United States Patent: 6,783,964
Issued: August 31, 2004
Inventors: Opara; Emmanuel C. (Durham, NC)
Assignee: Duke University (Durham, NC)
Appl. No.: 054796
Filed: January 23, 2002
Methods of treating and forming biocompatible microcapsules that contain living cells are provided, to improve the function of the microcapsules. In particular, methods of treating islet cells or microcapsules containing islet cells are provided. Culture of isolated islet cells prior to encapsulation, culture of encapsulated cells, and cryopreservation of islet cells prior to encapsulation, are described. Methods for harvesting viable islets that incorporates an anti-oxidant in the digestion medium are also disclosed.
SUMMARY OF THE INVENTION
A first aspect of the present invention is a method of treating isolated living cells, by first culturing the cells in a medium containing at least one of (or a combination of ): an antioxidant, an anti-cytokine, an anti-endotoxin, or an antibiotic. The cells are then microencapsulated in a biocompatible microcapsule that contains a hydrogel core and a semipermeable outer membrane, to provide a microcapsule containing living cells therein.
A further aspect of the present invention is a method of treating isolated living cells, by first cryopreserving the cells in a cryopreservation medium containing at least one of (or a combination of): an antioxidant, an anti-cytokine, an anti-endotoxin, or an antibiotic; then thawing the cells and encapsulating the cells in a biocompatible microcapsule having a hydrogel core and a semipermeable outer membrane.
A further aspect of the present invention is a method of treating biocompatible microcapsules containing living cells, where the microcapsule contains a hydrogel core and a semipermeable outer membrane. The microcapsules are cultured in a medium containing at least one of (or a combination of): an antioxidant, an anti-cytokine, an anti-endotoxin, or an antibiotic.
A further aspect of the present invention is a method of preparing microencapsulated cells by first culturing the cells in a cell culture medium containing at least one of (or a combination of): antioxidants, anti-cytokines, anti-endotoxins, and antibiotics. The cells are then encapsulated in a biocompatible microcapsule having a hydrogel core and a semipermeable outer membrane, where the living cells are present in the core. The microcapsules are then cultured in a medium containing at least one of (or a combination of): an antioxidant, an anti-cytokine, an anti-endotoxin, and an antibiotic.
A further aspect of the present invention is a method of preparing microencapsulated cells that includes a step of incubating the microencapsulated cells with a physiologically acceptable salt such as sodium sulfate or the like in order to produce a more durable, and therefore useful, biocompatible microcapsule.
A further aspect of the present invention is a method of isolating pancreatic islet cells in which an antioxidant is included in the digestion medium that is used to free the islet cells from pancreatic tissue.
DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
The present inventors have determined that biocompatible microcapsules that contain living cells, such as pancreatic islet cells, benefit from a period of culture prior to use. Such culture enhances the ability of microcapsules containing islet cells to produce insulin in response to glucose stimulation. Culture of cell-containing microcapsules in a medium containing any of, or a combination of, an antibiotic, an anti-oxidant, an anti-cytokine, and an anti-endotoxin, is preferred.
The present inventors have further determined that cryopreservation of isolated cells (such as pancreatic islet cells) prior to microencapsulation does not adversely affect the function of the cells when subsequently encapsulated. Cryopreservation in a medium containing any of (or a combination of) an antibiotic, an anti-oxidant, an anti-cytokine, and an anti-endotoxin is preferred.
The present inventors have further determined that pre-culturing isolated cells prior to microencapsulation is beneficial. Culture of isolated islet cells prior to microencapsulation improves the glucose-stimulated insulin response of the encapsulated islets. Culture in a medium containing any of (or a combination of) an antibiotic, an anti-oxidant, an anti-cytokine, and an anti-endotoxin is preferred.
The above methods of treating cells and microcapsules may be combined, for example, by culturing isolated cells prior to microencapsulation and then culturing the resulting microcapsules.
Methods of isolating pancreatic islet cells are known in the art. Field et al., Transplantation 61:1554 (1996); Linetsky et al., Diabetes 46:1120 (1997). Fresh pancreatic tissue can be divided by mincing, teasing, comminution and/or collagenase digestion. The islets are then isolated from contaminating cells and materials by washing, filtering, centrifuging or picking procedures. Methods and apparatus for isolating and purifying islet cells are described in U.S. Pat. Nos. 5,447,863, 5,322,790, 5,273,904, and 4,868,121. The isolated pancreatic cells may optionally be cultured prior to microencapsulation, using any suitable method of culturing islet cells as is known in the art. See e.g., U.S. Pat. No. 5,821,121. Isolated cells may be cultured in a medium under conditions that helps to eliminate antigenic components (Transplant. Proc.14:714-23 (1982)).
In general, a method of isolating pancreatic islet cells comprises (a) digesting pancreatic tissue with a digestion medium, the digestion medium containing an antioxidant, with the digesting step carried out for a time sufficient to produce free pancreatic islet cells suitable for subsequent harvesting; and then (b) collecting the free pancreatic islet cells to produce isolated pancreatic islet cells. The antioxidant is included in the digestion medium in an amount sufficient to inhibit reoxygenation injury of the isolated pancreatic islet cells (e.g., from about 0.05, 0.1, or 0.5 milliMolar to about 5, 10 or 20 milliMolar or more) The antioxidant is preferably one that is not itself digested in the digestion medium, such as a vitamin or organic chemical antioxidants. Examples of suitable antioxidants include, but are not limited to, vitamin C, vitamin E, vitamin K, lipoic acid, lazaroids, and butylated hydroxyanisole. Once collected, the isolated pancreatic islet cells may be cultured and microencapsulated as further described herein.
A preferred method for isolating viable islet cells, such as porcine islet cells, is as follows. Adult pigs (approximately 25-20 kg) are anesthetized, followed by intra-arterial infusion of ice-cold UW solution and complete pancreatectomy. The pancreatic duct is cannulated for infusion of digestion medium comprising an antioxidant. An exemplary digestion medium is Hanks' balanced salt solution containing collagenase type P (1.5 mg/ml), DNase 1 (10,000 units) and TROLOX.RTM. brand water soluble vitamin E antioxidant (1 mM). After 20 minutes incubation on ice, the pancreas is incubated at 37oC. for 20 minutes before being hand-shaken for one minute. Digested tissue is filtered, and islet clusters are separated using OPTIPREP.RTM. gradient, washed in Hanks' solution and identified by dithizone staining. This method utilizes an anti-oxidant in the digestion medium and hand-shaking, and does not require special isolation chambers or mechanical disruption of the pancreatic tissue.
Microencapsulation of islet cells generally involves three steps: (a) generating microcapsules enclosing the islet cells (e.g., by forming droplets of cell-containing liquid alginate followed by exposure to a solution of calcium chloride to form a solid gel), (b) coating the resulting gelled spheres with additional outer coatings (e.g., outer coatings comprising polylysine and/or polyornithine) to form a semipermeable membrane; and (c) liquefying the original core gel (e.g., by chelation using a solution of sodium citrate). The three steps are typically separated by washings in normal saline.
A preferred method of microencapsulating pancreatic cells is the alginate-polyamino acid technique. Briefly, islet cells are suspended in sodium alginate in saline, and droplets containing islets are produced. Droplets of cell-containing alginate flow into calcium chloride in saline. The negatively charged alginate droplets bind calcium and form a calcium alginate gel. The microcapsules are washed in saline and incubated with poly-L-lysine or poly-L-ornithine (or combinations thereof); the positively charged poly-l-lysine and/or poly-L-ornithine displaces calcium ions and binds (ionic) negatively charged alginate, producing an outer poly-electrolyte membrane. A final coating of sodium alginate may be added by washing the microcapsules with a solution of sodium alginate, which ionically bonds to the poly-L-lysine and/or poly-L-ornithine layer. See U.S. Pat. No. 4,391,909 to Lim et al (all US patents referenced herein are intended to be incorporated herein in their entirety). This technique produces what has been termed a "single-wall" microcapsule. Preferred microcapsules are essentially round, small, and uniform in size. Wolters et al., J. Appli Biomater. 3:281 (1992).
When desired, the alginate-polylysine microcapsules can then be incubated in sodium citrate to solubilize any calcium alginate that has not reacted with poly-1-lysine, i.e., to solubilize the internal core of sodium alginate containing the islet cells, thus producing a microcapsule with a liquefied cell-containing core portion. See Lim and Sun, Science 210:908 (1980). Such microcapsules are referred to herein as having "chelated", "hollow" or "liquid" cores.
A "double-wall" microcapsule is produced by following the same procedure as for single-wall microcapsules, but prior to any incubation with sodium citrate, the microcapsules are again incubated with poly-1-lysine and sodium alginate.
Alginates are linear polymers of mannuronic and guluronic acid residues. Monovalent cation alginate salts, e.g., Na-alginate, are generally soluble. Divalent cations such as Ca++, Ba++ or Sr++ tend to interact with guluronate, providing crosslinking and forming stable alginate gels. Alginate encapsulation techniques typically take advantage of the gelling of alginate in the presence of divalent cation solutions. Alginate encapsulation of cells generally involves suspending the cells to be encapsulated in a solution of a monovalent cation alginate salt, generating droplets of this solution, and contacting the droplets with a solution of divalent cations. The divalent cations interact with the alginate at the phase transition between the droplet and the divalent cation solution, resulting in the formation of a stable alginate gel matrix being formed. A variation of this technique is reported in U.S. Pat. No. 5,738,876, wherein the cell is suffused with a solution of multivalent ions (e.g., divalent cations) and then suspended in a solution of gelling polymer (e.g., alginate), to provide a coating of the polymer.
Chelation of the alginate (degelling) core solubilizes the internal structural support of the capsule, may adversely affect the durability of the microcapsule, and is a harsh treatment of the encapsulated living cells. Degelling of the core may also cause leaching out of the unbound poly-lysine or solubilized alginate, resulting in a fibrotic reaction to the implanted microcapsule. The effect of core liquidation on glucose-stimulated insulin secretion by the encapsulated cells has been studied. Fritschy et al., Diabetes 40:37 (1991). The present inventors examined the function of islets enclosed in microcapsules that had not been subjected to liquefaction of the core (i.e., `solid` or non-chelated microcapsules). It was found that culture of solid microcapsules prior to use enhanced the insulin response of the enclosed islets to glucose stimulation.
Alginate/polycation encapsulation procedures are simple and rapid, and represent a promising method for islet encapsulation for clinical treatment of diabetes. Many variations of this basic encapsulation method have been described in patents and the scientific literature. Chang et al., U.S. Pat. No. 5,084,350 discloses microcapsules enclosed in a larger matrix, where the microcapsules are liquefied once the microcapsules are within the larger matrix. Tsang et al., U.S. Pat. No. 4,663,286 discloses encapsulation using an alginate polymer, where the gel layer is cross-linked with a polycationic polymer such as polylysine, and a second layer formed using a second polycationic polymer (such as polyornithine); the second layer can then be coated by alginate. U.S. Pat. No. 5,762,959 to Soon-Shiong et al. discloses a microcapsule having a solid (non-chelated) alginate gel core of a defined ratio of calcium/barium alginates, with polymer material in the core.
U.S. Pat. Nos. 5,801,033 and 5,573,934 to Hubbell et al. describe alginate/polylysine microspheres having a final polymeric coating (e.g., polyethylene glycol (PEG)); Sawhney et al., Biomaterials 13:863 (1991) describe alginatelpolylysine microcapsules incorporating a graft copolymer of poly-1-lysine and polyethylene oxide on the microcapsule surface, to improve biocompatibility; U.S. Pat. No. 5,380,536 describes microcapsules with an outermost layer of water soluble non-ionic polymers such as polyethylene(oxide). U.S. Pat. No. 5,227,298 to Weber et al. describes a method for providing a second alginate gel coating to cells already coated with polylysine alginate; both alginate coatings are stabilized with polylysine. U.S. Pat. No. 5,578,314 to Weber et al. provides a method for microencapsulation using multiple coatings of purified alginate.
U.S. Pat. No. 5,693,514 to Dorian et al. reports the use of a non-fibrogenic alginate, where the outer surface of the alginate coating is reacted with alkaline earth metal cations comprising calcium ions and/or magnesium ions, to form an alkaline earth metal alginate coating. The outer surface of the alginate coating is not reacted with polylysine.
U.S. Pat. No. 5,846,530 to Soon-Shiong describes microcapsules containing cells that have been individually coated with polymerizable alginate, or polymerizable polycations such as polylysine, prior to encapsulation.
The methods of the present invention are intended for use with any microcapsule that contains living cells secreting a desirable biological substance (preferably pancreatic cells and more preferably islet cells), where the microcapsule comprises an inner gel or liquid core containing the cells of interest, or a liquid core containing the cells of interest, bounded by a semi-permeable membrane surrounding the cell-containing core. The inner core is preferably composed of a water-soluble gelling agent; preferably the water-soluble gelling agent comprises plural groups that can be ionized to form anionic or cationic groups. The presence of such groups in the gel allows the surface of the gel bead to be cross-linked to produce a membrane, when exposed to polymers containing multiple functionalities having a charge opposite to that of the gel.
Cells suspended in a gellable medium (such as alginate) may be formed into droplets using any suitable method as is known in the art, including but not limited to emulsification (see e.g., U.S. Pat. No. 4,352,883), extrusion from a needle (see, e.g., U.S. Pat. No. 4,407,957; Nigam et al., Biotechnology Techniques 2:271-276 (1988)), use of a spray nozzle (Plunkett et al., Laboratory Investigation 62:510-517 (1990)), or use of a needle and pulsed electrical electrostatic voltage (see, e.g., U.S. Pat. Nos. 4,789,550; 5,656,468).
The water-soluble gelling agent is preferably a polysaccharide gum, and more preferably a polyanionic polymer. An exemplary water-soluble gelling agent is an alkali metal alginate such as sodium alginate. The gelling agent preferably has free acid functional groups and the semi-permeable membrane is formed by contacting the gel with a polymer having free amino functional groups with cationic charge, to form permanent crosslinks between the free amino acids of the polymer and the acid functional groups. Preferred polymers include polylysine, polyethylenimine, and polyarginine. A particularly preferred microcapsule contains cells immobilized in a core of alginate with a poly-lysine coating; such microcapsules may comprise an additional external alginate layer to form a multi-layer alginate-polylysine-alginate microcapsule. See U.S. Pat. No. 4,391,909 to Lim et al, the contents of which are incorporated by reference herein in their entirety.
When desired, the microcapsules may be treated or incubated with a physiologically acceptable salt such as sodium sulfate or like agents, in order to increase the durability of the microcapsule, while retaining or not unduly damaging the physiological responsiveness of the cells contained in the microcapsules. By "physiologically acceptable salt" is meant a salt that is not unduly deleterious to the physiological responsiveness of the cells encapsulated in the microcapsules. In general, such salts are salts that have an anion that binds calcium ions sufficiently to stabilize the capsule, without substantially damaging the function and/or viability of the cells contained therein. Sulfate salts, such as sodium sulfate and potassium sulfate, are preferred, and sodium sulfate is most preferred. The incubation step is carried out in an aqueous solution containing the physiological salt in an amount effective to stabilize the capsules, without substantially damaging the function and/or viability of the cells contained therein as described above. In general, the salt is included in an amount of from about 0.1 or 1 milliMolar up to about 20 or 100 millimolar, most preferably about 2 to 10 millimolar. The duration of the incubation step is not critical, and may be from about 1 or 10 minutes to about 1 or 2 hours, or more (e.g., over night). The temperature at which the incubation step is carried out is likewise not critical, and is typically from about 4 degrees Celsius up to about 37 degrees Celsius, with room temperature (about 21 degrees Celsius) preferred.
When desired, liquefaction of the core gel may be carried out by any suitable method as is known in the art, such as ion exchange or chelation of calcium ion by sodium citrate or EDTA. Microcapsules useful in the present invention thus have at least one semipermeable surface membrane surrounding a cell-containing core. The surface membrane permits the diffusion of nutrients, biologically active molecules and other selected products through the surface membrane and into the microcapsule core. The surface membrane contains pores of a size that determines the molecular weight cut-off of the membrane. Where the microcapsule contains insulin-secreting cells, the membrane pore size is chosen to allow the passage of insulin from the core to the external environment, but to exclude the entry of host immune response factors.
As used herein, a "poly-amino acid-alginate microsphere" refers to a capsule of less than 2 mm in diameter having an inner core of cell-containing alginate bounded by a semi-permeable membrane formed by alginate and poly-1-lysine. Viable cells encapsulated using an anionic polymer such as alginate to provide a gel layer, where the gel layer is subsequently cross-linked with a polycationic polymer (e.g., an amino acid polymer such as polylysine. See e.g., U.S. Pat. Nos. 4,806,355, 4,689,293 and 4,673,566 to Goosen et al.; U.S. Pat. Nos. 4,409,331, 4,407,957, 4,391,909 and 4,352,883 to Lim et al.; U.S. Pat. Nos. 4,749,620 and 4,744,933 to Rha et al.; and U.S. Pat. No. 5,427,935 to Wang et al. Amino acid polymers that may be used to encapsulate islet cells in alginate include the cationic amino acid polymers of lysine, arginine, and mixtures thereof.
Culture of Isolated Cells
Generally, pancreatic islets are isolated by collagenase digestion of pancreatic tissue. This process involves subjecting the islet cells to a period of hypoxia which is then followed by reoxygenation. Hypoxia-reoxygenation produces an injury that is linked to excessive production of oxygen free radicals which impair the function, and cause the death, of islet cells, particularly those isolated from the pancreas of large mammals such as pigs and humans.
The present inventors have determined that culture of isolated islets or islet cells prior to microencapsulation is beneficial. The islets are cultured according to known cell culture techniques for a period of at least 3 hours, more preferably from 12-36 hours, and more preferably from 18-24 hours, in a culture medium containing an antioxidant compound. More preferably, the culture medium contains any one of, or any combination of, the following: an antioxidant, an anti-cytokine, an anti-endotoxin, and an antibiotic.
Culture of isolated pancreas cells to improve glucose-stimulated insulin secretion may utilize any suitable anti-oxidant as is known in the art. As used herein, an antioxidant is a compound that neutralizes free radicals or prevents or suppresses the formation of free radicals. Particularly preferred are molecules including thiol groups such as reduced glutathione (GSH) or its precursors, glutathione or glutathione analogs, glutathione monoester, and N-acetylcysteine. Other suitable anti-oxidants include superoxide dismutase, catalase, vitamin E, Trolox, lipoic acid, lazaroids, butylated hydroxyanisole (BHA), vitamin K, and the like. Glutathione, for example, may be used in a concentration range of from about 2 to about 10 mM. See, e.g., U.S. Pat. Nos. 5,710,172; 5,696,109; 5,670,545.
Culture of isolated pancreas cells to improve glucose-stimulated insulin secretion may utilize any suitable antibiotic as is known in the art. Suitable antibiotics include penicillins, tetracyclines, cephalosporins, macrolides, .beta.-lactams and aminoglycosides; examples of such suitable antibiotics include streptomycin and amphotericin B.
Culture of isolated pancreas cells to improve glucose-stimulated insulin secretion may utilize any suitable anti-cytokine as is known in the art. Cytokines are proteins secreted by many different cell types, that regulate the intensity and duration of immune responses. Cytokines include various growth factors, colony-stimulating factors, interleukins, lymphokines, monokines and interferons. Anti-cytokines are compounds that prevent or suppress the function of cytokines. Suitable anti-cytokines for use in culturing islet cells include dimethylthiourea (10 mM), citiolone (5 mM), pravastatin sodium (PRAVACHOL.RTM., 20 mg/kg), L-NG -monomethylarginine (L-NMMA, 2 mM), lactoferrin (100 .mu.g/ml), 4-methylprednisolone (20 .mu.g/ml), and the like.
Culture of isolated pancreas cells to improve glucose-stimulated insulin secretion may utilize any suitable anti-endotoxin as is known in the art. Endotoxins are bacterial toxins, complex phospholipid-polysaccharide molecules that form a part of the cell wall of a variety of Gram-negative bacteria. Anti-endotoxins are compounds that destroy or inhibit the activity of endotoxins. Endotoxins are intracellular toxins, and are complex phospholipid-polysaccharide macromolecules that form a part of the cell wall of a variety of Gram-negative bacteria, including enterobacteria, vibrios, brucellae and neisseriae. Suitable anti-endotoxins for use in culturing islet cells include L-NG -Monomethylarginine (L-NMMA, 2 mM), lactoferrin (100 .mu.g/ml), N-acetylcysteine (NAC, 1 mM), adenosine receptor antagonists such as bamiphylline (theophylline) and anti-lipopolysaccharide compounds such as echinomycine (10 nM), and the like.
Cryopreservation of Cells
Mammalian tissue remains viable in vitro only for short periods of time, usually days. Loss of islet cells suitable for transplantation may be avoided by viable cryopreservation and cold storage of the cells. The present inventors determined that microencapsulated islet cells respond poorly to cryopreservation. However, cryopreservation of naked (unencapsulated) islet cells did not adversely affect their later function in microcapsules when the cells were first cryopreserved, then thawed and microencapsulated. Frozen and thawed microencapsulated islets responded poorly to glucose stimulation; in comparison, `naked` islet cells that were cryopreserved and then thawed retained their ability to respond to glucose stimulation and were suitable for microencapsulation. Islet cells can thus be preserved by cryopreservation, thawed and microencapsulated just prior to use.
Methods of cryopreservation are well known in the art. In general terms, cryopreservation of animal cells involves freezing the cells in a mixture of a growth medium and another liquid that prevents water from forming ice crystals, and then storing the cells at liquid nitrogen temperatures (e.g., from about -80 to about -196oC.).
An aspect of the present invention is the cryopreservation of isolated mammalian cells in a cryopreservation medium containing an antioxidant, followed by microencapsulation of the cells prior to in vivo implantation. A preferred embodiment of the present invention is the cryopreservation of isolated islets or islet cells in a cryopreservation medium containing an antioxidant as described herein, followed by microencapsulation prior to in vivo implantation.
More preferably, the cells are cryopreserved in a medium containing at least one of, or a combination of, the following: an antioxidant, an anti-cytokine, an anti-endotoxin, and an antibiotic (each as described above). Preferably, the cells are cryopreserved in a medium containing at least one each of an antioxidant, an anti-cytokine, an anti-endotoxin, and an antibiotic (each as described above).
Culture of Microspheres.
The present inventors studied the response to glucose of islet-containing microcapsules, and found that culture of such microcapsules prior to use enhanced subsequent glucose-stimulated insulin production. The enclosed islets responded better to a glucose challenge than islets contained in fresh (non-cultured) microcapsules.
Culture of microencapsulated cells is carried out in a manner similar to the culture of isolated cells, as described herein and as generally known in the art. Accordingly, a method of the present invention is the culture of microcapsules (with either solid or liquid cores containing living cells) prior to implantation in vivo, to enhance or improve the ability of the microcapsule to produce a desired cell secretory product. A particularly preferred embodiment is the culture, prior to implantation, of gelled or solid-core alginate-polylysine microcapsules containing pancreatic islets or islet cells. Microcapsules are cultured for a period of at least 3 hours, more preferably from 12 to 36 hours, or 18 to 24 hours, prior to implantation.
Preferably the microcapsules are cultured in a medium containing at least one of, or a combination of, the following: an antibiotic, an antioxidant, an anticytokine, and an antiendotoxin (as described above). More preferably, the microcapsules are cultured in a medium containing at least one each of an antioxidant, an anti-cytokine, an anti-endotoxin, and an antibiotic (each as described above).
As noted above, microencapsulated islet cells are also an aspect of this invention. In general, such microencapsulated islet cells comprise a microcapsule containing living pancreatic islet cells therein, the microcencapsulated islet cells exhibiting a weight gain of not more than 1, 5 or 10 percent by weight over a period of one month in physiological saline solution at 37 degrees Celsius (exhibiting the durability thereof) and exhibiting at least 150, 200 or 250 percent basal insulin secretion in response to 16.7 millimolar glucose challenge in Krebs-Ringer physiological solution at pH 7.4 after said period of one month. Such microencapsulated cells may be produced by the procedures described herein, preferably including the step of incubating them with a physiologically acceptable salt to enhance the durability or stability of the capsule as described above.
Encapsulated islet cells produced according to the present invention may be transplanted into subjects as a treatment for insulin-dependent diabetes; such transplantation may be into the peritoneal cavity of the subject. An amount of encapsulated islet cells to produce sufficient insulin to control glycemia in the subject is provided by any suitable means, including but not limited to surgical implantation and intraperitoneal injection. The International Islet Transplant Registry has recommended transplants of at least 6,000 islets, equivalent to 150 .mu.m in size, per kilogram of recipient body weight, to achieve euglycemia. However, it will be apparent to those skilled in the art that the quantity of microcapsules transplanted depends on the ability of the microcapsules to provide insulin in vivo, in response to glucose stimulation. One skilled in the art will be able to determine suitable transplantation quantities of microcapsules, using techniques as are known in the art.
Claim 1 of 12 Claims
1. A microencapsulated islet cell product comprising microcapsules containing isolated living pancreatic islet cells therein, said microcencapsulated islet cells exhibiting a weight gain of not more than 10 percent by weight over a period of one month in physiological saline solution at 37 degrees Celsius and exhibiting at least 150 percent basal insulin secretion in response to 16.7 milliMolar glucose challenge in Krebs-Ringer physiological solution at pH 7.4 after said period of one month.