was at least 85.0%, an osmolality sample was evaluated as a control. The RPMI 1640 was then added to PoIySFH-P polymerized hemoglobin (lg/lOOmL) at 4-8⁰C and thoroughly mixed to homogeneity prior to determining the osmolality of the mixture.

[0074] Because these procedures produced a hyperosmotic solution, a buffer solution of RPMI (10.10g/1.0L) was formulated. The buffer solution was used to carry out a four- volume wash (diafiltration) of the 20OmL PoIySFH-P polymerized hemoglobin. Upon completion of the diafiltration, the Cooximetry and osmolality of the sample was tested.

TABLE III A- Osmolality Results During Oxygenation and RPMI Addition

TABLE III B- Osmolality Results of RPMI Diafiltration

[0075] Addition of RPMI to PoIySFH-P polymerized hemoglobin resulted in an osmolality of 600mmol/kg. PoIySFH-P polymerized hemoglobin used with RPMI media in this fashion resulted in a hyperosmotic solution which had the potential to negatively impact islet cells. Consequently, this solution would be inappropriate for islet cell harvesting. However, when the RPMI was formulated into a buffer solution with ascorbic acid and used for diafiltration, the resulting solution of PoIySFH-P polymerized hemoglobin + RPMI had an osmolality of 268mmol/kg. With a slight adjustment to the osmolality of the solution, this mixture would be acceptable for use in islet cell harvesting. EXAMPLE 3 ISLET ISOLATION

[0076] Pancreatic islets were isolated from experimental animals (rats) using in vitro culture media containing collagenase and with or without the addition of PoIySFH-P prepared as described in Example 1. All animal procedures involving animals were performed in accordance with the guidelines of the National Institutes of Health and the Animal Care Committee (ACC) at the University of Illinois Chicago. Male Lewis rats (Harlan Industries, Indianapolis, IN), weighing between 175-200 g were used as pancreas donors for islets. Animals were anesthetized by isoflurane inhalation using a vaporizer and masks (Viking Medical, Medford Lakes, NJ.). There were 2 experimental groups: PoIySFH-P Group (PolySFH-P/RPMI solution containing collagenase, n=40 rats) and Control Group (RPMI 1640 medium containing collagenase, n= 40 rats). [0077] Rat islet isolation was performed following a conventional technique previously described in Lacy & Kostanovsky (1967, Diabetes 16:35-39), modified by using the warm ischemia model described in Avila et al. (2003, Cell Transplant 12:877-881). Briefly, after the animal was anesthetized, a laparotomy incision was performed followed by incision into the thoracic cavity and section of the heart for euthanasia by exsanguination. The abdominal cavity was closed, covered with gauze and left for 30 minutes before pancreas perfusion. [0078] Collagenase type XI (Sigma Chemical Co., St. Louis, MO) was reconstituted to a final concentration of 1 mg/mL in either PolySFH-P/RPMI solution (Treatment) or RPMI 1640 medium (Control), and both Treatment and Control were oxygenated by bubbling the solutions with 100% O₂ for 15 minutes. The effect of collagenase on the stability of polymerized hemoglobin was determined by HPLC analysis. PolySFH-P/RPMI solution was incubated with or without collagenase under different conditions, before and after oxygenation, at 4⁰C and 37⁰C. HPLC analysis did not reveal any degradation of PoIySFH-P. In addition, the formation of Methemoglobin (MetHb) and carboxyhemoglobin (COHb) was analyzed after various oxygenation times. No significant MetHb or COHb formation was found.

[0079] The oxygenated enzyme solutions were injected via the bile duct and into the main pancreatic duct for distention of the pancreas. The pancreas was then excised, and each pancreas placed in a 50 mL conical tube with 7.5 mL of its respective perfusion solution. This was followed by incubation in a 37⁰C water bath (digestion phase) for 18 minutes. After this step, each pancreas was gently shaken in the tubes, washed with cold RPMI 1640 medium, and transferred into a 500 mL beaker. Islets were purified from the exocrine tissue by discontinuous Ficoll density gradients (Mediatech Inc., Herndon, VA). In this procedure, the islet/exocrine tissue mixtures were applied to the Ficoll density gradients and then centrifuged for 15 minutes at 1,500 rpm; the islet cell portion of the gradient was identified by visual inspection from the middle layer of the Ficoll gradient and handpicked. Isolated islets were then washed and cultured in RPMI 1640 medium containing 10% fetal calf serum (FBS), 10% Penicillin/Streptomycin (Invitrogen) and without glutamine, for 24 hours culture at 37⁰C.

[0080] O₂ tension and pH were measured in the pancreas perfusion medium (PoIySFH-

P and Control) before and after digestion using a blood gas analyzer (ABL/700 Radiometer, Copenhagen, Denmark). O₂ tension was higher in PoIySFH-P compared to the Control in the perfusion solution (containing distended pancreata) before the digestion phase (Table III). Moreover, PoIySFH-P maintained the pH in physiological range, whereas in the Control group the pH fell significantly during the digestion phase (Table III). These results were not the result of differences in the buffering capacities of the treatment and control solutions, which were determined to be similar (data not shown).

Table III

O₂ Tension O₂ Tension pH PH PH PH (mniHg) (mniHg) Initial Initial Pre- Post- Pre-digestion Post-digestion (without O2) (with O₂) digestion digestion

PoIySFH-P 381.7+35.3* 184.3+39.8 7.4+0.04** 7.4+0.03^f 7.4+0.03¹¹ 7.2+0.06***

Control 202.3+28.2 128.3+27.8 7.1+0.03 7.8+0.01 6.9+0.04 6.6+0.11

[0081] In Table III, oxymetry values (O₂ and pH) are shown for perfusion media

(PolySFH-P/RPMI solution ("PoIySFH-P") and RPMI 1640 medium ("Control")) before and after digestion. Values are means±SEM, n=12 rats per group. *p=0.01; **p=0.009; tp=0.006; ItP=O-OOl; ***p=0.009.

EXAMPLE 4 IN VITRO ASSESMENT OF ISLET YIELD, VIABILITY, AND FUNCTION

[0082] The results of islet isolation using a collagenase IX/RPMI 1640 solution with or without PoIy-SFH-P as described in Example 3 were analyzed for yield, viability and islet cell function. To determine islet yield, dithizone stained islets from a representative sample were counted under a stereoscopic microscope (Leica Microsystems, Bannockburn, IL). Islet viability was assessed by staining with trypan blue dye (Sigma). Islets stained more than 25% of its surface were considered dead. Live versus dead islets were assessed in a representative sample, where a minimum of 50 islets were counted per sample. [0083] Perfusion of rat pancreata with PoIySFH-P did not have a significant impact on post-isolation islet yields when compared to the control group (207 ±33 vs. 172 ±32 islets/rat respectively, p=0.46). The results on islet viability, on the other hand, surprisingly showed that viability was significantly increased in isolates prepared in the presence of PoIySFH-P as compared with the control collagenase/RPMI 1640 media without PoIySFH-P (Figure 1). [0084] Cell death was further characterized as follows. The level of apoptotic cell death was measured using a living cell fluorescein active caspase-3 staining kit (Biovision, Mountain View, CA). In these assays, an aliquot of 1,200 islets per group was counted and divided into four Eppendorf tubes with 300 μL of media (RPMI 1640 supplemented with 10% FBS and 10% Pen/Strep). A fluorescent dye for Caspase-3 (FITC-DEVD-FMK; 1 μL per tube) was added into two of the tubes of each group and the other two tubes were left untreated as a control. The tubes were incubated for 1 hour at 37⁰C under a 5% CO₂ atmosphere. Cells were pelleted from the suspension by centrifugation at 1,100 rpm for 1 min and supernatant removed. The pelleted cells were then resuspended using the wash buffer in the kit according to the manufacturer's instructions and washed twice in this buffer by centrifugation and resuspension. The cells were then resuspended in 100 μL of the wash buffer and the contents of each tube transferred into individual wells of a black microtiter plate. Fluorescence intensity was measured using an excitation wavelength of 485 nm and emission wavelength of 535 nm in a fluorescent plate reader (GENios, Tecan US Inc., Durham, NC). As shown in Figure 2, isolated islets from PoIySFH-P perfused pancreata showed fewer apoptotic cells compared to the control as detected by lower caspase 3 activity.

[0085] Next islet cell function was assayed by incubation with varying amounts (5, 8 and 14 mM) of glucose. Intracellular divalent calcium ion concentration during glucose stimulation was measured for functional evaluation in isolated islets, using standard wide- field fluorescence imaging with dual-wavelength excitation fluorescent microscopy. In these assays, islets were loaded with a calcium-specific dye (Fura-2/AM; Molecular Probes, Eugene OR) by incubating the islets for 25 min at 37⁰C in Krebs solution supplemented with 2 rnM glucose (KRB2), containing 5 μM Fura-2/AM. After loading, the islets were placed into a temperature-controlled perfusion chamber (Medical Systems Inc, Paola, KS) mounted on an inverted epifluorescence microscope (TE-2000U, Nikon, Inc.) and perfused by a continuous flow (rate 2.5 mL/min) with 5% CO₂-bubbled KRB2 buffer at 37⁰C (pH 7.4). Krebs buffers containing different glucose concentrations (5, 8, and 14 mM) were administered to the islets and resulting fluorescence followed for 15 min each, rinsing with KRB2 in between. Multiple islets were imaged with 10x-20x objectives for each sample. Fura-2 dual-wavelength excitation was set at 340 nm and 380 nm (excitation wavelengths), and fluorescence detected at 510 nm (emission wavelength). Fluorescence was analyzed using Metafluor/Metamorph imaging acquisition and analysis software (Universal Imaging Corporation, West Chester, PA) and images collected using a high-speed, high-resolution charge-coupled device (Roper Cascade CCD, Tucson, AZ). Estimation of Ca²⁺ levels was accomplished using an in vivo calibration method. The percentage change of intracellular Ca²⁺ between both groups was calculated by the maximum increase after glucose stimulation, minus the basal (2 mM glucose) Ca²⁺ level for each group. [0086] Intracellular calcium ion concentration was also assessed in these islet cells in the presence of tolbutamide, an inhibitor of K⁺-ATP channels. In these experiments, tolbutamide was added to the perfusion media at a final concentration of 100 μM in Krebs perfusion media containing 2mM glucose (basal levels) and used to perfuse islet cells in the absence of glucose stimulation. These measurements were performed on islets as described above.

[0087] As shown in Figure 3, improved islet responsiveness to glucose was shown by increased intracellular Ca²⁺ levels in islets after stimulation with glucose at different concentrations (Figure 3A). In all three concentrations (5, 8, and 14 mM) of glucose tested, PolySFH-P-treated islets demonstrated significantly higher intracellular Ca²⁺ values than control in a dose-response manner (Figure 3B). Further, addition of tolbutamide (an inhibitor of ATP-dependent K⁺ channels) showed that when mitochondrial ATP regulation in these channels was by-passed, there was no significant difference in intracellular Ca²⁺ levels between both groups (Figure 4 A and B).

[0088] Islet cell function was also assessed for glucose-induced insulin secretion. Static glucose incubation was used to compare glucose induced insulin secretion (stimulation index, SI) between islets isolated in the presence or absence of PoIy-SFH-P as described in Example 1. SI as used herein was defined by the ratio of stimulated versus basal insulin secretion. Briefly, for each experiment, groups of 5 handpicked islets with similar size (approximately 100 μm) were placed in five different wells of a 12 well-plate (5 replicates), then pre-incubated with 1 mL of Krebs buffer at low glucose concentration (1.6 mM glucose final concentration) for 30 min, after which the supernatant was collected and discarded. Islets were then incubated for 1 hour in low glucose Krebs (1.6 mM glucose final concentration) at 37⁰C and 5% CO₂, and supernatants were collected under a microscope without removing any islets from the well. The same step was repeated with addition of Krebs-high glucose solution (16.7 mM glucose final concentration) and incubation of the islets under these conditions for 90 min. Supernatants were collected and frozen at -2O⁰C for later measurement using an ELISA kit immunologically-specific for rat insulin (obtained from Mercodia, Uppsala, Sweden). All samples are measured in duplicates. As shown in Figure 5, insulin secretion in response to glucose stimulation was significantly increased in islet cells isolated from rat pancreata in the presence of PoIySFH-P compared to the control group.

[0089] Isolation in the presence of O₂ created the potential for reactive oxygen species (ROS) to have injured the functional integrity of islet cells, particularly at the mitochondrial and cell membranes, which could be disrupted inter alia by ROS-peroxidation. Functional integrity of islet cells isolated in the presence or absence of PoIy-SFH-P as disclosed in Example 1 was further assessed by analyzing mitochondrial membrane integrity. In these assays, mitochondrial membrane potential were assessed using the fluorescent dye Rhodamine 123 (Rhl23), a lipophilic cation that integrates selectively into the negatively- charged mitochondrial membranes and can be used as a probe of mitochondrial transmembrane potential. In cells pre-loaded with Rhl23, membrane potential increase (hyper-polarization) that occurs after glucose stimulation in functional islet cells causes more Rh 123 to be concentrated in the mitochondrial membrane, leading to aggregation of dye molecules and a decrease (quenching) of the fluorescence signal. Rhl23 was used as previously described.(Zhou et al., 2000, Am J Physiol Endocrinol Metab 278: E340-E351). Briefly, islets were incubated for 20 min at 37⁰C in Krebs solution containing 2mM glucose and supplemented with 10 μg/mL Rhl23 (Molecular Probes, Eugene, OR), then placed into a temperature-controlled perfusion chamber (Medical Systems Inc.) mounted on an inverted epifluorescence microscope (TE-2000U, Nikon Inc, Melville, NY.) The islets were perfused with a continuous flow (rate 2.5 mL/min) of 5% CO₂-bubbled Krebs buffer at 37⁰C (pH 7.4). Islets were then stimulated with 14 mM glucose and the changes in fluorescence measured for 15 min after glucose stimulation. Rhl23 fluorescence was determined using 540 nm as excitation wavelength and 590 nm as emission wavelength, and images collected with a charged coupled device camera (Roper Cascade CCD). Data were normalized to the average fluorescence intensity recorded during a five-minute period prior to glucose stimulation. The percentage change in fluorescence intensity between both islet isolation groups (i.e., isolated in the presence or absence of PoIy-SFH-P) was calculated as the maximum reduction in fluorescence intensity after 14 mM glucose stimulation, minus the basal fluorescence intensity for each group.

[0090] In addition, Rh 123 was used to assay islet cells for changes in mitochondrial morphology. In these assays, islets from PoIySFH-P and control groups were incubated for 15 minutes in Krebs buffer containing 2.5 μM Rhl23 and visualized using a Carl Zeiss LSM 510 confocal microscopy equipped with 60 X water immersion objective. The 488 nm line from an argon-krypton laser used for excitation and Rh 123 emission was detected through an LP 505 filter. The intensity and the distribution of fluorescence were used to morphologically characterize mitochondrial integrity in these islet cells. [0091] The results of experiments to assess whether ROS were present during islet isolation and to what extent these species caused oxidative damage to the islet cells are shown in Figures 6 and 7. Measurements of mitochondrial membrane potential indicated a better functional integrity of PoIySFH-P islets than in the control group as shown by an increased percentage of the change (decrease) in Rh 123 fluorescence, representative of undamaged electrochemical potential as a response to glucose stimulation (14 mM) (Figures 6 A and 6B).

[0092] In addition, morphological assessment of mitochondria in islets from the control group appeared swollen and fragmented, showing decreased staining with Rh 123 around the nuclei with loss of the continuity of the staining. In contrast, PoIySFH-P treatment showed improved islet cell mitochondrial morphology, with reduced swelling and fragmentation and increased staining around the nuclei (Figure7). These results are consistent with islet isolation in the presence of PoIySFH-P showing less ROS-generated oxidative damage that in the control group isolated in the absence of PoIySFH-P.

[0093] Another assay of ROS-caused injury was assessment of oxidative stress by assaying reduced glutathione (GSH) levels. These assays were performed on islet cells 12 hours post-isolation using the monochlorobimane (mcbm) method (Avila et al., 2003, Cell Transplant 12: 877-881). Briefly, 500 islets were cultured for 30 min at 37°C in one well of a 12 well-plate in 5 mL CMRL culture medium containing 10 μL mcbm (a final concentration of 50 mM) (Molecular Probes). Islets were collected, washed with phosphate buffered saline (PBS) at pH 7.5, resuspended in 500 μL of 50 mM TRIS buffer containing 1 mM EDTA and then sonicated. The sonicated islet cell mixture was centrifuged to clear the supernatant of debris and the fluorescence from the cleared supernatant detected using a fluorescence plate reader (GENios, Tecan US Inc., Durham, NC) with an excitation wavelength of 380 nm and an emission wavelength of 470 nm.

[0094] Cell membrane damage from lipid peroxidation by ROS was used as a marker of oxidative injury. The extent of lipid peroxidation in islets isolated in the presence or absence of PoIy-SFH-P as disclosed in Example 1 was determined by detecting malondialdehyde (MDA), a product of lipid peroxidation. MDA levels were assessed using thiobarbituric acid (TBA) according to the method of Yagi (1998, Methods MoI Biol 108: 101-106). Briefly, a reaction mixture was prepared containing 0.1 M HCl, 0.67% TBA, 10% phosphotungstic acid and 7% sodium dodecylsulphate (SDS) (all obtained from Sigma). 500 islets were sonicated in 700 μL PBS into a cell lysate. After centrifugation at 15,000 rpm to clear the lysate of debris, 500 μL of the supernatant were extracted and mixed with 875 μL of the reaction mixture, then boiled at 95-98°C for 1 hour. After this process, samples were cooled and mixed with 750 μL of n-butanol in order to extract MDA and avoid interference of other compounds. After a brief centrifugation, 100 μL of this supernatant were extracted and fluorescence assessed in duplicate on a 96 well plate with a fluorometer (GENios, Tecan US Inc. Durham, NC) at an excitation wavelength of 530/25 and an emission wavelength of 575/15. Samples were assayed in comparison with MDA standards (obtained from Sigma) prepared at different concentrations (2, 4, and 8 mM).

[0095] Whether O₂ delivery by PoIySFH-P increased oxidative stress or injury was examined by assaying GSH and MDA levels in islet cells isolated as disclosed in Example 1. Oxygenated PoIySFH-P did not decrease glutathione levels (7.1+2.9 nmol/mg protein for PoIySFH-P and 6.8+2.4 for control; p=0.93). Similarly, lipid peroxidation as measured by MDA levels was not statistically significantly different between PoIySFH-P and control group (1.8 +0.9 nmol/mg protein vs. 6.2 +2.4, respectively; p=0.19) indicating the there was no increased oxidative stress by the presence of higher O₂ levels. [0096] The foregoing observations indicated that, surprisingly, intraductal perfusion of ischemic pancreata with PoIySFH-P improved islet viability and function associated with maintenance of mitochondrial integrity, and that isolating pancreatic islets in the presence of PoIySFH-P did not lead to increased oxidative stress in isolated islets. [0097] These results illustrate significant advantages in using PoIySFH-P in isolating pancreatic islets. These results demonstrated that mitochondria, which are a major contributor to apoptotic cell death under ischemic conditions, maintain improved function and integrity in the presence of oxygenated PoIySFH-P. Higher O₂ availability to PoIySFH- P-treated islets was shown by higher O₂ tensions in the perfusion media compared to the control. The availability of O₂ substrate for mitochondria may be responsible for the improved viability observed in islets from the PoIySFH-P group. Islets are exposed to significant oxidative stress during the islet isolation and transplantation procedure. Surprisingly, increased O₂ provided in the form of oxygenated PoIySFH-P did not result in significant production of ROS as assessed by analysis of mitochondria, both structurally and functionally as shown above. Indeed, the results shown above support the conclusion that mitochondrial function and integrity were improved by oxygenated PoIySFH-P treatment, leading to both improved glucose-stimulated insulin secretion and decreased cell death. [0098] The results shown above indicated that increased O₂ availability resulting from the use of oxygenated PoIySFH-P protected islets from apoptosis, measured by lower levels of caspase-3 than in the control group. This result is consistent with the observation that hypoxia has been shown to initiate apoptosis, mainly through the release of mitochondrial mediators into the cytosol. Mitochondrial functional integrity was shown to be superior in PolySFH-P-treated islets with improved membrane electrochemical potential in response to glucose stimulation. Functional integrity was complemented by the conservation of mitochondrial structure in the PoIySFH -P -treated islets, determined by less swelling and more elongated mitochondria. Enhanced mitochondrial staining, representative of improved perinuclear localization in the PolySFH-P-treated islets, was also observed. [0099] The foregoing results also indicate that in vitro function of isolated islets was improved by intraductal administration of PoIySFH-P to the ischemic pancreas. Higher stimulation indices were obtained in PolySFH-P-treated islets compared to the control in response to a static glucose challenge. The enhanced function for PoIySFH-P treated islets was supported by higher intracellular Ca²⁺ levels in response to glucose. These results demonstrate that the capacity of islet mitochondria to increase cytosolic Ca²⁺, necessary for insulin secretion in beta cells, is greater in islets isolated in the presence than in the absence of oxygenated PoIySFH-P. The specificity of this improvement was shown in experiments where islets were incubated in the presence of tolbutamide, a K⁺-ATP channel inhibitor. Under these conditions, cells depolarize and raise calcium levels, directly promoting insulin secretion. After the addition of tolbutamide, intracellular Ca²⁺ response to glucose was similar between both groups. These results suggest that the provision of O₂ by PoIySFH-P protected the mitochondrial pathway in the process of insulin secretion in response to glucose.

[00100] These in vitro results all supported the conclusion that pancreatic islets isolated in the presence of oxygenated PoIySFH-P were structurally and functionally superior to islets isolated without oxygenated PoIySFH-P.

EXAMPLE 5 IN VIVO ASSESSMENT OF ISLET YIELD, VIABILITY, AND FUNCTION

[00101] Islet function was assessed in vivo by transplantation under the kidney capsule of diabetic athymic nude mice (Harlan Industries), using animals treated as set forth in Example 1 with the exception that these animals were housed and surgeries performed under a laminar flow hood located in "barrier" rooms to prevent adventitious infection. [00102] Diabetes was induced in these animals by a single intraperitoneal (IP) injection of streptozotocin (Sigma) at a dose of 220 mg/kg body weight. Diabetes was considered induced in treated animals after three or more non- fasting blood glucose levels of >300 mg/dL taken from the tail vein, which generally occurred after a maximum of 72 hours post injection.

[00103] For transplantation, animals were anesthetized by isoflurane inhalation using a vaporizer and masks (Viking Medical). In these experiments, islets were transplanted without culture fresh after isolation. 250 islets from PolySFH-P/RPMI solution-treated pancreata (PoIySFH-P) or RPMI 1640 medium-treated pancreata (Control) were transplanted into each mouse under the left kidney capsule as described in Oberholzer et al. (1999, Immunology 97:173-180). It was expected using this procedure that transplantation of 250 ischemic rat islets would reverse diabetes in less than 50% of recipients. Successful transplantation was defined by reduction of glycemia to below 200 mg/dL. Normoglycemic recipients underwent graft-bearing nephrectomy 5-7 weeks post-transplantation. Return to hyperglycemia was interpreted as indirect proof of islet graft function rather than spontaneous recovery of the native pancreas.

[00104] Graft function was also assessed by the lag period required to achieve normoglycemia, using an Intraperitoneal Glucose/ Arginine tolerance test (IPG/ ATT) one week post-transplantation. Briefly, in these assays glucose (at 2 mg/kg body weight) and arginine (3 mg/kg) were injected intraperitoneally (IP) in 0.5 cc using a representative sample of randomly selected euglycemic animals (n=5 for PoIySFH-P and n=3 for Control; in the Control group only 4 animals achieved normoglycemia). Blood glucose levels were detected by tail puncture at serial time-points (0, 5, 15, 30, 45 and 60 minutes) after injection.

[00105] The results of these experiments were evaluated statistically, using Student's t test and Pearson Chi-Square test, where p values <0.05 were regarded as statistically significant.

[00106] The results of the foregoing experiments revealed that the percentage of cured mice transplanted with PoIySFH-P or Control islets was similar (6 out of 10 and 4 out of 9 respectively, p=0.4). Surprisingly, mice transplanted with islets treated with PoIySFH-P achieved normoglycemia and reversed diabetes in a significantly shorter time than the mice transplanted with islets from the Control group (Figure 8). Moreover, the mice receiving PolySFH-P-treated islets showed better graft function with lower glucose levels during IPG/ATT (Figure 9).

[00107] These results indicated that PoIySFH-P perfusion of the ischemic rat pancreas improved islet graft function in vivo, as shown by a better response to IPG/ AT stress test and a shortened lag time to reach normoglycemia after transplantation. These in vivo results confirmed the improved function of Poly SFH-P -treated islets observed in vitro. [00108] In order to determine the effect of PoIySFH-P perfusion specifically on the beta cell population, fractional beta cell viability was assessed using the method of Ichii et al. (2005, Am J Transplant 5: 1635-1645). This method involved assessing cell membrane stability and mitochondrial membrane stability of beta and non-beta cells. In these experiments, islets were dissociated and the cells staining with the following dyes: 7- aminoactinomycin D (7aad, specific for cell membrane stability), teramethylrhodamine ethyl ester (TMRE, mitochondrial membrane stability) and Newport Green (NG, wherein NG high populations were beta cells and NG low populations were non-beta cells). A single cell suspension was created by incubating 1000 islets per condition in 2mL Accutase (Innovative Cell Technologies Inc. San Diego) for 7 minutes at 37⁰C followed by gentle pipetting. Cells were then incubated with IuM Newport green PDX; (Invitrogen, Molecular Probes) and lOOng/mL TMRE (Invitrogen, Molecular Probes) in PBS for 30 min at 37⁰C. After washing with PBS, cells were stained with 5μg/mL 7AAD (Invitrogen, Molecular Probes). The cells were analyzed using Cell Quest software and the LSR by Becton Dickinson (Mountainview, CA). Gating for NG was performed by side scatter and FLl .

[00109] The results of these experiments are shown graphically in Figure 10. PoIySFH-P improved integrity of both beta and non-beta cells. Fractional islet cell viability assessment indicated that beta cells were more vulnerable to ischemic damage than non-beta cells in the islets, and thus benefited to a greater extent from the presence of oxygenated PoIySFH-P in the culture media.

EXAMPLE 6 PURIFICATION OF ISOLATED PANCREATIC ISLETS

[00110] Improved methods for isolating human pancreatic islets were developed using an optimized mixture of University of Wisconsin solution (UW solution) and Biocoll for density gradient centrifugation as follows.

[00111] The islets used in these studies were obtained from donors screened at the time of offer and having the characteristics set forth in Table IV. The cold ischemia time was less than 12 h. Islet isolations were performed at the University of Illinois at Chicago (UIC) following informed consent by donor relatives. All isolations were performed by an expert team using identical techniques with the only difference being the modifications to the purification and density gradient centrifugation methods set forth herein. [00112] Isolation Process:

[00113] All steps were performed under aseptic conditions in clean rooms and Class II biohazard hoods with solutions comprised of sterile components.

[00114] a) Enzymatic Digestion of the Pancreas to Obtain Free Islets [00115] Pancreata were removed from the sterile interior of its transport jar by a sterile gowned team member. The spleen and duodenum were dissected away from the pancreas and the pancreas decontaminated by immersion into three solutions: Betadine (300 cc, 5%) Kefzol/Fungizone (150 cc HBSS, lgr Kefzol, lOOmg Fungizone) solution and finally Hank's Balanced Salt Solution (HBSS, 300 cc).

[00116] The pancreata were digested using a modified automated method as described in Ricordi et al. (1998, Diabetes 37: 413-420). Briefly, pancreatic ducts were perfused in controlled fashion with 300 mL of cold enzyme solution (0.5 g Liberase-HI, Roche Molecular, Indianapolis, IN, or Collagenase NBl, Serva, EU) in indicator- free HBSS, supplemented with HEPES and calcium chloride. A 6OmL syringe and two 16 gauge angiocatheters were used to infuse the enzyme solution. After delivery of enzyme solution through the pancreatic ducts, the pancreas was divided into 6-10 sections and transferred to a sterile container designed to facilitate tissue degradation. The islets were then separated by gentle mechanical dissociation provided by manually shaking the stainless-steel chamber (Ricordi chamber), with a closed circulation flow-through system to bathe the tissue in the digestive enzymes at 37°C. At intervals during the digestion, samples were taken to assess the progress of the dissociation of islets from the acinar tissue. Free floating islets were detected at 10-20 min of digestion. When an adequate number of acinar- free islets were detected (>50% free islets), the enzymatic process was stopped by cooling and dilution with 6-10 L of M199 medium (Media Tech) supplemented with 250 mL of 20% human albumin. The crude cellular fraction was then collected in 500 mL conical tubes and concentrated by centrifugation for 1 minute at 1,000 rpm. The number and size of the islets were determined by dithizone staining, the volume of the pellet was measured and the total number of islet equivalents (IE) units calculated.

[00117] b) Purification of Islets by Density Gradient Centrifugation [00118] After incubation in UW solution (commercially-available from DuPont Pharma, Bad Homburg, Germany) for at least 30 minutes, islets were purified by one of two different continuous gradients in a comparative study. One gradient was a conventional (standard) method (SM) using Biocoll, essentially as described by Berney et al.(2002, Methods of Tissue Engineering, Epithelia cell culture: Pancreatic Islets, Atala A and Lanza RP, eds., Academic Press, San Diego, CA, pp. 203-218). The second was the inventive (UIC-UB) method, using an optimized mixture of Biocoll and UW solution as set forth below). Both gradients were assessed using a cell separator (Cobe 2991™, Cobe Laboratories Inc., Lakewood, CO) as described in Lake et al. (1989, Diabetes 38(Supp 11): 143-5) at 4°C. Islets were then cultured at 37°C for up to 72 h. [00119] A series of 164 islet isolations were compared based upon different techniques used for purification of islets (n=32 pancreas processed with the SM and n=132 with UIC- UB method). Using the SM, the Cobe device was pre-loaded with 150 mL Biocoll having a density of 1.10 g/mL and centrifuged at 1500 rpm. Thereafter, 130 mL Biocoll (density 1.10 g/mL) was added to the front beaker as the "heavy" gradient and 140 mL 1.077 Biocoll (density 1.077 g/mL) was added to the rear beaker as the "light" gradient. The tissue was loaded after loading the gradient mixture and islets separated at 2000 rpm. In this method, up to 20 mL tissue was used to isolate islets at each iteration of the gradient separation. After 5 minutes, the tissue was collected in 12 fractions (250 mL tubes pre-fϊlled with 200 wash media).

[00120] Using an embodiment of the inventive method (also termed the UIC-UB gradient method), the Cobe device was loaded with 110 mL Biocoll having a density of 1.10 g/mL and centrifuged at 1500 rpm. Then, 130 mL "heavy gradient" (49% Biocoll, 51% UW solution, density 1.079 g/mL) was added to the front beaker and 140 mL "light" gradient (30% Biocoll, 70% UW, density 1.068 g/mL) to the rear beaker (UW solution has density of 1.046 g/mL). After first incubating in UW solution, the unpurifϊed pancreatic islet cells in UW solution were loaded onto the gradient once the gradient is almost completed. The pancreatic islet cells were centrifuged and separated at 3000 rpm. Using this method, up to 50 mL of tissue (packed volume) can be loaded to each run of the gradient separation. After 5 minutes, tissue was collected in 12 fractions (30 mL into tubes pre-fϊlled with 200 wash media). Wash media used was HBSS-based solution (Hank's Buffered Salt Solution). In both methods, fractions with purity > 60% were collected as "top" and < 60% as "bottom" layers (up to 7 mL tissue).

[00121] Isolation outcome was assessed through quantification of islet mass by dithizone staining, expressed in equivalent islet numbers (EIN) according to the method of Ricordi et al. (1990, Acta Diabetol Lat.. 27: 185-95). Islet recovery rate was calculated as ratio of the islets number after purification to the islets number after digestion x 100. Islet viability was determined by fluorescent staining with Syto-Green/ Ethidium Bromide as described in Yang et al. (1998, Cell Transplant 7: 443-51); percentage of dead and live cells was estimated according to the methods of Avila et al. (2003, Cell Transplant 12: 877-81). In vitro islet function was assessed by static glucose incubation, challenged with low and high glucose conditions, expressed as stimulation index according to Tsujimura et al. (2002, Transplantation 74: 1687-91). Statistical analysis was carried out by Student's t test and Pearson Chi-Square test where applicable. P-values < 0.05 were regarded as statistically significant.

[00122] These comparative results are shown in Table V and Figure 11. Pre-purification islet counts were similar between the groups (359,425±40,794 EIN in the samples to be isolated using the standard method versus 370,682±l 7,579 EIN in the samples to be isolated using the UIC-UB method; Table V). The gradient density in each fraction for SM was greater than the UIC-UB gradient; ranging 1.077-1.10 g/mL in SM and 1.068-1.079 in UIC- UB; Figures 1 IA and 1 IB). The density curve was thus less steep in UIC-UB compared to conventional methods (as shown in Figures 11C and 1 ID). The islets purified with SM were collected in only two fractions (average purity of 68.9% and 36.3%); in contrast, using the UIC-UB method, highly purified islets were consistently collected in 6 separate fractions (with purity of 84.8%, 82.5%, 72.0%, 59.3%, 46.8% and 36.2%). Overall purity of the "top" layer was 77.5 ±13.4% in SM and 82.9 ±11.4% in UIC-UB method. The UIC-UB method yielded more tissue volume than SM (1.37 ±0.08 mL vs 0.88 ±0.19 niL, p=0.015) in the top layer (which had greater purity). The UIC-UB method resulted in significantly higher yield compared to SM (368,419 ±18,245 total islets with the UIC-UB method compared with 260,908 ± 37,835 total islet using the SM, p = 0.017). Islet recovery rate was superior using the UIC-UB method (84.9% vs 64.5%, p = 0.04), and the rate of clinically applicable preparations (having EIN>300,000) was greater using the UIC-UB method compared to SM (46.7% vs 34.2%, P<0.05). Significantly, the ratio of equivalent islet number to actual islet count in the top layer was markedly greater in preparations made using the UIC-UB method compared to SM (p=0.037), indicating either recovery of larger islets or less fragmentation. The mean EIN of the bottom layer was 86,771 ±1,327 using SM and 76,810 ±5,694 using the UIC-UB method (p=0.55), and islet viability was higher using the UIC-UB method (80.9 ±1.8 for SM vs 86.1 ±2.4 for UIC-UB, p=0.003). The stimulation index showed equivalence between these groups (5.3 ±0.7 versus 4.5 ±0.6).

Table IV. Characteristics of pancreas donors for both Standard Method (SM) and UIC- UB gradient) groups

Standard UIC-UB gradient P-value

Method Method

Total Numbers 32 132

Age (year) 49.8±1.9 48.2±1.7 P>0.05

Sex (male/female) 19/13 71/61

BMI (Kg/m²) 28±0.9 29.7±0.7 P>0.05

Cold ischemia (hr) 8.65±0.39 8.1 1±0.32 P>0.05

Pancreas weight (g) 115.6±5.2 112.5±3.9 P>0.05

Table V. Islet isolation outcomes for both Standard Method (SM) and UIC-UB gradient groups

Standard UIC-UB gradient P-valu

Method Method

Pre purification EIN ^™359i425±40J94

Post purification EIN 260,908±37835 368,419±18,245 0.017

EIN per gram of pancreas 2492±344 3212±279 0.045

Islet Recovery rate 64.5% 84.9% 0.04

Rate of successful isolations* 34.2% 46.7% 0.039

Top layer EIN 194,022±39771 310,607±17198 0.022

Top layer actual islet count 169,531 ±44758 210,050±11 ,069 0.049

EIN / Actual count ratio 1.12±0.2 1.56±0.06 0.037

Isolations resulted in a yield of over 300,000 EIN.

[00123] Although certain presently preferred embodiments of the application have been described herein, it will be apparent to those of skill in the art to which the application pertains that variations and modifications of the described embodiment may be made without departing from the spirit and scope of the application. Accordingly, it is intended that the application be limited only to the extent required by the following claims and the applicable rules of law. References

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Claims

We claim:

1. A method of isolating pancreatic islet cells from a mammal, comprising a. treating pancreatic tissue from the mammal with one or a plurality of enzymes for producing unpurifϊed pancreatic islet cells therefrom; b. collecting the unpurifϊed pancreatic islet cells from the tissue treated with the enzyme; c. incubating the unpurifϊed pancreatic islet cells in a tissue preservation solution; and d. subjecting the unpurifϊed pancreatic islet cells to a continuous density gradient to isolate pancreatic islet cells, wherein the density gradient is formed in a mixture of Biocoll and the tissue preservation solution, and wherein the density gradient formed has a density range of from about 1.06 g/mL to about 1.08 g/niL.

2. The method of claim 1 , wherein the tissue preservation solution is University of Wisconsin (UW) solution.

3. The method of claim 1 , wherein the density gradient has a density range of from about 1.068 g/mL to about 1.079 g/mL.

4. The method of claim 1 , wherein the density differential is not more than 0.011 g/mL.

5. The method of claim 1 , wherein the pancreatic islet cells are isolated by centrifugation in the density gradient at 3000 rpm for 5 min.

6. The method of claim 1, wherein the pancreatic tissue from the mammal is incubated in the presence of polymerized hemoglobin at 4⁰C prior to enzymatic treatment and at 37⁰C during enzymatic treatment.

7. The method of claim 6, wherein the unpurifϊed pancreatic islet cells are produced from the pancreatic tissue by enzymatic treatment in the presence of polymerized hemoglobin.

8. The method of claim 1 , wherein the unpurifϊed pancreatic islet cells are collected by spinning down the cells and resuspending the cells in the tissue preservation solution.

9. The method of claim 8, wherein the resuspended unpurifϊed pancreatic islets are incubated in the tissue preservation solution for at least 30 minutes.

10. The method of claim 9, wherein the tissue preservation solution is UW solution.

11. The method of claim 1 , further comprising the step of separating the unpurified pancreatic islet cells by gentle physical dissociation after treatment of the pancreatic tissue with the one or a plurality of enzymes and before subjecting the unpurified pancreatic islet cells to the density gradient.

12. The method of claim 1 , wherein the mammal is a human.

13. The method of claim 1 , wherein the enzyme is a collagenase.

14. The method of claim 6, wherein the polymerized hemoglobin is derived from human blood.

15. The method of claim 14, wherein the polymerized hemoglobin is pyridoxylated.

16. Purified pancreatic islet cells produced according to the method of claim 1.

17. A method for treating a mammal that suffers from a defect, disorder, disease or deficiency of pancreatic islet cells, comprising a step of administering to the mammal pancreatic islet cells according to claim 16.

18. The method of claim 17, wherein the defect, disorder, disease or deficiency of pancreatic islet cells is diabetes.

19. The method of claim 17 wherein the mammal is a human.

20. A pharmaceutical composition comprising a therapeutic effective amount of the pancreatic islet cells of claim 16 and at least one pharmaceutically acceptable excipient, diluent or carrier.

21. A composition comprising the pancreatic islet cells of claim 16 and at least one pharmaceutically acceptable excipient, diluent or carrier.

22. A kit for isolating pancreatic islet cells comprising (a) a first Biocoll and a tissue preservation solution gradient mixture having a density of about 1.06 g/mL; and (b) a second Biocoll and the tissue preservation solution gradient mixture having a density of about 1.08 g/mL.

23. The kit of claim 22, wherein the tissue preservation solution is UW solution.

24. The kit of claim 22 further comprising at least one enzyme that is capable of digesting pancreatic tissue and liberating pancreatic islet cells from the associating stromal components.

25. The kit of claim 22, further comprising polymerized hemoglobin.

26. The kit of claim 22, wherein the first Biocoll and a tissue preservation solution gradient mixture has a density of about 1.068 g/mL and the second Biocoll and the tissue preservation solution gradient mixture has a density of about 1.079 g/niL.

27. The kit of claim 26, wherein the tissue preservation solution is UW solution.