MULTI-MEMBRANE IMMUNOISOLATION SYSTEM FOR CELLULAR

TRANSPLANT.

FIELD OF THE INVENTION

[0001] This invention relates to a multi-membrane immunoisolation system for cellular transplant that can be used in large animals and humans without immunosuppression.

FEDERAL FUNDING LEGEND

[0002] This invention was produced in part using funds from the Federal government under NASA contract NAG 5-12429. Accordingly, the Federal government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] The World Health Organization estimates that, as of the year 2000, over 176 million people suffer from diabetes mellitus worldwide. It is predicted that this number will more than double by the year 2030. In patients with insulin-dependent or type 1 diabetes mellitus, autoimmune processes destroy the insulin-producing beta cells of the pancreatic islets. Injection of human insulin, while somewhat effective, does not precisely restore normal glucose hoemostasis, which can lead to serious complications such as diabetic nephropathy, retinopathy, neuropathy and cardiovascular disease.

[0004] Recently, cellular transplantation has generated enthusiasm for treating a number of human diseases characterized by hormone or protein deficiencies, such as diabetes, Parkinson disease, Huntington disease, and others. However, a number of technical and logistical challenges have prevented cellular transplantation from working effectively. In particular, transplanted cells must be protected from immune attack by the transplant recipient. This often requires potent immunosuppressive agents having considerable toxicity that can expose the patient to a wide variety of serious side effects.

[0005] An alternative approach is to enclose the transplanted cells within a semipermeable membrane. In theory, the semi-permeable membrane is designed to protect cells from immune attack while allowing for both the influx of molecules important for cell function/survival and the efflux of the desired cellular product. This immunoisolation approach has two major potentials: i) cell transplantation without the need for immunosuppressive drugs and their accompanying side effects, and ii) use of cells from a variety of sources such as autografts (host stem-cell derived), allografts (either primary cells

or stem-cell derived), xenografts (porcine cells or others), or genetically engineered cells. While this technique has been effective in treating small mammals, such as rodents, the techniques were found to be ineffective when used to treat larger mammals.

[0006] Certain immunoisolation systems have been tested in large animal models, but many of those experiments were performed on spontaneous diabetic subjects or utilized immunosuppressive agents. See Sun et al. "Normalization of diabetes in spontaneously diabetic cyπomolgus monkeys by xenografts of microencapsulated porcine islets without immunosuppressant," J. Clin. Invest. 98:1417-22 (1996); Lanza et ah, "Transplantation of islets using microencapsulation: studies in diabetic rodents and dogs," J. MoI. Med. 77(1): 206-10 (1999); Calafiore R., 'Transplantation of minimal volume microcapsules in diabetic high mammalians," Ann NY Acad. ScL 875: 219-32 (1999); Hering et al., "Long term (>100 days) diabetes reversal in immunosuppressed nonhuman primate recipients of porcine islet xenographs," American J. Transplantation, 4: 160-61 (2004); and Soon-Shiong et al., "Insulin independence in a Type 1 diabetic patient after encapsulated islet transplantation," Lancet 343:950-951 (1994). Moreover, many of these experiments could not be reproduced to acceptable scientific standards. The lack of experimental control and consistency of those experiments has complicated scientific interpretation and limited their applicability.

[0007] Clearly, the promise of immunoprotection of living cells to treat hormone- deficient diseases has not been realized. Accordingly, what is needed in the art is a reproducible and effective cell therapy treatment that can be used in large mammals without the use of immunosuppressive drugs. This invention answers that need.

BRIEF SUMMARY OF THE INVENTION

[0008] This invention relates to a multi-membrane composition for encapsulating biological material, comprising (a) an inner membrane that is biocompatible with the biological material and possesses sufficient mechanical strength to hold the biological material within the membrane and provide immunoprotection from antibodies in the immune system of a host; (b) a middle membrane that possesses sufficient chemical stability to reinforce the inner membrane from the chemicals in the host; and (c) an outer membrane that is biocompatible with the host and possesses sufficient mechanical strength to shield the inner and middle membranes from non-specific immune response systems in the immune system of the host. The middle membrane also binds the inner membrane with the outer membrane.

[0009] This invention also relates to a multi-membrane composition capable of encapsulating biological material, that includes (a) a membrane comprising sodium alginate,

cellulose sulfate, poly(methylene-co-guanidine), and calcium chloride; (b) a membrane comprising a polycation; and (c) a membrane comprising a carbohydrate polymer having carboxylate or sulfate groups. The polycation is a poly-L-lysine, poly-D-lysine, ρoly-L,D- lysine, polyethylenimine, polyallylamine, poly-L-ornithine, poly-D-ornithine, poly-L,D- ornithine, poly-L-aspartic acid, poly-D-aspartic acid, poly-L,D-aspartic acid, polyacrylic acid, poly-L-glutamic acid, poly-D-glutamic acid, poly-L,D-glutamic acid, succinylated poly-L- lysine, succinylated poly-D-lysine, succinylated poly-L,D-lysine, chitosan, polyacrylamide, poly( vinyl alcohol) or combination thereof.

[0010] This invention also relates to a method of treating a subject suffering from diabetes or related disorders, comprising administering to the subject sufficient amounts of a composition containing insulin-producing islet cells, wherein the composition is a multi- membrane capsule that includes (a) an inner membrane that is biocompatible with the biological material and possesses sufficient mechanical strength to hold the biological material within the membrane and provide immunoprotection from antibodies in the immune system of the subject; (b) a middle membrane that possesses sufficient chemical stability to reinforce the inner membrane from the chemicals in the subject; and (c) an outer membrane that is biocompatible with the host and possesses sufficient mechanical strength to shield the inner and middle membranes from non-specific immune response systems in the immune system of the subject.

[0011] The invention also relates to a method of treating a subject suffering from diabetes or related disorders, comprising administering to the subject sufficient amounts of a composition containing insulin-producing islet cells, wherein the composition is a multi- membrane capsule that includes (a) a membrane comprising sodium alginate, cellulose sulfate, poly(methylene-co-guanidine), and calcium chloride; (b) a membrane comprising a polycation; and (c) a membrane comprising a carbohydrate polymer having carboxylate or sulfate groups. The polycation is poly-L-lysine, poly-D-lysine, poly-L,D-lysine, polyethylenimine, polyallylamine, poly-L-ornithine, poly-D-ornithine, poly-L,D-ornithine, poly-L-aspartic acid, poly-D-aspartic acid, poly-L.D-aspartic acid, polyacrylic acid, poly-L- glutamic acid, poly-D-glutamic acid, poly-L,D-glutamic acid, succinylated poly-L-lysine, succinylated poly-D-lysine, succinylated poly-L,D-lysine, chitosan, polyacrylamide, poly( vinyl alcohol), or combination thereof.

[0012] The invention also relates to a method of treating a large-mammal subject suffering from diabetes or related disorders with a cell therapy treatment that does not involve immunosuppression. The method comprises administering to the subject a cell therapy

treatment of a composition containing insulin-producing islet cells that provides a sustained release of insulin for at least 30 days. The composition does not exhibit significant . degradation during the sustained-release period.

[0013] This invention also relates to a capsule containing a biological material that, when introduced into a large mammal having a functioning immune system, secretes a bioactive agent for at least 30 days without incurring significant degradation caused by immune attack from the immune system.

[0014] This invention also relates to a method of stabilizing the glucose level in a patient for at least 30 days, comprising administering to a patient suffering from diabetes or related disorders a cell therapy treatment of a composition containing insulin-producing islet cells. The cell therapy treatment is not administered in conjunction with an additional treatment involving immunosuppression.

BRIEF SUMMARY OF THE DRAWINGS

[0015] FIG. 1: Biocompatibility of single-membrane capsules. Two single-membrane capsules prepared under identical formula and processing steps were photographed 30 days after being transplanted into intraperitonealJy into a normal mouse (left) and a normal mongrel dog (right).

[0016] FIG. 2: Biocompatibility of multi-membrane capsules in a large animal. The omentum of normal dog is shown more than six months after treatment having capsules loosely adhered to the omentum.

[0017] FIG. 3: Permeability of capsule membrane. The chart illustrates normalized retention time as a function of pore size distribution of capsule membrane.

[0018] FIG. 4: Capsule mechanical stability. The chart illustrates the mechanical strength of capsules of two different polymer concentrations by plotting the rupture load versus the capsule membrane thickness and size.

[0019] FIG. 5: Capsule stability. The chart illustrates the mechanical strength of two capsules as a function of time with different chemical compositions and membrane thickness.

[0020] FIG. 6: Perifusion of encapsulated islets. The secretion level of insulin- releasing islets was assessed in a cell perifusion system. Free islets (not encapsulated), islets encapsulated in a single-membrane system (encapsulated islets), and islets encapsulated in a multi-membrane system (encapsulated with layer) were independently assessed.

[0021] FlG. 7: Insulin secretion by retrieved encapsulated islets. Islets encapsulated in a multi-membrane capsule retrieved after being transplanted in a dog at 100 days post

transplantation were tested in a cell perifusion system.

[0022] FIG. 8: Blood glucose analysis of canine allotransplantation. The figure is an example of a canine model that has undergone a total pancreatectomy. The top paneJ illustrates the venous plasma glucose concentrations collected 12-18 hours following a meal. The lower panel illustrates the daily dosage of subcutaneous porcine insulin administered.

[0023] FIG. 9: Body weight analysis of canine allotransplantation. The top and bottom panels have been imported from FIG. 8. The middle panel shows the animal body weight monitored during the testing period.

[0024] FIG. 10: Fructosamine analysis of canine allotransplantation. The top and bottom panels have been imported from FIG. 8. The middle panel shows fructosamine measurements, an indicator of blood glucose level averaged over 2-3 weeks in diabetic subjects.

[0025] FIG. 1 1 : Re-transplantation of encapsulated islets in canine. This chart illustrates an initial allotransplantation and re-transplantation on a canine of islets encapsulated in a multi-membrane system.

[0026] FIG. 12: Intravenous Glucose Tolerance Test (IVGTT). The chart evaluates intravenous dextrose (300 mg/kg) administration in a canine having previously received a transplantation of islets encapsulated in multi-membrane system.

DETAILED DESCRIPTION OF THE INVENTION

[0027] Immυnoisolation systems have been developed that allow for the effective and sustained encapsulation of bioJogicaJ materia! in cellular therapy treatments. Any disease best treated by the release of a cellular product (hormone, protein, neurotransmitter, etc.) is a candidate for transplantation of immunoisolated cells. Potential cell types for immunoisolation include pancreatic islets, hepatocytes, neurons, parathyroid cells, and cells secreting clotting factors. When using encapsulating pancreatic islets in a cell therapy system, the system offers a surrogate bio-artificial pancreas and a functional treatment to a patient suffering from diabetes.

[0028] This invention relates to a multi-membrane composition for encapsulating biological material, comprising (a) an inner membrane that is biocompatible with the biological material and possesses sufficient mechanical strength to hold the biological material within the membrane and provide immunoprotection from antibodies in the immune system of a host; (b) a middle membrane that possesses sufficient chemical stability to reinforce the inner membrane from the chemicals in the host; and (c) an outer membrane that is biocompatible with the host and possesses sufficient mechanical strength to shield the inner and middle membranes from non-specific immune response systems in the immune system of the host. The middle membrane also binds the inner membrane with the outer membrane.

[0029] The multi-membrane composition is a composition that contains at least three membranes. The composition is preferably either a capsule or a composition that has the ability to encapsulate biological material. However, other systems may also work.

[0030] The inner membrane should be biocompatible with the biological material. That is, the biological material should not interact with the biological material in a manner that would kill or otherwise be detrimental to the biological material. The inner membrane should also possess sufficient mechanical strength to hold the biological material within the membrane and provide immunoprotection from antibodies in the immune system of a host.

[0031] The middle membrane possesses sufficient chemical stability to reinforce the inner membrane from the chemicals in the host. The chemical stability provided by the middle membrane also assists both the inner membrane and outer membrane in withstanding the effects of the chemicals in the host. Common chemicals in the host include sodium, calcium, magnesium, and potassium ions, as well as other chemicals in the bloodstream. The middle membrane is chemically stabile against those chemicals, which allows it to retard the deterioration of the membranes. This prolongs the life of the membranes and consequently the biological material that is being enclosed by the inner membrane. The middle membrane

also binds the inner membrane with the outer membrane, preferably through affinity binding. Binding the membranes together in this manner provides a crosslinking effect that creates a tighter and more cohesive multi-membrane composition, and eliminates or reduces the possibility of membrane separation.

[0032] The outer membrane should be biocompatible with the host. Because the outer membrane is the portion of the multi-membrane composition that is contact with the host, it should be sufficiently biocompatible that the host does not treat the composition as a foreign object and reject it or attempt to destroy it. The term "biocompatible" as used in this context refers to the capability of the implanted composition and its contents to avoid detrimental effects of the host's various protective systems, such as the immune system or foreign body fϊbrotic response, and remain functional for a significant period of time. In addition, "biocompatible" also implies that no specific undesirable cytotoxic or systemic effects are caused by the composition and its contents such as would interfere with the desired immunoisolation functionality.

[0033] The outer membrane also should possess sufficient mechanical strength to shield the inner membranes from the non-specific innate immune system of the host. The innate immune system, which includes neutrophils, macrophages, dendritic cells, natural killer cells, and others, when activated, can attack the multi-membrane composition or capsule by engulfing it. It can also stimulate the activities of antibodies to attack the islets inside of the composition.

[0034] The combination of these features in the separate membranes allows the composition to jointly function in a manner not afforded by a single. membrane. In particular, each membrane performs at least one function in a manner that allows the multi-membrane composition to meet the dichotomy goals of a large-animal transplantation. Each membrane is designed to allow optimal mass transport while maintaining islet health and functionality.

[0035] For instance, increasing the membrane pore sizes to improve mass transfer can jeopardize capsule stability. Likewise, increasing polymer concentration to improve capsule stability can decrease the mass transport. These dichotomies can lead to compromises on capsule design and performance. In the preferred system, no single membrane is required to compromise its design to meet the multi-faceted dichotomy goals. Each membrane is designed to perform only one or two specific tasks. Together, the multiple membranes meet most or all of the dichotomy goals of cellular transplants in a large animal model without the need for immunosuppression.

[0036] The membrane thickness of the inner membrane preferably ranges from about

5 to about 100 microns. More preferably, the membrane thickness ranges from about 10 to about 60 microns, and most preferably, the thickness ranges from about 20 to about 40 microns. Generally, the thicker the membrane, the more mechanical strength is provided. However, when a, membrane becomes too thick, mass transport capabilities start to diminish.

[0037] The middle membrane typically has a thickness of less than about 5 microns, preferably about 1-3. The outer membrane typically has a thickness ranging from about 5 to about 500 microns, preferably ranging from about 100 to about 300 microns; however, a outer membrane thickness ranging from about 10 to about 30 microns is also suitable.

[0038] The multi-membrane composition has a porosity that is sufficiently large enough to allow for the release of bioactive agents from the biological material but sufficiently small enough to prevent the entry of antibodies from an immune system. There are known antibodies that destroy living cells that should, when possible, be prevented from entering the multi-membrane composition. For instance, the antibody IgM, which has a molecular weight of about 300 kilodaltons, can be particularly deadly when exposed to islet- containing capsules. In view of these known antibodies, the porosity cutoff (i.e., the considerable drop off of the number of pores larger than the cutoff size) of the multi- membrane composition should be less than 300 kilodaltons. Additionally, because membranes are often formulated as a random network system, the porosity cutoff is preferably below about 250 kilodaltons. This better assures that the designed membrane contains very few or no pores larger than 300 kilodaltons.

[0039] On the other hand, the porosity cutoff should be larger than about 50 kilodaltons to ensure that the biological material has the ability to be freely released from the multi-membrane composition. The porosity cutoff preferably ranges from about 50 kilodaltons to about 250 kilodaltons to permit the passage of molecules having a molecular weight less than about 50 kilodaltons while preventing the passage of molecules having a molecular weight greater than about 250 kilodaltons. More preferably, the porosity cutoff ranges from about 80 kilodaltons to about 150 kilodaltons.

[0040] In one embodiment of the invention, each membrane has a different porosity, with the inner membrane having a porosity cutoff ranging from about 50 to about 150 kilodaltons; the middle membrane having a porosity cutoff ranging from about 100 to about 200 kilodaltons; and the outer membrane having a porosity cutoff ranging from about 150 to about 250 kilodaltons. Having membranes of varying porosity assists, among other areas, in mass transport and immunoprotection.

[0041] The biological material may be any material that is a capable of being encapsulated by a membrane. Typically, the biological material is a cell or group of cells that can provide a subject with some therapeutic result when introduced into the subject. Preferably, the biological material is selected from the group consisting of pancreatic islets, hepatocytes, choroid plexuses, neurons, parathyroid cells, and cells secreting clotting factors. Most preferably, the biological material is pancreatic islets or other insulin-producing islets capable of treating a patient suffering from diabetes.

[0042] The bioactive agent is any agent that can be released or secreted from the biological material. For example, pancreatic islets have the capability of secreting the bioactive agent insulin; choroid plexuses have the capability of secreting cerebral fluids; neurons have the capability of secreting agents such as dopamine that can effect the nervous system; and parathyroid cells have the capability of secreting agents that can effect metabolism of calcium and phosphorus in a subject. Preferably, the bioactive agent is insulin.

[0043] The host can include any subject that is in need or otherwise capable of receiving an encapsulated multi-membrane composition. While the host can include small mammals, such as rodents, the multi-membrane composition is particularly suitable for large mammals. Preferably, the host is a human.

[0044] While the multi-membrane composition should contain an inner, middle, and outer membrane, it may contain one or more additional membranes. Additional membranes may be desirable to provide better or more enhanced features to those provided by the three- membrane system. For instance, the additional membranes can, independently or jointly, provide additional immunoprotection, mechanical strength, chemical stability, and/or biocompatibility to the multi-membrane composition.

[0045] This invention also relates to a multi-membrane composition capable of encapsulating biological material, comprising (a) a membrane containing sodium alginate, cellulose sulfate, and a multi-component polycation; (b) a membrane containing a polycation; and (c) a membrane comprising a carbohydrate polymer having carboxylate or sulfate groups.

[0046] One membrane should contain sodium alginate, cellulose sulfate, and a multi- component polycation. The polycation is preferably contains a combination of poly(methy]ene-co-guanidine) and either calcium chloride, sodium chloride, or a combination thereof. This membrane may be the encapsulation system described in U.S. Patent No. 5,997,900, herein incorporated by reference in its entirety.

[0047] A second membrane should contain a polycation. Preferably, the polycation is selected from the group consisting of poly-L-lysine, poly-D-lysine, poly-L,D-lysine,

polyethylenimine, polyallylamine, poly-L-ornithine, poly-D-ornithine, poly-L,D-ornithine, poly-L-aspartic acid, poly-D-aspartic acid, poly-L,D-aspartic acid, polyacrylic acid, poly-L- glutamic acid, poly-D-glutamic acid, poly-L,D-gJutamic acid, succinylated poly-L-]ysine, succinylated poly-D-lysine, succinylated poly-L,D-]ysine, chitosan, polyacryl amide, poly(vinyl alcohol) and combinations thereof. More preferably, the polycation is selected from the group consisting of poly-L-lysine, poly-D-lysine, poly-L,D-lysine, poly-L-ornithine, poly-D-ornithine, ;poly-L,D-ornithine, chitosan, polyacrylamide, poly(vinyl alcohol), and combinations thereof. Most preferably, the polycation is poly-L-]ysine.

[0048] The second membrane also preferably contains at least one compound selected from the group consisting of sodium alginate, cellulose sulfate, and poly(methylene-co- guanidine). More preferably, the second membrane contains a polycation and all three compounds. Most preferably, the second membrane contains poly-L-lysine, sodium alginate, cellulose sulfate, and poly(methylene-co-guanidine).

[0049] The third membrane contains a carbohydrate polymer having carboxylate or sulfate groups. The carbohydrate polymer preferably is selected from the group consisting of sodium carboxymethyl cellulose, low methoxy pectins, sodium alginate, potassium alginate, calcium alginate, tragacanth gum, sodium pectate, kappa carrageenaπs, and iota carrageenans. More preferably, the carbohydrate polymer is selected from the group consisting of sodium alginate, potassium alginate, and calcium alginate. Most preferably, the carbohydrate polymer is sodium alginate.

[0050] The third membrane also preferably contains an inorganic metal salt. Suitable metal salts include calcium chloride, magnesium sulfate, manganese sulfate, calcium acetate, calcium nitrate, ammonium chloride, sodium chloride, potassium chloride, choline chloride, strontium chloride, calcium gluconate, calcium sulfate, potassium sulfate, barium chloride, magnesium chloride, and combinations thereof. Preferably, the inorganic metal salt is selected from the group consisting of calcium chloride, ammonium chloride, sodium chloride, potassium chloride, calcium sulfate, and combinations thereof. Most preferably, the inorganic metal salt is calcium chloride.

[0051] In the multi-membrane composition, the first membrane is preferably the inner membrane, the second membrane is preferably an inner or middle membrane, and the third membrane is preferably the outer membrane. The multi-membrane composition may also contain one or more additional membranes.

[0052] Preferably, the multi-membrane composition is a five-component/three- membrane capsule system. The five components are sodium alginate (SA), cellulose sulfate

(CS), poly(methylene-co-guanidine) (PMCG), calcium chloride (CaCIa), and poly-L-Lysine (PLL). The inner membrane is the same PMCG-CS /CaCl ₂ -Alginate membrane successfully tested in small-animal models. This membrane is designed to provide a proper balance between immunoisolation and mass transport. The middle membrane is a preferably a thin interwoven PMCG-CS/ PLL-Alginate membrane that reinforces the inner membrane. Strong ionic bonds, for example those present in the PMCG-CS/ PLL-Alginate system, can assist in providing chemical stability. Additionally, having a thin membrane with a relatively large pore size can assist in allowing the membrane to not upset the balance between immunoisolation and mass transport of the inner membrane. The middle membrane can also provide impedance match for the inner and outer membranes by gradually increasing the PLL concentration of the middle membrane outwardly to bind the inner membrane with the outer membrane. An outer membrane of CaCl ₂ /A]ginate shields the PMCG and PLL of the two inner membranes from the host immune system. This membrane improves the biocompatibility of the capsule and can also provide additional mechanical strength for stability as well as immune protection.

[0053] This invention also relates to a method of treating a subject suffering from diabetes or related disorders, comprising administering to the subject sufficient amounts of a composition containing insulin-producing islet cells. The composition is a multi-membrane capsule comprising: (a) an inner membrane that is biocompatible with the biological material and possesses sufficient mechanical strength to hold the biological material within the membrane and provide immunoprotection from antibodies in the immune system of the subject; (b) a middle membrane that possesses sufficient chemical stability to reinforce the inner membrane from the chemicals in the subject; and (c) an outer membrane that is biocompatible with the host and possesses sufficient mechanical strength to shield the inner and middle membranes from non-specific immune response systems in the immune system of the subject.

[0054] Diabetes and related disorders include, but are not limited to, the following disorders: Type 1 diabetes, Type 2 diabetes, maturity-onset diabetes of the young (MODY), latent autoimmune diabetes adult (LADA), impaired glucose tolerance (IGT), impaired fasting glucose (IFG), gestational diabetes, and metabolic syndrome X. Preferably, the method is used to treat Type 1 diabetes or Type 2 diabetes.

[0055] The subject may be any animal thai suffers from diabetes or related disorders. Preferably, the subject is a large mammal, such as a human.

[0056] Insulin-producing islet cells are preferably pancreatic islets, however, other cells capable of producing insulin are suitable. Porcine or human pancreatic islets are preferred, especially if the subject is a human.

[0057] This invention also relates to a method of treating a subject suffering from diabetes or related disorders, comprising administering to the patient sufficient amounts of a composition containing insulin-producing islet cells. The composition is a multi -membrane capsule comprising: (a) a membrane containing sodium alginate, cellulose sulfate, and a multi-component polycation; (b) a membrane containing a polycation; and (c) a membrane containing a carbohydrate polymer having carboxylate or sulfate groups. The polycation is in membrane (b) is selected from the group consisting of poly-L-lysine, poly-D-lysine, poly- L,D-lysine, polyethylenimine, polyallylamine, poly-L-ornithine, poly-D-ornithine, poly-L,D- ornithine, poly-L-aspartic acid, poly-D-aspartic acid, poly-L,D-aspartic acid, polyacrylic acid, poly-L-glutamic acid, poly-D-glutamic acid, poly-L,D-glutamic acid, succinylated poly-L- lysine, succinylated poly-D-lysine, succinylated poly-L,D-lysine, chitosan, polyacrylamide, poly(vinyl alcohol), and combinations thereof. The multi-membrane composition may also contain one or more membranes in addition to the three discussed above.

[0058] This invention also relates to a method of treating a large-mammal subject suffering from diabetes or related disorders with a cell therapy treatment that does not involve immunosuppression. The method comprises administering to the subject a cell therapy treatment of a composition containing insulin-producing islet cells that provides a sustained release of insulin for at least 30 days. The composition does not exhibit significant degradation during the sustained-release period.

[0059] As known in the art, cell therapy is the transplantation of human or animal cells to replace or repair damaged or malfunctioning tissues, and/or cells. The types of cells that are administered correspond in some way with the organ or tissue in the patient that is failing. In the context of a subject suffering from diabetes or related disorders, cell therapy treatment involves the transplantation of insulin-producing cells that can replicate the function of pancreatic cells and release insulin into the subject upon the advent of certain conditions, namely an elevated glucose level in the subject.

[0060] A cell therapy treatment typically involves the introduction of either xenogenic (animal) cells (e.g., from sheep, cows, pigs, and sharks) or cell extracts from human tissue. The cells can be introduced through implantation, transplantation, injection or other means known in the art. Cells can be directly introduced into the host or introduced through cell encapsulation or special coatings on the cells designed to trick the immune

system into recognizing the new cells as native to the host. Two general cell encapsulation methods have been used: microencapsulation and macroencapsulation. Typically, in microencapsulation, the cells are sequestered in a small permseJective spherical container, whereas in macroencapsulation the cells are entrapped in a larger non-spherical membrane. Various polymeric materials have been used to form the membrane of the capsules in the encapsulation methods.

[0061] The cell therapy treatment preferably involves the transplantation of the encapsulated cells into the body cavity of the subject. This may be performed by creating a surgical opening in the body cavity and introducing the encapsulated cells into the body cavity through the opening. This may be accomplished through plausibly simple techniques, such as pouring the encapsulated cells into a funnel-type device that carries them through the opening and introduces them into the body cavity. Other techniques known in the art, such as hypodermic injections, may also be used.

[0062] Once inside the body cavity, the encapsulated cells are then able to freely move in the body cavity. Typically, the encapsulated cells will end up on the omentum of the subject. The omentum is a preferable place for the encapsulated cells because there is little danger of the cells interfering with the functions of the omentum. In contrast, if the encapsulated cells were to attach themselves to the outer walls of another organ, such as the liver or kidney, there is a chance that the encapsulated cells could disrupt the function of those organ, leading to other medical concerns.

[0063] The encapsulated cell therapy treatment is not administered with immunosuppressive agents designed to suppress a functioning immune system or otherwise prevent the immune system of the subject from rejecting the cell therapy treatment. Many cell therapy treatments require the use of immunosuppressive agent to ensure that the biological material being transplanted is not attacked and rejected by the immune system of the host. While immunosuppressive agents increase the chance that the host will accept the cell therapy treatment, it has been well documented that immunosuppressive drugs can cause deleterious effects to the host. In particular, immunosuppressive agents lower a subject's resistance to infection, make infections harder to treat, and increase the chance of uncontrolled bleeding. The drugs may also be harmful to the islets.

[0064] The term "sustained release," as used herein, refers to the continual release of the biological agent from the biological material during instances when the release should take place. For instance, if the biological material is a pancreatic islet and the biological agent is insulin, the pancreatic islets should, after transplantation, continually release insulin

into the host any time the pancreatic islets recognize that the glucose level of the host has reached a certain point. After the glucose level in the host has been maintained, the pancreatic islets will temporarily cease secreting additional insulin. However, when the glucose levels in the host again reach a point where insulin is needed, the temporarily- dormant pancreatic islets will again begin to secrete insulin. This type of continual release is an example of sustained release.

[0065] The sustained-release period should last at least 30 days. Preferably, it lasts at least 60 days; more preferably, at least 120 days; and most preferably, at least 180 days. The longer the composition is able to provide a sustained release of insulin, the longer the patient will be functioning on the cell therapy treatment alone without needing additional treatment. For instance, if the cell therapy treatment is able to last for at least 180 days, a patient will only need to receive a booster treatment approximately once every six months. This allows a diabetic patient a significantly increased amount of freedom to pursue daily activities without having to continually monitor their disorder and correct for high or low blood sugars and take insulin by injection or otherwise to counterbalance carbohydrate intake and regular and continual release of glucose into the bloodstream by the liver. This will also allow for overall greater glycemic control by reducing the occurrence of insulin shock or ketoacedosis as well as preventing or delaying the onset of diabetic related complications.

[0066] When a composition containing cells is effectively attacked by the immune system of a host, the immune system can damage or destroy the composition, causing significant degradation to the composition. There are two main avenues the immune system of a host can attack a foreign material, in this case a composition containing cells. First, the while blood cells in a host can either consume the composition containing the cells or adhere to the surface and suffocate the biological material inside. Second, the immune system of a host can generate specific antibodies that have the ability to penetrate the pores of a composition and attack the biological material inside. Either of these attacks will cause some form of degradation of the composition. However, if the composition contains sufficient biocompatibility, chemical stability, and mechanical strength the damage caused by the immune system and the degradation of the composition will be minimal. On the other hand, if the composition is not sufficiently biocompatible and chemically stable, and does not possess sufficient mechanical strength, the composition will be susceptible to attacks and the corresponding destruction caused by those attacks. Effective attacks wiJ] damage or destroy the biological material in the composition and leave the composition in a degraded state.

[0067] Conventional cell therapy systems, when introduced into large mammals, such as the canine model, were found to be stable for less than one month. An example of a conventionally produced encapsulated cell that experienced significant degradation in the canine model may be found in FIG. 1. FIG. 1 depicts two single-membrane capsules prepared under identical formula and processing steps were transplanted into intraperitoneally into a normal C57/B16 mouse (left) and a normal mongrel dog. Capsules were retrieved 30 days later and photographed. The rodent capsule shows no degradation while the canine capsule shows significant degradation due to breakage in the capsule and destruction of the biological material by the immune system of the host.

[0068] This invention also relates to a capsule containing a biological material that, when introduced into a large mammal having a functioning immune system, secretes a bioactive agent for at least 30 days without incurring significant degradation caused by immune attack from the immune system.

[0069] The term "capsule" refers to any type of encapsulation device used in an encapsulation system, including microencapsulation and macroencapsulation. Preferably, the capsule is a spherical capsule, such as those used in microencapsulation techniques. The capsule may be formed using special apparatuses and reactors, such as those described in U.S. Patent Nos. 5,260,002 and 6,001 ,312, herein incorporated by reference in their entirety.

[0070] This invention also relates to a method of stabilizing the glucose level in a patient for at least 30 days, comprising administering to a patient suffering from diabetes or related disorders a cell therapy treatment of a composition containing insulin-producing islet cells. The cell therapy treatment is not administered in conjunction with an additional treatment involving immunosuppression.

[007J] As is well known in the art, patients suffering from diabetes or related disorders have glucose levels in their bodies that are not stabilized through a properly functioning pancreas. Stabilizing the glucose levels in diabetic patients or any other type of patient suffering from an instable glucose level, can be achieved through a cell therapy treatment of a composition containing insulin-producing islet cells. The cell therapy treatment can stabilize the glucose level for at least 30 days; preferably, at least 60 days; more preferably, at least 120 days; and most preferably, at least 180 days.

[0072] There are several known processes to prepare cells for encapsulation that may be utilized. One form involves extracting cells from the patient they are to be used on and then culturing them in a laboratory setting until they multiply to the level needed for transplant back into the patient. However, cell multiplicity has not yet been achieved for all

types of cells, such as pancreatic cells. Another procedure uses freshly removed animal tissue, which has been processed and suspended in a saline solution. The preparation of fresh cells then may be either injected immediately into the patient, or preserved by being freeze- dried or deep-frozen in liquid nitrogen before being injected. Cells may be tested for pathogens, such as bacteria, viruses, or parasites, before use.

[0073] Porcine pancreatic islet cells may be harvested from the pancreases of pigs or piglets obtained from research laboratories or local slaughterhouses. Preferably, the pigs or piglets are specific pathogen free (SPF) animals that have been bred and monitored for the purpose of islet donation. Alternatively, neonatal islets, which contain nascent or not fully developed immune systems, fetal pig islets, which contain islets that are matured in the laboratory, or embryonic cells from stem cell research, which contain cells that may be regenerated in the laboratory, may also be used for supplying islets. Human islets that are donated from healthy patients theoretically represent a good source of islets and tend to have less immune problems. However, currently not enough human islets are donated per year, effectively preventing, as a practical measure, human islets from being used as a sole source of islets.

[0074] The following examples are intended to illustrate the invention. These examples should not be used to limit the scope of the invention, which is defined by the claims.

EXAMPLES

[0075] Capsule Design: The following examples utilize a five-component/three- membrane capsule system. This system provides design flexibility to conduct systematic tradeoff studies to optimize capsule performance in large animals. The five components of the system are sodium alginate (SA), cellulose sulfate (CS), poly(methylene-co-guanidine) (PMCG), calcium chloride (CaCl ₂ ), and poly-L-Lysine (PLL). The inner membrane is the PMCG-CS /CACL ₂ -Alginate (porosity of approximately 10O kDa, thickness of 20-40 micron); the middle membrane is a thin interwoven PMCG-CS/ PLL-Alginate membrane (porosity of approximately 150 kDa, thickness of 1 -3 micron); and the outer membrane is CaCl ₂ /Alginate (porosity of approximately 250 kDa, thickness of 100-300 micron).

[0076] Capsule Optimization: The following tests were performed to optimize the capsule. Because all the membranes should work together, it is difficult to predict how one membrane will affect another after the capsule has been fabricated. For instance, the process of forming the middle membrane can alter the performance of the inner membrane. Likewise,

the process of forming the outer membrane can alter the performances of the middle and inner membranes. Additionally, advance characterization of each membrane individually does not predict how the multi -membrane capsules will function together inside transplantation hosts. For these reasons, capsule formation was treated as a total system with multiple parameters listed in the table below, with each membrane as a component. The desired function of each component (membrane) was listed, and the total performance of the system (capsule) was measured after fabrication.

Reasents/Steos in < Capsule Formation and optimization

# 1 2 3 4 5 6 7 8 9 10 1 1 12

Reagent/Step PA PA PC RT PC RT PC RT PA PC RT PA 1 2 1 1 2 2 3 3 3 4 4 4

PA = polyanioπ; RT = reaction time; PC = polycatioπ;

Capsule Design Parameter Membrane Formation Parameters (see #1 -12 above) Mass Transport (T) 1, 2, 3, 4, 5, 6, 7, 8 Immune Protection (P) All Biocompatibility 9, 10, 1 1, 12 Sphericity/Centering 1, 2, 3, 4, Stability (S) 5, 6, 7, 8

[0077] Capsule performance: The multi-membrane composition was designed to be biocompatible, achieve effective mass transport, provide immune protection, provide mechanical strength to the biological material, and provide chemical stability.

[0078] Biocompatibility: Biocompatibility of the capsules depends on shielding the immune-genesis components of the capsules from the transplantation host. Long-term biocompatibility of the capsule membrane was demonstrated when examination of encapsulated islets transplanted into a healthy dog for six and a half months revealed no complications. See FIG. 2.

[0079] FIG. 2 depicts the omentum of normal dog shown more than six months after treatment (dog received encapsulated islets on 2/14/01 and was sacrificed on 8/14/01). Before sacrifice, no complications were observed in the animal, and post sacrifice, no abnormalities were observed in or on the organs. The figure shows minimal inflammatory response and mild vascularization of the omentum. A few capsules (less than 1 %), were observed to contain a scant amount of fibrin and rare mononuclear cells adherent to the surface. The surface of the vast majority of capsules retrieved from the dog were clean and transparent, and barely visible with the naked eye but readily apparent under microscope. Evidence of tissue reactivity has been minimal. There was no observed involvement of any other organ system in the splanchnic bed. The capsules loosely adhered to the omentum and were easily washed off, indicating that the capsules were anchored but not imbedded in the omentum. Capsule integrity was excellent with minimal capsule "breakage" observed. The retrieved encapsulated islets removed after six and a half months were still alive.

[0080] Mass Transport: Using interwoven pipes model, mass transport is proportional to R ⁴ ZD, where R is average pore size, and D is the membrane thickness. See Wang T., "New Technologies for Bϊoartifical Organs,'Mrfi/: Organs, 22, 1 : p. 68-74 (1998), herein incorporated by reference in its entirety. The membrane pore size can be measured using the size exclusion chromatography method. See Brissova et a]., "Control and measurement of permeability for design of microcapsule cell delivery system," J. Biomed. Mat. Res., 39:61-70 (1998), herein incorporated by reference in its entirety.

[0081] FIG. 3 demonstrates the pore size distribution of a capsule membrane with a cutoff of 80 KDa (about 12 nanometers in diameter). This pore size is large enough for the glucose and insulin to enter and exit, and small enough to keep the immune system from penetrate all the way to the core of the capsules where islets reside. The chart illustrates normalized retention time as a function of pore size distribution of capsule membrane. Pore size of the capsular membrane was determined by size exclusion chromatography (SEC) that measures the exclusion of dextran solutes from the column packed with microcapsules. The measured values of solute size exclusion coefficients (K _SEC ) and known size of solute molecules allow the membrane pore size distribution and capsule permeability to be estimated.

[0082] Immune protection: Using random walk model, immune protection is proportional to D ² / R ² , where D is the membrane thickness, and R is the average pore size. See Wang T., "New Technologies for Bioartifical Organs," Artif. Organs, 22, 1 : p. 68-74 (1998). In general, the immune protection goal is inversely proportional to the mass transport goals. However, their power dependences on membrane thickness and pore size are sufficiently different that it is possible to adjust the parameters to satisfy both goals simultaneously.

[0083] Mechanical strength: Mechanical strength of the capsules was measured by placing an increasing uniaxial load on the capsule until the capsule burst. The capsule mechanical strength, a function of membrane thicknesses, can be adjusted anywhere from a fraction of a gram to many tens of gram load to meet the transplantation goals without altering the permeability of the capsule.

[0084] FlG. 4 illustrates the mechanical strength of capsules of two different polymer concentrations by plotting the rupture load versus the capsule membrane thickness and size. The slope of the curve represents the rupture stress and thereby indirectly the inherent strength of the capsular membrane. The chart measures mechanical burst strength of capsules by placing them on a uniaxial load. The solid circles represent 0.6-0.6 alginate-CS capsules,

the open circles represent 0.9-0.9 alginate-CS capsules, and the solid square represents a PLL-alginate system. As can be seen in the chart, while certain polymers are stronger than others, it is generally observed that thicker membranes tend to be stronger membranes.

[0085] Stability: Stability of the capsules depends largely on the stability of chemical bonds and the membrane thickness. The intra peritonea fluid of a large animal such as dog can react chemically with the capsule membrane thus weaken the mechanical strength.

[0086] FlG. 5 illustrates the mechanical strength of two capsules with different chemical compositions and membrane thickness. The stability was experimentally determined by measuring the length of time for the capsules to loss its mechanical strength by a factor of 1/e incubated in dog serum at 40° C. It is believed that a properly designed capsule system can last years in a hostile environment of peritonea of a large animal. In FIG. 5, capsule mechanical strength was measured as a function of time as the capsules were incubated in dog serum at 40° C. The solid diamond represent 0.6-0.6 alginate-CS capsules, the solid squares represent 0.9-0.9 alginate-CS capsules, and the open squares represent 0.6- 0.6 alginate-CS capsules. Stability is shown by the least amount of fluctuation over time. In the chart, the 0.6-0.6 alginate-CS capsules showed the least amount of fluctuation and would thus be considered the most stable capsules of the three tested.

[0087] The biocompatibility and functional capacity of the multi-membrane encapsulated islets has been studied in a pancreatectomized canine model. The animal's size and hence blood volume permits the daily evaluations of plasma glucose and insulin, clinical assessments of glucose tolerance and evaluations of biocompatibility and safety. In addition, the canine model is widely utilized model of human glucose homeostasis and diabetes. Total pancreatectomy in the canine results in complete absence of endogenous insulin and thus assessments of insulin concentration can be directly assessed to the function and responsiveness of the encapsulated islets.

[0088] Canine preparation: mongrel canines of either sex with a mean wt of 7.6 kg were studied. The animals were housed in a facility that met the American Association for the Accreditation of Animal care guidelines. All animal care procedures were reviewed and approved by Vanderbilt's Institutional Animal Care and Use Committee. Seventeen to twenty four days prior to encapsulated islet intraperitoneal administration, a total pancreatectomy was performed as described below. In the posi-operative period animals are fed a standard diet of chow and canned diet (34% protein, 14.5% fat, 46% carbohydrate, and 5.5% fiber) based on dry weight. Exocrine pancreatic enzymes, lipase (70,000 U), amylase (210,000 U)

and protease (210,000U) were administered along with their meal in order to assist in food digestion and compensate for the absence of exocrine pancreatic function. Animals received daily insulin injections in adjusted dosages to maintain euglycemia at 12 hours post feeding without glycosuria during 24 hours. The insulin requirements generally range from 0.6-0.9 U/kg Regular Pork and 1.0-1.3 U/kg NPH Pork, q 24hr.

[0089] Encapsulated Islet Administration: After pancreatectomy, daily insulin requirements were allowed to stabilize. Animals were fasted 12 hours and placed under general anesthesia using propofol (4.4 mg.kg, FV) and lsoflurane (2.0% with O ₂ , inhalation). A 1.5 cm midline laparotomy was performed and a 7.0 mm LD. cannula is inserted into the peritoneal space. A funnel is connected to the free end of the cannula. Encapsulated islets suspended in modified Hanks solution containing canine albumin were administered into the abdominal space at room temperature. Total administered packed volume of capsules was 150-200 ml. The intraperitoneal cannula was immediately removed and the laparotomy incision closed. The animal was allowed to recover and immediately fed her/his daily ration. The ration was consumed within 2 hours from the time of the encapsulated islet administration. Six to eight hours post administration of food, blood was collected for the assessment of glycemic status and daily collections were performed thereafter for 3 days.

[0090] Daily and clinical assessments: Following the immediate post-administration period, animals were fed the standard daily rations and blood collections were performed on 2-3 day intervals for the determination of glucose and insulin. At the time of blood collections, animals were weighed and general physical conditions were assessed. An oral glucose tolerance test was performed at 2-4 weeks following encapsulated islet administration. Following an 18-hour fast, an 18-gauge angiocath was placed into either the left or right jugular vein for the collection of blood. Dextrose (0.7 gm/kg) was administered orally following the collection of a baseline blood sample. Blood samples were collected at 2.5-minute intervals for the first 20 minutes and 5 and 10-minute intervals thereafter for the 3-hour duration of the test. Plasma glucose levels were determined by the glucose oxidase method using a Beckman Glucose Il analyzer (Beckman Instruments Palo Alto CA.). Plasma insulin was determined by radioimmunoassay using a double-antibody system.

[0091] On the day of encapsulated islet administration, exogenous insulin is withheld and blood-glucose levels were monitored. No immune-suppressive drugs were administered to the animals.

[0092] Pancreatic islet isolation and evaluation: For the isolation of pancreatic islets, mongrel canines (20-28 kg body weight) were placed under general anesthesia following an

18-hour fast. A midline laparotomy was performed. The gastroduodenal, splenic and pancreaticoduodenal veins and arteries were isolated and a ligature was placed around each vessel. The main pancreatic duct was identified at the point of duodenal entry and dissected. A ligature was placed around the duct. An 18-gauge angiocath was inserted into the duct and the tip advanced 2-3 mm such that it remained in the main ductal architecture just prior to ductal branching in the pancreas. The catheter was sutured to the duct to secure its position. Immediately prior- to harvest, the previously placed vascular ligatures were tightened and the animal was euthanized. The pancreas was transected from all peritoneal and vascular attachments and dissected from the duodenum. Once excised, the pancreas was immediately perfused with ice-cold University of Wisconsin (UW-D) perfusion solution via the previously placed ductal catheter.

[0093] A visual inspection was performed to ensure that the entire pancreas is perfused. The harvested glands were transported on ice to the laboratory where the UW-D solution was replaced by a solution of collagenase in UW-D (Crescent Chemical). The glands were then placed in a shaking water bath and digested at 40° C for approximately 35 minutes. The dissociated tissue was filtered through a 400-μm mesh screen and washed several times with ice-cold media to remove and inactivate the coMagenase. Based on density differences between islets and exocrine tissue, a discontinuous ficoll gradient was used to separate the islets and exocrine tissue. After density centrifugation, the islets were collected, washed, and transferred to tissue culture M 199 media supplemented with 10% FBS (Fetal Bovine Serum) and antibiotics. During culture for 48-72 hours, isolated islets maintained their compact appearance and the capsule surface remained smooth.

[0094] Islet isolations were performed on 56 canine pancreases. A profile of the average isolation results per pancreas is shown below (islets fragments that are smaller than 50 μm are not quantified). In addition to the number of islets isolated, the quality of isolations was evaluated by determining the islet diameter, purity, islet viability, and islet function. Since the average islet diameter will vary, the isolation yield is normalized by computing the ratio of the average islet volume and the volume of a "standard" islet of 150 μm in diameter. The resulting value is referred to as the Equivalent Islet Number (EIN) and allows a yield-comparison for different isolations. Islet purity was determined from a sample that was stained with the islet-specific dye dithizone. The dye stains islets red but leaves exocrine tissue unstained. Most of the exocrine tissue dies during the first 24 hrs of culture, resulting in an increase in purity during culture to approximately 95%.

[0095] Islet viability is determined from a sample that was stained with a combination of CaJcein AM (stains live cells fluorescent green) and Ethidium Bromide (stains the nuclei of dead cells fluorescent red). Viability is scored on a scale of 1 (all cells dead) to 4 (all cells alive). The average of five typical isolations is tabulated below.

Islets per pancreas 435 ± 38 K

Islet Diameter 106 ± 3.8 μm

Islet Equivalent Number 0.48 ± 0.04

Purity 87.3 ± 1.2%

Viability 3.5 ± 0.1

[0096] Capsule formation and characterization: Capsules can be made with a droplet generator and a chemical reaction chamber, such as that described in U.S. Patent Nos. 5,260,002 or 6,001,312, both of which are herein incorporated by reference in their entirety.

[0097] Another droplet generator system is a duo syringe system in which two or more syringes are connected in parallel and submerged in a temperature bath to keep the living cells healthy. The temperature bath containing the syringes may be an ice water bath having a temperature at about 4° C, which aids in keeping the cells in a dormant state. It has been found that islets, when in a dormant state, incur Jess damage during the transplantation process. This duo syringe system provides continuous operation by allowing for the refilling of one syringe while the experiment is ongoing with the other syringe. The syringes may also contain slow-turning propellers located inside the syringes that assist in maintaining islet density uniformity; i.e., more even distribution of the islets in the syringe.

[0098] The chemical reaction apparatus includes a multi-loop chamber reactor that is filled with solution, such as a cation solution. This cation solution bath is fed by a cation stream, which continuously replenishes the solution and carries away the anion drops being introduced into the chamber. Continuous SA/CS droplets can stream from the drop generator, with pancreatic islets enclosed, and enter the cation stream at a designated height and angle; so as to reduce or minimize islet decentering, drop deformation, and air bubble entrainment problems associated with impact. The droplets are then carried into the multi-loop reactor by the polycation stream. The reactor assists in controlling the time of complex formation as well as negating certain gravitational sedimentation effects. The capsules are carried into a second loop reactor with the same or different polycation solution for continuous operation. This facilitates tighter control of capsule diameter and sphericity as well as membrane thickness and uniformity.

[0099] Capsules may be produced with diameters ranging from about 0.5 mm to about 3.0 mm and membrane thicknesses ranging from about 0.006 mm to about 0.125 mm.

[00100] The mechanical strength of capsules may be measured by placing an increasing uniaxial load on the capsule until the capsule burst or totally compressed to a flat disc, as discussed .previously and depicted in FIG. 4. The mechanical strength of the capsule, a function of membrane composition and thicknesses, can be adjusted anywhere from a fraction of a gram;to many tens of grams load to meet the transplantation goals without significantly altering the permeability of the capsule.

[00101] A series of capsules having a range of permeability (porosity cutoff ranging from 40 kDa-230 kDa, based on dextran exclusion measurement) was developed and characterized. Capsule permeability can be measured by utilizing size exclusion chromatography (SEC) with dextran molecular weight standards. Measuring permeability and component concentration allows for the better control and manipulations of capsule permeability. The apparent pore size of the capsular membrane was determined by size exclusion chromatography (SEC) that measures the exclusion of dextran solutes from a column packed with microcapsules. By using neutral polysaccharide molecular weight standards, it is possible to evaluate the membrane properties under the conditions when solute diffusion is controlled only by its molecular dimension. Based on the measured values of solute size exclusion coefficients (K _S EC) and known size of solute molecules, the membrane pore size distribution (PSD) can be estimated.

In vivo function of new capsules

[00102] Encapsulated islets insulin secretion in response to stimuli: Following islet isolation, diameter, purity, and viability testing, the islets were cultured for 48-72 hours and encapsulated with a multi-membrane capsule. The insulin secretory capacity of the free islets and encapsulated islets was determined in a cell perifusion system, as described below. Insulin secretion by encapsulated islets was evaluated in a cell perifusion apparatus with a flow rate of 1 ml/minute with RPMI 1640 with 0.1 % BSA as a perifusate. Encapsulated islets were perifused with 2 mM glucose for 30 minutes and the column flowthrough discarded. Three minute samples of perifusate were collected during a 30 minute perifusion of 2 mM glucose, a 30 minute perifusion of 20 mM glucose + 0.045 mM IBMX (a nutrient), and a 60 minute perifusion of 2 mM glucose. Samples were assayed in duplicate for insulin using Coai-a-Count kits (Diagnostic Products Corporation, Los Angeles, CA) with a canine insulin standard. The amount of insulin secreted was normalized for the number of islets.

[00103] As assessed by the dynamic response to insulin secretagogues, insulin secretion by encapsulated islets bad a similar profile as unencapsulated free islets with a slightly delay in insulin secretion. See FIG. 6. This delay in insulin secretion and the cessation of insulin secretion following removal of the stimulus reflects (a) the time for the secretagogue to enter the capsule and reach the islet and (b) the time for insulin to exit the capsule.

[00104] FIG. 6 depicts a cell perifusion system measuring the secretion level of insulin-releasing islets. Free islets (not encapsulated), islets encapsulated in a single- membrane system (encapsulated islets), and islets encapsulated in a multi-membrane system (encapsulated with layer) were independently assessed. Stimuli for insulin secretion are shown in the black bars at the top of the graph. Insulin in perifusion fractions collected every 3-mintues was quantified by radioimmunoassay. The number of islets was not normalized, so the focus of the chart should lie on the response time rather than the height of the graphs. The similarity of the response time in the three graphs with only minute delays suggests that the islets encapsulated in the multi-membrane system will function normally inside transplanted animals.

[00105] Encapsulated Islet Function and Safety: Using the total pancreatectomy dog model, the function and safety of the intra-peritoneally administered encapsulated canine islets (allograft) was assessed in 10 diabetic animals. The recurrence of diabetes, as determined by a glucose level of greater than 180mg/dl for 4 consecutive days, occurred in dog 1 at approximately 100 days post transplantation. Encapsulated islets retrieved and tested in the cell perifusion system using the same stimuli as used in the previous transplantation shown in FIG. 6. See FlG. 7.

[00106] The chart in FIG. 7 indicates that the encapsulated islets are still viable as evidenced by the response to a high glucose plus IBMX, but have reduced insulin secretory capacity. These results suggest that the diabetes recurred because of inadequate islet mass and further suggest that this is due to reduced islet mass or function that is not the result of an allograft reaction.

[00107] Fasting glucose concentrations, body weight, and fructosamine measurements of dog 10 are shown in FIGS. 8-10 as representative data. The retrieved capsules were clean and intact, suggesting that the longevity of the transplant is no longer limited by the capsule stability, but rather the loss of islet mass.

[00108] FlG. 8 depicts blood glucose analysis of canine allotransplantation.

Transplantation of islets encapsulated in a mulii-membrane system has demonstrated the

efficacy in reversing diabetes in a canine model (dog no. 10) that has undergone a total pancreatectomy. The top panel illustrates the venous plasma glucose concentrations collected 12-18 hours following a meal. The lower panel illustrates the daily dosage of subcutaneous porcine insulin administered. The upper portion of bar in the lower panel indicates NPH insulin and the lower portion of bar indicates regular insulin. In days 18 and 19, treatments ceased to verify that the dog was diabetic. As seen in the top panel, glucose level rose dramatically wheη insulin treatments ceased. Insulin treatments resumed on day 20. On the morning of day 25, insulin treatments again ceased. In the afternoon of day 25, islets encapsulated in multi-membrane system were transplanted into the canine, as indicated by the vertical line. As illustrated in the top panel, glucose levels remained stabilized past day 200 at levels comparable or better than those observed during the period of insulin treatment. The bottom panel confirms that no additional insulin treatments were administered during this time period.

[00109] FIG. 9 depicts body weight analysis of canine allotransplantation. The top and bottom panels have been imported from FIG. 8. The middle panel shows the animal body weight monitored during the testing period. As can be seen in this chart, the body weight of the canine remained stable throughout the testing period.

[00110] FlG. 10 depicts a fructosamine analysis of canine allotransplantation.

The top and bottom panels have been imported from FIG. 8. The middle panel shows fructosamine measurements, an indicator of blood glucose level averaged over 2-3 weeks in diabetic subjects. A fructosamine level of 400 is roughly equivalent to an A lC measurement of 8.0, which is a similar indicator. The shaded area in the middle panel shows acceptable fructosamine levels. As can be seen in this chart, the fructosamine level on the tested days falls within the acceptable level. The tested fructosamine level is equivalent to an A lC level ranging from 6.0 (days 1 10-120) to 8.0 (days 195-200).

[00111] Re-transplantation: When fasting hyperglycemia recurs in animal, the transplant procedure may be repeated to maintain normoglycemia. For example, dog 7 received 40,000 EIN/kg, but was only able to maintain some semblance of glucose control for approximately 90 days. The dog was then given a second dosage of encapsulated islets of 63,000 EIN/kg total in two transplants (the transplants were administered a month apart due Io the availability of the islets). The normoglycemia lasted approximately 1 10 days. These results are similar to those observed in the transplantation of dog 6 with 100,000 EIN/kg, and of comparable effectiveness in providing fasting glucose control.

[00112] FlG. 1 1 shows the daily fasting blood glucose of dog 7 at 90-110 mg/dl without any supplemental insulin or immunosuppression. The vertical lines show the day of islet transplantation. The top panel shows data points that indicate the venous plasma glucose concentrations collected 12-18 hours following a meal. The lower panel indicates the daily dosage of subcutaneous pork insulin administered, with the upper portion of bar indicating NPH insulin, and the lower portion of bar indicating regular insulin. This figure illustrates the effectiveness of re-transplantation, as evidenced by the glucose levels stabilizing immediately after the second transplantation.

[00113] These results suggest additional transplants perform just as well if not better than initial transplantation. It is believed that the subsequent transplant performs better than the initial transplant because of the subject's ability to acclimate to the treatment and minimize vascularization. Re-transplantation provides improved glucose control and was well tolerated in the animal in terms of biocompatibility. Four successful re-transplantations have been performed on one subject; however, there is no practical limit to the number of re- transplantations that can be performed on a subject.

[00114] Intravenous glucose tolerance test (IVGTT): Intravenous glucose tolerance test (IVGTT) were performed on all animals to assess the in vivo function of encapsulated islets. FIG. 12 illustrates the IVGTT results of dog 5. Intravenous dextrose (300 mg/kg) was administered at t=o in a canine having previously received a transplantation of islets encapsulated in multi-membrane system. Venous samples were collected from the jugular vein to determine plasma glucose and insulin.

[00115] The subject's blood-glucose level returned to normal at approximately

105 minutes, which is longer, but not unreasonably longer, than the 50-minute average exhibited by 6 control dogs. The rate of glucose clearing (The K value) was high, yet within normal range. Circulating insulin values for all the transplanted animals increased an average of 40% above basal in 75 minutes of the IVGTT and stayed at that level for the remainder of the test. Dogs with encapsulated islets did nol demonstrate a first-phase insulin release that is often seen in the control animals. The lack of an insulin spike in response to glucose challenge (likely due to dilution effect of IP transplantation site) may have contributed to the islets gradually losing their ability to secrete sufficient insulin to maintain normoglycemia.