WO2001019962A2

WO2001019962A2 - Method of preserving tissue viability during mechanical separation process

Info

Publication number: WO2001019962A2
Application number: PCT/US2000/025372
Authority: WO
Inventors: Anton-Lewis Usala; Richard Chris Klann
Original assignee: Encelle, Inc.
Priority date: 1999-09-15
Filing date: 2000-09-15
Publication date: 2001-03-22
Also published as: WO2001019962A3; AU7382300A

Abstract

The present invention provides a method of preserving tissue viability during separation, the method including placing intact tissue in a cell culture medium. The cell culture medium includes at least one nitric oxide inhibitor. The tissue is mechanically separated into a plurality of physiologic tissue units, each unit maintaining physiologic cell-to-cell connections within the unit. The mechanical separation step may comprise cutting the tissue into slices and tearing the slices of tissue into a plurality of tissue units. Thereafter, the tissue units may be embedded in a hydrogel matrix for storage.

Description

METHOD OF PRESERVING TISSUE VIABILITY DURING MECHANICAL

SEPARATION PROCESS

FIELD OF INVENTION The present invention relates to methods of preserving tissue viability. More particularly, the present invention relates to a method for preserving tissue viability during a mechanical separation process.

BACKGROUND OF THE INVENTION

Isolating tissue from a tissue source, such as a whole organ or gland, is one of the first steps in preparing tissue for transplant. Conventional tissue isolation techniques involve enzymatic cleavage of connective tissue that binds cells together. Most commonly, the enzyme used for tissue separation is a form of collagenase. Collagenase is used to isolate a variety of tissue. For example, in the case of pancreatic tissue, collagenase is applied to isolate islet tissue from acinar tissue. In the case of enzymatic cleavage of liver tissue, the collagenase is applied to obtain hepatocytes.

Enzymatic cleavage or digestion of tissue has proven to be problematic due to difficulty in maintaining cell viability and/or differentiation over time. For example, isolated islet tissue does not remain viable for extended periods of time. Additionally, hepatocytes lose their ability to metabolize pharmacological agents and adipocytes de- differentiate into a fibroblast-like pre-adipocyte. The loss of specialized function of cells has hindered the development of biologic delivery products. There remains a need in the art for a method of preserving tissue viability, particularly during and after a separation process.

SUMMARY OF THE INVENTION The present invention provides a method of preserving tissue viability during separation. The method of the present invention involves use of a mechanical separation process, rather than enzymatic digestion, in order to maintain the physiologic cell-to-cell connections found in intact tissue. Tissue separated using the present invention maintains in vivo functional response to specific stimuli without loss of specialized function or cell viability.

In one embodiment, the method of the present invention includes placing intact tissue, such as a whole organ, in a cell culture medium. The cell culture medium comprises at least one nitric oxide inhibitor. Thereafter, the intact tissue is mechanically separated into a plurality of physiologic tissue units, each unit maintaining physiologic cell-to-cell connections within the unit. In a preferred embodiment, the mechanical separation step comprises cutting the tissue into slices and tearing the slices into a plurality of physiologic tissue units. For example, the slices of tissue may be torn into units using a blender or similar device. The method of the present invention may further include concentrating the physiologic units within a solution of the cell culture medium, aspirating the supernatant of the solution, and collecting the resulting tissue pellet.

The present invention will find use with a variety of tissue, including lung, liver, kidney, thymus, thyroid, heart, brain, and pancreatic tissue. In one embodiment, the tissue is pancreatic tissue, such that the tissue units created during the mechanical separation step maintain physiologic cell-to-cell connections between acinar tissue and islet tissue.

The nitric oxide inhibitor of the cell culture medium may be selected from the group consisting of L-cysteine, L-arginine analogues, cystine, and heparin. Preferably, the nitric oxide inhibitor or inhibitors are present in an amount of about 15 to about 600 μM, preferably about 100 to about 500 μM, and most preferably about 200 to about 350 μM. In one embodiment, the nitric oxide inhibitor comprises about 15 to about 250 μM aminoguanidine and about 50 to about 300 μM cysteine.

Following separation, the tissue may be imbedded within a hydrogel matrix for storage. Preferably, the matrix comprises gelatin, dextran, at least one nitric oxide inhibitor, and an effective amount of polar amino acids. Preferably, the tissue slices are about 0.25 mm (1/100 inch) to about 6.4 mm (% inch) across and the physiologic units are about 1.0 to about 1.5 mm across. The present invention also provides a tissue suspension, wherein the suspension comprises a hydrogel matrix as described above, and a plurality of physiologic tissue units embedded in the matrix, each unit maintaining physiologic cell-to-cell connections within the unit. DETAILED DESCRIPTION OF THE INVENTION One or more preferred embodiments of the present invention will be described more fully hereinafter. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

The present invention is directed to a method of preserving tissue viability and function during and after a separation process. Using the method of the present invention, cellular viability and specialized function is maintained for extended periods of time following tissue separation. Additionally, the separated tissue provided by the present invention maintains immediate cell-to-cell contacts that allow the tissue to continue to exhibit functional response as found in vivo.

As used herein, tissue includes any cellular and intercellular biological material, such as autologous tissue, xenogeneic tissue, allogeneic tissue, artificial organs, hormone-producing tissue suitable for implantation into individuals who suffer from conditions such as diabetes, thyroid deficiency, growth hormone deficiency, congenital adrenal hyperplasia, Parkinson's disease, and the like. Likewise, tissue includes any type of biological material useful for transplants involving therapeutic conditions benefiting from implantable delivery systems for biologically active and gene therapy products for the treatment of central nervous system diseases and other chronic disorders. More specifically, tissue includes cells useful in various transplantation therapies, including without limitation, cells secreting insulin for treatment of diabetes, cells secreting human nerve growth factors for preventing the loss of degenerating cholinergic neurons, satellite cells for myocardial regeneration, striatal brain tissue for Huntingdon's disease, liver cells, bone marrow cells, dopamine-rich brain tissue and cells for Parkinson's disease, cholinergic-rich nervous system tissue for Alzheimer's disease, adrenal chromaffin cells for delivering analgesics to the central nervous system, cultured epithelium for skin grafts, and cells releasing ciliary neurotropic factor for amyotrophic lateral sclerosis, and the like. As would be appreciated by one of ordinary skill in the art, the term tissue includes cells derived from a variety of organs and glands, such as lung, liver, kidney, thymus, thyroid, heart, brain, pancreas (including acinar and islet cells), and the like, as well as various cultured cell populations.

Autologous refers to tissue that is derived from the transplant recipient. Allogeneic or allograft refers to tissue that is derived from the same species as the recipient. Xenogeneic and xenograft refers to tissue that is derived from a species other than that of the recipient.

The present invention involves placing intact tissue in a cell culture medium, the cell culture medium comprising at least one nitric oxide inhibitor. Intact tissue refers to any tissue suitable for use in an isolation or separation process, such as whole organs or glands or substantial pieces thereof. Preferably, the cell culture medium is a standard culture medium supplemented with additional ingredients as described below.

The preferred standard culture medium is Medium 199 lx liquid. However, other standard culture media known in the art would be suitable for use with the present invention. Standard culture media which may be employed in accordance with the present invention are standard culture media for growing cells that typically provide an energy source, such as glucose, substantially all essential and nonessential amino acids and vitamins and/or other cell growth supporting organic compounds required at low concentrations. When combined with a buffering agent and a salt solution, the standard culture medium provides many of the nutrients required for normal metabolic functioning of cultured cells. The preferred salt solution is Earle's Balanced Salts. The salt solution helps to maintain pH and osmotic pressure and also provides a source of energy. The preferred buffering agent is Hepes. Other salt solutions and buffering agents known in the art may be used without departing from the present invention. The nitric oxide inhibitors of the cell culture medium are present in an amount of about 15 to about 600 μM, preferably about 100 to about 500 μM, more preferably about 200 to about 350 μM. Nitric oxide inhibitor is broadly defined as including any composition or agent that inhibits the production of nitric oxide or scavenges or removes existing nitric oxide. Nitric oxide is a pleiotropic mediator of inflammation. Nitric oxide is a soluble gas produced by endothelial cells, macrophages, and specific neurons in the brain, and is active in inducing an inflammatory response. Nitric oxide and its metabolites are known to cause cellular death from nuclear destruction and related injuries. Suitable nitric oxide inhibitors for the cell culture medium include, but are not limited to, L-cysteine, L-arginine analogues, cystine, and heparin. Aminoguanidine is a preferred L-arginine analogue. Other L-arginine analogues, such as N-monomethyl L-arginine, N-nitro-L-arginine or D-arginine may also be used in the present invention. Since nitric oxide is generally produced when cells are experiencing stress, such as the trauma caused by tissue separation, the cell culture medium is useful in preventing cellular death during periods of cell stress. The nitric oxide inhibitors also serve to inhibit the release of enzymatic moieties that may be deleterious to the tissue as they undergo separation. For example, the cell culture medium inhibits the release of digestive enzymes from acinar tissue that can damage islet tissue during separation. Thus, the cell culture medium allows use of pancreatic tissue without the conventional step of separating acinar tissue from the insulin- producing islet tissue.

In a preferred embodiment, aminoguanidine is provided at a concentration of about 15 to about 250 μM, preferably about 30 to 180 μM, most preferably about 80 to about 120 μM. In one embodiment, the concentration of aminoguanidine is about 100 _μM.

The culture medium also preferably includes L-cysteine. L-cysteine acts as a scavenger of already formed nitric oxide and thereby prevents nitric oxide induced cellular damage. Additionally, L-cysteine may obscure immune recognition sites on the cultured cells by sulfhydryl bond formation to integral surface proteins containing sulfur groups. Further, L-cysteine provides sulfhydryl bonds which strengthen cell membranes. The preferred final concentration of L-cysteine is about 50 to about 300 μM, preferably about 80 to about 250 μM, most preferably about 150 to about 200 μM. In one embodiment, the final concentration is about 180 μM. The cell culture medium may also include an effective amount of polar amino acids. The preferred polar amino acids are selected from the group consisting of arginine, lysine, histidine, glutamic acid, and aspartic acid, although other chemicals containing polar amine and carboxyl groups may be used. An effective amount is the amount necessary to strengthen cellular membranes and bond to immune recognition sites on the cell surface. In one embodiment, the concentration of polar amino acids is raised to a final concentration of between about 5 to about 150 mM, preferably about 10 to about 65 mM, and more preferably about 15 to about 40 mM. Advantageously, supplemental amounts of L-arginine and L-glutamic acid are added to the buffer medium. Preferably, the final concentration of L-arginine is about 2 to about 60 mM, preferably about 5 to about 30 mM, most preferably about 5 to about 15 mM. The final concentration of L-glutamic acid is about 2 to about 60 mM, preferably about 5 to about 30 mM, most preferably about 10 to about 20 mM. In one embodiment, the final concentration of L-arginine is about 10 mM and the final concentration of L-glutamic acid is about 15 mM.

Additionally, the cell culture medium may include a superoxide inhibitor. A preferred superoxide inhibitor is ethylenediaminetetraacetic acid (EDTA). Superoxide is a highly toxic reactive oxygen species, whose formation is catalyzed by divalent transition metals, such as iron, manganese, cobalt, and sometimes calcium. Highly reactive oxygen species such as superoxide (O₂") can be further converted to the highly toxic hydroxyl radical (OH-) in the presence of iron. By chelating these metal catalysts, EDTA serves as an antioxidant. The concentration range for the superoxide inhibitor is about 0 to about 10 mM, preferably 1 to about 8 mM, most preferably about 2 to about 6 mM. In a preferred embodiment, the superoxide inhibitor is present at a concentration of about 4 mM.

Other additives known in the art, such as antibiotics, may be added to the cell culture medium without departing from the present invention.

Table 1 below lists the particularly preferred additives and supplemental ingredients for the cell culture medium of the present invention and summarizes the final concentration ranges and preferred final concentrations for each ingredient.

Table 1

After placing the tissue in the above-described cell culture medium, the intact tissue is then mechanically separated into a plurality of physiologic tissue units, each unit maintaining physiologic cell-to-cell connections within the unit. Physiologic cell-to-cell connections refer to intact cell-to-cell interconnections existing in vivo. Although not bound by any particular theory, it is believed that the loss of specialized function and viability in enzymatically digested tissue is due, in part, to the loss of cell-to-cell connection found in vivo. It is believed that the maintenance of cell-to-cell interconnections found in vivo is necessary to maintain in vivo function or response to specific stimuli. For example, when cell-to-cell connections are maintained between acinar tissue and islet tissue in separated pancreatic tissue, the insulin response of the islet tissue is pulsatile, unlike the insulin response exhibited by purified islet tissue. The pulsatile insulin response is very similar to the insulin response found in vivo.

Mechanical separation refers to any separation process that involves physical division of tissue, rather than enzymatic digestion or separation. One method of mechanically separating the tissue comprising cutting the tissue into slices and then tearing the slices of tissue into a plurality of physiologic tissue units. Preferably, the slices are about 0.25 mm (1/100 inch) to about 6.4 mm (^lA inch) across, more preferably about 0.25 mm (1/100 inch) to about 3.2 mm (1/8 inch) across. The physiologic tissue units are preferably about 0.5 mm to about 3.0 mm across, more preferably about 1.0 mm to about 1.5 mm across. The slices of tissue may be torn in any manner known in the art. For example, the slices may be torn using a blender or any similar device capable of rending or tearing the tissue into fairly uniform units. In one embodiment, the tissue slices are placed in a blender and subjected to three five- second bursts at a medium speed in order to tear the tissue into the preferred size range.

Each physiologic tissue unit maintains a significant number of immediate cell- to-cell connections. Unlike tissue separated by enzymatic digestion, each tissue unit closely approximates the conditions and environment that cells within the unit experience in vivo. In essence, each physiologic tissue unit functions as a smaller version of the intact tissue, such as the whole organ or gland.

After the mechanical separation, the physiologic tissue units may be concentrated within a solution of the cell culture medium. Preferably, the tissue is allowed to gravity settle or concentrate in a large excess of the above-described cell culture medium. Thereafter, the supernatant of the solution is aspirated one or more times. After aspirating the supernatant, the tissue units are collected in pellet form, the tissue pellet comprising a plurality of the physiologic units adhered together. It is also recognized that the above-described cell culture medium may be used as a buffer medium for cell suspensions. The cell culture medium protects the cells from trauma in the culture medium environment. The cell culture medium also strengthens and protects cellular membranes and inhibits the production of nitric oxide, thereby promoting cell viability and function. The tissue pellet may be placed in a hydrogel matrix for long-term storage. A preferred matrix is described in U.S. Application Ser. No. 09/113,437, filed July 10, 1998, which is herein incorporated by reference in its entirety. The preferred matrix has been demonstrated to preserve complex cell cultures, such as pancreatic acinar tissue/islet tissue units for up to four weeks at refrigerated temperatures, without loss of insulin or amylase production. In addition, tissue embedded in the preferred matrix has been maintained at frozen temperatures for weeks without the need for other cryopreservation compounds such as DSMO.

The present invention also provides a tissue suspension comprising a hydrogel matrix and a plurality of physiologic tissue units embedded in the matrix. As described above, each physiologic tissue unit maintains physiologic cell-to-cell connections within the unit. Preferably, the tissue units are adhered together in pellet form prior to encapsulation by the matrix. A preferred hydrogel matrix includes a gelatin component having exposed polar groups. For example, the exposed polar groups may be amine and carboxyl groups. The gelatin component provides scaffolding for cellular attachment. The preferred gelatin component is denatured collagen. Denatured collagen contains polar and non-polar amino acids that readily form a gel based on amine, carboxyl group, hydroxyl group, and sulfhydryl group interactions.

The gelatin is present at a concentration of about 0.01 to about 40 mM, preferably about 0.05 to about 30 mM, most preferably about 1 to 5 mM. Advantageously, the gelatin concentration is approximately 1.6 mM. The above concentrations provide a solid phase at storage temperature and a liquid phase at transplant temperature. In order to increase cell binding, intact collagen may be added in small amounts to provide an additional binding network for the cells contained in the matrix. The final concentration of intact collagen is from about 0 to about 5 mM, preferably 0 to about 2 mM, most preferably about 0.05 to about 0.5 mM. In one embodiment, the concentration of intact collagen is about 0.11 mM. The gelatin component of the matrix of the present invention is mixed with a liquid composition. The liquid composition is preferably based upon a standard culture medium, such as Medium 199, supplemented with additives as described below.

Dextran is loosely polymerized around the gelatin component and facilitates cell attachment by preventing movement of the scaffolding provided by the gelatin. For tissue-containing transplants, this allows the cells within the tissue of the transplant to firmly attach to the matrix. Dextran is present at a concentration of about 0 to about 2 mM, preferably 0 to about 1 mM, most preferably about 0 to about 0.1 mM. In one embodiment, dextran is present in a concentration of about 0.086 mM.

The matrix also includes an effective amount of polar amino acids, such as arginine, lysine, histidine, glutamic acid, and aspartic acid, which further enhance the bioadhesiveness of the matrix. An effective amount is the amount necessary to increase the rigidity of the matrix and allow direct injection of the matrix with the transplant encapsulated therein into a host mammal without the need for further immunosuppression. In one embodiment, the concentration of polar amino acids is about 3 to about 150 mM, preferably about 10 to about 65 mM, and more preferably about 15 to about 40 mM.

Advantageously, the added polar amino acids comprise L-glutamic acid, L- lysine, and L-arginine. The final concentration of L-glutamic acid is about 2 to about 60 mM, preferably about 5 to about 40 mM, most preferably about 10 to about 20 mM. In one embodiment, the concentration of L-glutamic acid is about 15 mM. The final concentration of L- lysine is about 0.5 to about 30 mM, preferably about 1 to about 15 mM, most preferably about 1 to about 10 mM. In one embodiment, the concentration of L-lysine is about 5.0 mM. The final concentration of L-arginine is about 1 to about 40 mM, preferably about 1 to about 30, most preferably about 5 to about 15 mM. In one embodiment, the final concentration of L-arginine is about 10 mM.

Additionally, the matrix contains at least one nitric oxide inhibitor. Preferred nitric oxide inhibitors include L-cysteine, L-arginine analogues, cystine, and heparin. Preferably, the nitric oxide inhibitors include L-cysteine. The final concentration of L-cysteine is about 5 to about 500 μM, preferably about 10 to about 100 μM, most preferably about 15 to about 25 μM. In one embodiment, the final concentration is about 20 _μM.

Advantageously, aminoguanidine is also added to the matrix of the present invention. As indicated above, aminoguanidine is an L-arginine analogue and acts as a nitric oxide inhibitor. Other L-arginine analogues could also be used in the present invention. The final concentration of aminoguanidine is about 5 to about 500 μM, preferably about 10 to about 100 μM, most preferably about 15 to about 25 μM. In one embodiment, the final concentration is about 20 μM.

Additionally, the matrix of the present invention may include a superoxide inhibitor. A preferred superoxide inhibitor is ethylenediaminetetraacetic acid (EDTA). The concentration range for the superoxide inhibitor is about 0 to about 10 mM, preferably 1 to about 8 mM, most preferably about 2 to about 6 mM. In a preferred embodiment, the superoxide inhibitor is present at a concentration of about 4 mM.

Other additives known in the art may be included in the matrix. For example, although serum is not required, albumin or other nutrient sources may be added. If used, the serum or albumin is preferably derived from the same species as the cells to be encapsulated within the matrix.

Table 2 below lists particularly preferred components of the matrix of the present invention along with suitable concentrations as well as preferred concentrations for each component.

Table 2

The following examples are offered by way of illustration and not by way of limitation.

Experimental

Matrix Preparation

Place 835 ml of Medium 199 into a beaker. While stirring, heat the solution to 50°C. Using a syringe, add 20 ml of albumin to the stirred solution. Pipette 63.3 μl of L-cysteine, 1 ml of L-glutamine and 200 μl of aminoguanidine into the stirred beaker. Add the following gamma irradiated dry raw materials: 120 grams of denatured collagen, 50 grams of dextran, and 0.1 grams of intact collagen. Use a glass stirring rod to aid mixing of the dry materials into solution. Pipette 8 ml of EDTA into the solution. Pipette 5 ml of L-glutamic acid, 5 ml of L- lysine acetate, and 5 ml of L-arginine HC1 into the stirred beaker. Note that the solution will turn yellow. Use 10% NaOH to adjust the pH of the matrix solution to a final pH of 7.40 ± 0.05.

Tissue may be embedded in the matrix of the present invention using the following procedure. Aspirate the supernatant from centrifuged tissue pellets. Add a volume of cell culture medium and matrix to the tissue pellets. Add a volume of matrix approximately equal to about 4 times the pellet volume. Add a volume of cell culture medium to the tissue pellets equaling approximately 0.05 times the matrix volume added. Preferably, the cell culture medium is added to the tissue pellets prior to adding the matrix. Store the encapsulated tissue at refrigerated temperatures if not using immediately.

Example 1

Porcine liver was freshly harvested and mechanically separated according to the method described above and stored in the above-described hydrogel matrix. After eight weeks of storage at 4°C, H and E staining indicated that the separated hepatocytes had maintained normal morphology.

Example 2

A porcine pancreas was freshly harvested and mechanically separated according to the method described above. The freshly isolated tissue units were stained red with DTZ, indicating the presence of insulin within the islets. After 3.5 weeks at 4°C storage in the above-described hydrogel matrix, DTZ staining still indicated the presence of insulin within the islets. This indicates that the islet tissue remains functional for extended periods of time, despite the presence of digestive enzyme-containing acinar tissue. Using conventional separation techniques, the acinar tissue is enzymatically separated from the islet tissue in order to prevent digestion of the islet cells.

Many modifications and other embodiments of the invention will come to mind to one skilled in the art to which this invention pertains having the benefit of the teachings presented in the foregoing descriptions and associated drawings. Therefore, it is to be understood that the invention is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

THAT WHICH IS CLAIMED:

1. A method of preserving tissue viability during separation, comprising: providing intact tissue; placing the tissue in a cell culture medium, the cell culture medium comprising at least one nitric oxide inhibitor; and mechanically separating the tissue into a plurality of physiologic tissue units, each unit maintaining physiologic cell-to-cell connections within the unit.

2. A method according to Claim 1, wherein said step of mechanically separating the tissue comprises: cutting the tissue into slices; and tearing the slices of tissue into a plurality of physiologic tissue units.

3. A method according to Claim 2, wherein said tearing step comprises separating the tissue in a blender.

4. A method according to Claim 1, further comprising the steps of: concentrating the physiologic units within a solution of said cell culture medium; aspirating the supernatant of the solution; and collecting a tissue pellet, the tissue pellet comprising a plurality of physiologic units.

5. A method according to Claim 4, wherein said concentrating step comprises gravity settling the physiologic units.

6. A method according to Claim 1, wherein the tissue is selected from the group consisting of lung, liver, kidney, thymus, thyroid, heart, brain, and pancreas.

7. A method according to Claim 1 , wherein the tissue is organ tissue.

8. A method according to Claim 7, wherein the organ tissue is pancreatic tissue such that the physiologic units maintain physiologic cell-to-cell connections between acinar tissue and islet tissue.

9. A method according to Claim 2, wherein the slices are about 0.25 mm to about 6.4 mm across.

10. A method according to Claim 9, wherein the slices are about 0.25 mm to about 3.2 mm across.

11. A method according to Claim 1, wherein the physiologic tissue units are about 0.5 to about 3.0 mm across.

12. A method according to Claim 11 , wherein the physiologic tissue units are about 1.0 to about 1.5 mm across.

13. A method according to Claim 1, wherein the nitric oxide inhibitor is selected from the group consisting of L-cysteine, L-arginine analogues, cystine, and heparin.

14. A method according to Claim 1 , wherein the nitric oxide inhibitor is present in an amount of about 15 to about 600 μM.

15. A method according to Claim 14, wherein the nitric oxide inhibitor is present in an amount of about 200 to about 350 μM.

16. A method according to Claim 1, wherein the nitric oxide inhibitor comprises about 15 to about 250 μM of aminoguanidine and about 50 to about 300 μM of L- cysteine.

17. A method according to Claim 16, wherein the nitric oxide inhibitor comprises about 80 to about 120 μM of aminoguanidine and about 150 to about 200 _μM of L- cysteine.

18. A method according to Claim 1 , wherein the cell culture medium further comprises about 5 to about 150 mM of polar amino acids.

19. A method according to Claim 1, wherein the cell culture medium further comprises about 5 to about 150 mM of polar amino acids.

20. A method according to Claim 1 , wherein the cell culture medium further comprises about 1 to about 8 mM of a superoxide inhibitor.

21. A method according to Claim 20, wherein the superoxide inhibitor is EDTA.

22. A method according to Claim 1, further comprising embedding the tissue units within a hydrogel matrix.

23. A method according to Claim 22, wherein the hydrogel matrix comprises gelatin, dextran, at least one nitric oxide inhibitor, and an effective amount of polar amino acids, the polar amino acids selected from the group consisting of arginine, lysine, histidine, glutamic acid, and aspartic acid.

24. A method according to Claim 23, wherein the effective amount of polar amino acids comprises about 3 to about 150 mM of polar amino acids.

25. A method according to Claim 23, wherein the effective amount of polar amino acids comprises: about 2 to about 60 mM of L-glutamic acid; about 0.5 to about 30 mM of L-lysine; and about 1 to about 40 mM of L-arginine.

26. A method of preserving tissue viability during separation, comprising: providing intact tissue; placing the tissue in a cell culture medium, the cell culture medium comprising at least one nitric oxide inhibitor, the nitric oxide inhibitor comprising about 15 to about 250 μM of aminoguanidine and about 50 to about 300 _μM of L- cysteine; and mechanically separating the tissue into a plurality of physiologic tissue units, each unit maintaining physiologic cell-to-cell connections within the unit, said separating step comprising cutting the tissue into slices and tearing the slices of tissue into a plurality of physiologic tissue units.

27. A method according to Claim 26, wherein the tissue is pancreatic tissue such that the physiologic units maintain physiologic cell-to-cell connections between acinar tissue and islet tissue.

28. A method according to Claim 26, wherein the slices are about 0.25 mm to about 6.4 mm across.

29. A method according to Claim 26, wherein the physiologic units are about 1.0 to about 1.5 mm across.

30. A method according to Claim 26, further comprising embedding the tissue units within a hydrogel matrix.

31. A method of preparing insulin-producing cells for use in a transplant, said method comprising: providing intact pancreatic tissue, the pancreatic tissue comprising acinar tissue and islet tissue; placing the pancreatic tissue in a cell culture medium, the cell culture medium comprising at least one nitric oxide inhibitor, the nitric oxide inhibitor comprising about 15 to about 250 μM of aminoguanidine and about 50 to about 300 μM of L- cysteine; mechanically separating the pancreatic tissue into a plurality of physiologic tissue units, said separating step comprising cutting the tissue into slices about 0.25 mm to about 6.4 mm across and tearing the slices of tissue into a plurality of physiologic tissue units, each unit being about 0.5 to about 3.0 mm across and maintaining physiologic cell-to-cell connections between acinar tissue and islet tissue.

32. A method according to Claim 31 , further comprising: concentrating the physiologic units within a solution of said cell culture medium; aspirating the supernatant of the solution; and collecting a pancreatic tissue pellet, the tissue pellet comprising a plurality of physiologic units.

33. A tissue suspension, comprising: a hydrogel matrix, said matrix comprising gelatin, dextran, at least one nitric oxide inhibitor, and an effective amount of polar amino acids, the polar amino acids selected from the group consisting of arginine, lysine, histidine, glutamic acid, and aspartic acid; and a plurality of physiologic tissue units embedded in said matrix, each unit maintaining physiologic cell-to-cell connections within the unit.

34. A method according to Claim 33, wherein the effective amount of polar amino acids comprises about 3 to about 150 mM of polar amino acids.

35. A method according to Claim 33, wherein the effective amount of polar amino acids comprises: about 2 to about 60 mM of L-glutamic acid; about 0.5 to about 30 mM of L-lysine; and about 1 to about 40 mM of L-arginine.

36. A method according to Claim 33, wherein said tissue units comprise pancreatic tissue, said pancreatic tissue comprising physiologic cell-to-cell connections between acinar tissue and islet tissue.

37. A method according to Claim 33, wherein said tissue units are adhered together in pellet form.