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Encapsulation Methods and Compositions

20170252304 · 2017-09-07

    Inventors

    Cpc classification

    International classification

    Abstract

    This invention provides methods for the formation of biocompatible membranes around biological materials using photopolymerization of water soluble molecules. The membranes can be used as a covering to encapsulate biological materials or biomedical devices, as a “glue” to cause more than one biological substance to adhere together, or as carriers for biologically active species. Several methods for forming these membranes are provided. Each of these methods utilizes a polymerization system containing water-soluble macromers, species, which are at once polymers and macromolecules capable of further polymerization. The macromers are polymerized using a photoinitiator (such as a dye), optionally a cocatalyst, optionally an accelerator, and radiation in the form of visible or long wavelength UV light. The reaction occurs either by suspension polymerization or by interfacial polymerization. The polymer membrane can be formed directly on the surface of the biological material, or it can be formed on material, which is already encapsulated.

    Claims

    1. A composition comprising: encapsulating devices comprising a micro-bulk coating, and cell aggregates, wherein said composition has a cell density of at least about 100,000 cells/ml, wherein the micro-bulk coating for the encapsulating devices comprises a polymerizable high density ethylenically unsaturated polyethylene glycol (PEG) having a molecular weight between 900 and 20,000 Daltons, and a sulfonated comonomer, and wherein the micro-bulk coating comprises salt, MOPS (3-(N-morpholino)propanesulfonic) acid, co-monomer, a diol containing compound, an x-ray contrast agent and a photo-initiator.

    2. The composition of claim 1, wherein the encapsulating devices are micro-bulk capsules.

    3. The composition of claim 1 where the PEG is selected from the group consisting of a diacrylate of PEG with a molecular weight in the range of 2 kD to 16 kD, a triacrylate of PEG with a molecular weight in the range of 3 kD to 16 kD, a tetra-acrylate of PEG with a molecular weight in the range of 4 kD to 20 kD, and combinations thereof.

    4. The composition of claim 1 where the co-monomer is selected from the group consisting of AMPS (2-Acrylamido-2-methylpropane sulfonic acid), ammonium AMPS, 2-methyl-2-((1-oxo-2-propenyl)amino)-monoammomium salt, nVP (N-Vinylpyrrolidone), polyvidone, polyvinylpolypyrrolidone and similar types of co-polymers.

    5. The composition of claim 1 where the x-ray contrast agent is selected from the group consisting of nycodenz, iohexol, omnipaque and similar low-osmolality agents.

    6. The composition of claim 1 where the diol containing compound is selected from the group consisting of PEG-diol, beta propylene glycol, propylene-1,3,diol, bisphenol A, 1,4-butanediol and similar compounds.

    7. The composition of claim 2, wherein the micro-bulk capsule envelopes the cell aggregate.

    8. The composition of claim 7, wherein the cell aggregate is pancreatic islets.

    9. The composition of claim 7, wherein the cell density is at least about 6,000,000 cells/ml.

    10. The composition of claim 1, where the cell is selected from the group consisting of neurologic, cardiovascular, hepatic, endocrine, skin, hematopoietic, immune, neurosecretory, metabolic, systemic, and genetic.

    11. The composition of claim 10, where the cell is selected from the group consisting of autologous, allogeneic, xenogeneic and genetically-modified.

    12. The composition of claim 10, where the endocrine cell is an insulin producing cell.

    13. The composition of any one of claim 1, where the polymerizable high density ethylenically unsaturated PEG is a high density acrylated PEG.

    14. The composition of claim 13, where the polymerizable high density acrylated PEG has a molecular weight of 2 kD to 20 kD.

    15. The composition claim 1, further comprising a cocatalyst selected from the group consisting of triethanolamine, triethylamine, ethanolamine, N-methyl diethanolamine, N,N-dimethyl benzylamine, dibenzyl amino, N-benzyl ethanolamine, N-isopropyl benzylamine, tetramethyl ethylenediamine, potassium persulfate, tetramethyl ethylenediamine, lysine, omithine, histidine and arginine.

    16. The composition of claim 15, where the cocatalyst is triethanolamine.

    17. The composition of claim 1, further comprising an accelerator selected from the group consisting of N-vinyl pyrrolidinone, 2-vinyl pyridine, 1-vinyl imidazole, 9-vinyl carbazone, 9-vinyl carbozol, acrylic acid, n-vinylcarpolactam, 2-allyl-2-methyl-1,3-cyclopentane dione, and 2-hydroxyethyl acrylate.

    18. The composition of claim 17, where the accelerator is N-vinyl pyrrolidinone.

    Description

    BRIEF DESCRIPTION OF THE DRAWING

    [0137] FIG. 1A is a photomicrograph of Alginate capsules.

    [0138] FIG. 1B is a photomicrograph of encapsulated islets.

    [0139] FIG. 1C is a photomicrograph of PEG Conformal Coated Islets.

    [0140] FIG. 2A is a photomicrograph of Empty Alginate Capsules in Nu/Nu Mice—Histology without fragmentation.

    [0141] FIG. 2B is a photomicrograph of Empty Alginate Capsules in C57Bl6 mice—Histology without fragmentation.

    [0142] FIG. 2C is a photomicrograph of IP Empty PEG-Diacrylate Capsules in Nu/Nu Mice at 2 weeks—In vivo.

    [0143] FIG. 2D is a photomicrograph of IP Empty PEG-Diacrylate Capsules in Nu/Nu Mice at 2 weeks—Ex vivo.

    [0144] FIG. 2E is a photomicrograph of cell histology.

    [0145] FIG. 2F is a photomicrograph of cell histology.

    [0146] FIG. 2G is a photomicrograph of IP Empty PEG-Diacrylate Capsules in C57Bl6 Mice at 2 weeks—In vivo.

    [0147] FIG. 2H is a photomicrograph of IP Empty PEG-Diacrylate Capsules in C57Bl6 Mice at 2 weeks—Ex vivo.

    [0148] FIG. 2I is a photomicrograph of cell histology.

    [0149] FIG. 2J is a photomicrograph of cell histology.

    [0150] FIG. 3 is a photograph of an Unconstrained Compression Testing to Measure PEG Capsule Strength.

    [0151] FIG. 4 shows the Test Results of Elasticity of PEG-Diacrylate Capsules.

    [0152] FIG. 5 shows Capsule Size Distributions.

    [0153] FIG. 6A is a photomicrograph of PEG 5% after transplant from nude mice.

    [0154] FIG. 6B is a photomicrograph of PEG 5% after transplant from BL6 mice.

    [0155] FIG. 6C is a photomicrograph of 5% PEG-DA Histology—Nu/Nu Mouse Implants.

    [0156] FIG. 6D is a photomicrograph of 5% PEG-DA Histology—C57Bl6 Mouse Implants.

    [0157] FIG. 6E is a photomicrograph of PEG 7.5% after transplant from nude mice.

    [0158] FIG. 6F is a photomicrograph of PEG 7.5% after transplant from BL6 mice.

    [0159] FIG. 7 is a photomicrograph of PEG-Acrylates under testing.

    [0160] FIG. 8 shows the elasticity of different PEG-Acrylates.

    [0161] FIG. 9A is a photomicrograph of 5% 1 kDa PEG Diacrylate with 150 sec light exposure (dead islets).

    [0162] FIG. 9B is a photomicrograph of 7.5% 1 kDa PEG Diacrylate with 150 sec light exposure (dead islets).

    [0163] FIG. 9C is a photomicrograph of 5% 10 kDa PEG-tetra-Acrylate with 20 sec light exposure (viable islets).

    [0164] FIG. 9D is a photomicrograph of 10% 10 kDa PEG-tetra-Acrylate with 20 sec light exposure (viable islets).

    [0165] FIG. 10A is a photomicrograph of Nu/Nu mice Implants empty capsules.

    [0166] FIG. 10B is a photomicrograph of Nu/Nu mice Implants empty capsules.

    [0167] FIG. 10C is a photomicrograph of C57Bl6 mice Implants empty capsules.

    [0168] FIG. 10D is a photomicrograph of C57Bl6 mice Implants empty capsules.

    [0169] FIG. 10E is a photomicrograph of Nu/Nu mice implants+human islets.

    [0170] FIG. 10F is a photomicrograph of C57Bl6 mice implants+human islets.

    [0171] FIGS. 11A, B & C are Black & White photomicrographs of micro-bulk PEG Capsules (Bar=1000 microns) First micro-bulk PEG capsules containing human islets produced at 1-2 mm size. (dark spots).

    [0172] FIG. 11D shows the Viability test (EB/FDA stain) of micro-bulk PEG encapsulated human islets. (Green=Viable).

    [0173] FIG. 11E shows the Functional Glucose Stimulated Insulin Release (GSIR) test results on these first, large micro-bulk PEG capsules. 1.sup.st Stim Index=12 mM/3 mM, 2.sup.nd Stim Index=25 mM/3 mM, 3.sup.rd Stim Index=25 mM+IBMX/3 mM).

    [0174] FIGS. 11F & G is a photomicrograph of Starting Size of micro-bulk PEG capsules with human islets >1000 microns (Bar=1000 microns).

    [0175] FIGS. 11H, I & J is a photomicrograph of First Size Reduction Step to the 1000 to the 500 micron range with human islets. (Bar=1000 microns).

    [0176] FIGS. 11K, L & M is a photomicrograph of Second Size Reduction Step to the <500 micron range with human islets. (Bar equals 400 micron).

    DETAILED DESCRIPTION OF THE INVENTION

    [0177] This invention provides novel methods for the formation of biocompatible membranes around biological materials using photopolymerization of water-soluble molecules. The membranes can be used as a covering to encapsulate biological materials or biomedical devices, as a “glue” to cause more than one biological substance to adhere together, or as carriers for biologically active species.

    [0178] Several methods for forming these membranes are provided. Each of these methods utilizes a polymerization system containing water-soluble macromers, species, which are at once polymers and macromolecules capable of further polymerization. The macromers are polymerized using a photoinitiator (such as a dye), optionally a cocatalyst, optionally an accelerator, and radiation in the form of visible or long wavelength UV light. The reaction occurs either by suspension polymerization or by interfacial polymerization. The polymer membrane can be formed directly on the surface of the biological material, or it can be formed on material, which is already encapsulated.

    [0179] Ultrathin membranes can be formed-by the methods described herein. These ultrathin membranes allow for optimal diffusion of nutrient and bioregulator molecules across the membrane, and great flexibility in the shape of the membrane. Such thin membranes produce encapsulated material with optimal economy of volume. Biological material thus coated can be packed into a relatively small space without interference from bulky membranes.

    [0180] The thickness and pore size of membranes formed can be varied. This variability allows for “perm-selectivity”—membranes can be adjusted to the desired degree of porosity, allowing only preferred molecules to permeate the membrane, while acting as a barrier against larger undesired molecules. Thus, the membranes are immunoprotective in that they prevent the transfer of antibodies or cells of the immune system.

    [0181] When the encapsulated biological material is cellular in nature, the absence of small monomers in the polymerization solution prevents the diffusion of toxic molecules into the cell. In this manner the present invention provides a polymerization system which is more biocompatible than any available in the prior art.

    [0182] Additionally, the polymerization method utilizes short bursts of visible or long wavelength UV light, which is nontoxic to biological material. Bioincompatible polymerization initiators employed in the prior art are also eliminated.

    [0183] According to the present invention, membrane formation occurs under physiological conditions. Thus, no damage is done to the enclosed biological material due to harsh pH, temperature, or ionic conditions.

    [0184] Because the membrane adheres to the biological material, the membrane can be used as an adhesive to fasten more than one biological substance together. The macromers are polymerized in the presence of these substances which are in close proximity. The membrane forms in the interstices, effectively gluing the substances together.

    [0185] Additionally, utilizing the tendency of the membrane to adhere to biological material, a membrane can be formed around or on a biologically active substance to act as a carrier for that substance.

    [0186] In one embodiment, the invention is directed to a composition for cellular therapy, which includes a plurality of encapsulating devices comprising a micro-bulk coating including a polyethylene glycol (PEG) coating, said PEG having a molecular weight between about 900 and about 20,000 Daltons; and a plurality of cells encapsulated in the encapsulating devices, wherein said composition has a cell density of at least about 100,000 cells/ml and a sulfonated comonomer, and wherein the micro-bulk coating comprises salt, MOPS (3-(N-morpholino)propanesulfonic) acid, co-monomer, a diol containing compound, an x-ray contrast agent and a photo-initiator.

    [0187] In one embodiment, the encapsulating devices are microcapsules. In a one embodiment, the microcapsules are micro-bulk coated cell aggregates.

    [0188] In one embodiment, the cell aggregates are pancreatic islets with a cell density which is at least about 100,000 cells/ml.

    [0189] In one embodiment, the cell is neurologic, cardiovascular, hepatic, endocrine, skin, hematopoietic, immune, neurosecretory, metabolic, systemic, or genetic. In one embodiment, the cell is autologous, allogeneic, xenogeneic or genetically-modified. In one embodiment, the cell is an insulin producing cell.

    [0190] In one embodiment, the PEG is a diacrylate of PEG with a molecular weight in the range of 2 kD to 16 kD. In one embodiment, the PEG is a triacrylate of PEG with a molecular weight in the range of 3 kD to 16 kD. In one embodiment, the PEG is a tetra-acrylate of PEG with a molecular weight in the range of 4 kD to 20 kD.

    [0191] In one embodiment, the PEG is a combination of a diacrylate of PEG with a molecular weight in the range of 2 kD to 16 kD, a triacrylate of PEG with a molecular weight in the range of 3 kD to 16 kD and a tetra-acrylate of PEG with a molecular weight in the range of 4 kD to 20 kD.

    [0192] In one embodiment, the co-monomer is AMPS (2-Acrylamido-2-methylpropane sulfonic acid), ammonium AMPS, 2-methyl-2-((1-oxo-2-propenyl)amino)-monoammomium salt, nVP (N-Vinylpyrrolidone), polyvidone, polyvinylpolypyrrolidone or similar types of co-polymers.

    [0193] In one embodiment, the x-ray contrast agent is nycodenz, iohexol, omnipaque or similar low-osmolality agents.

    [0194] In one embodiment, the salt has the formula of XCl.sub.2(H.sub.2O).sub.a; where X=calcium, magnesium, barium or strontium; and a=0, 1, 2, 4 or 6.

    [0195] In one embodiment, the photo-initiator is eosin Y, tetrabromo derivative of fluorescein, methylated eosin Y, ethylated eosin Y, eosin yellowish, bromofluoresceic acid, acid red 87, bromoeosine, eosin B, dibromo dinitro derivative of fluorescein, or similar compounds.

    [0196] In one embodiment, the diol containing compound is PEG-diol, beta propylene glycol, propylene-1,3,diol, bisphenol A, 1,4-butanediol or similar compounds.

    [0197] In one embodiment, the micro-bulk capsule envelopes the cell aggregate.

    [0198] In another embodiment, the invention is directed to a therapeutically effective composition which includes a plurality of encapsulating devices having an average diameter of less than 400 micron, where the encapsulating devices include encapsulated cells in an encapsulation material, and the composition comprises at least about 500,000 cells/ml.

    [0199] In one embodiment, the average diameter of the encapsulating device is less than 300 micron. In one embodiment, the average diameter of the encapsulating device is less than 200 micron. In one embodiment, the average diameter of the encapsulating device is less than 100 micron. And in one embodiment, the average diameter of the encapsulating device is less than 50 micron.

    [0200] In on embodiment, the invention is directed to a therapeutically effective composition including a plurality of encapsulating devices having an average diameter of less than 400 micron, where the encapsulating devices include encapsulated cells in an encapsulation material, and the composition has a ratio of volume of encapsulating device to volume of cells of less than about 20:1.

    [0201] In one embodiment, the composition has a ratio of volume of encapsulating device to volume of cells of less than about 10:1. In one embodiment, the composition has a ratio of volume of encapsulating device to volume of cells of less than about 2:1.

    [0202] In another embodiment, the invention is directed to using a therapeutic composition as described herein in a method which includes the step of implanting the composition into an implantation site in an animal in need of treatment for a disease or disorder.

    [0203] In one embodiment, the invention is directed to a method of using the therapeutic composition which includes encapsulating devices with a polyethylene glycol (PEG) coating having a molecular weight between 900 and 20,000 Daltons, where the composition has a cell density of at least about 100,000 cells/ml in a method which includes the step of implanting the composition into an implantation site in an animal in need of treatment for a disease or disorder.

    [0204] In one embodiment, the implanting is an injection.

    [0205] In one embodiments, the disease or disorder is neurologic, cardiovascular, hepatic, endocrine, skin, hematopoietic, immune, neurosecretory, metabolic, systemic, or genetic.

    [0206] In one embodiment, the disease is an endocrine disease which is diabetes.

    [0207] In one embodiment, the animal is from an Order of Subclass Theria which is Artiodactyla, Carnivora, Cetacea, Perissodactyla, Primate, Proboscides, or Lagomorpha. In one embodiment, the animal is a Human.

    [0208] In one embodiment, the implantation site is subcutaneous, intramuscular, intraorgan, arterial/venous vascularity of an organ, cerebro-spinal fluid, or lymphatic fluid. In one embodiment, the implantation site is subcutaneous.

    [0209] In one embodiment, the method includes implanting encapsulated islets in a subcutaneous implantation site.

    [0210] In one embodiment, the method of implanting the composition into an implantation site in an animal in need of treatment for a disease or disorder also includes the step of administering an immunosuppressant or anti-inflammatory agent.

    [0211] In one embodiment, the immunosuppressant or anti-inflammatory agent is administered for less than 6 months. In one embodiment, the immunosuppressant or anti-inflammatory agent is administered for less than 1 month.

    [0212] In another one embodiment, the invention is directed to using a therapeutic composition which includes a plurality of encapsulating devices having an average diameter of less than 400 micron, where the encapsulating devices include encapsulated cells in an encapsulation material and the composition has at least about 500,000 cells/ml, in a method which includes the step of implanting the composition into an implantation site in an animal in need of treatment for a disease or disorder.

    [0213] In one embodiment, the implantation is an injection.

    [0214] In one embodiment, the animal is from an Order of Subclass Theria which is Artiodactyla, Carnivora, Cetacea, Perissodactyla, Primate, Proboscides, or Lagomorpha. In one embodiment, the animal is a Human.

    [0215] In one embodiment, the implantation site is subcutaneous, intramuscular, intraorgan, arterial/venous vascularity of an organ, cerebro-spinal fluid, or lymphatic fluid. In one embodiment, the implantation site is subcutaneous.

    [0216] In one embodiment, the method includes implanting encapsulated islets in a subcutaneous implantation site. In one embodiment, the method of implanting the composition into an implantation site in an animal in need of treatment for a disease or disorder also includes the step of administering an immunosuppressant or anti-inflammatory agent.

    [0217] In one embodiment, the immunosuppressant or anti-inflammatory agent is administered for less than 6 months. In one embodiment, the immunosuppressant or anti-inflammatory agent is administered for less than 1 month.

    [0218] In one embodiment, the encapsulated biological material is a PEG micro-bulk coated islet allograft. In one embodiment, the biological material is an organ, tissue or cell. In one embodiment, the tissue is a cluster of insulin producing cells. In one embodiment, the cell is an insulin producing cell.

    [0219] In one embodiment, the photoinitiator is carboxyeosin, ethyl eosin, eosin Y, fluorescein, 2,2-dimethoxy, 2-phenylacetophenone, 2-methoxy, 2-phenylacetophenono, camphorquinone, rose bengal, methylene blue, erythrosin, phloxine, thoionine, riboflavin or methylene green.

    [0220] In one embodiment, the photoactive polymer solution includes a polymerizable high density ethylenically unsaturated PEG and a sulfonated comonomer.

    [0221] In a one embodiment, the polymerizable high density ethylenically unsaturated PEG is a high density acrylated PEG. In a one embodiment, the polymerizable high density acrylated PEG has a molecular weight of 1.1 kD.

    [0222] In one embodiment, the sulfonated comonomer is 2-acrylamido-2-methyl-1-propanesulfonic acid, vinylsulfonic acid, 4-styrenesulfonic acid, 3-sulfopropyl acrylate, 3-sulfopropyl methacrylate, or n-vinyl maleimide sulfonate. In a one embodiment, the sulfonated comonomer is 2-acrylamido-2-methyl-1-propanesulfonic acid.

    [0223] In one embodiment, the photoactive polymer solution also includes a cocatalyst which is triethanolamine, triethylamine, ethanolamine, N-methyl diethanolamine, N,N-dimethyl benzylamine, dibenzyl amino, N-benzyl ethanolamine, N-isopropyl benzylamine, tetramethyl ethylenediamine, potassium persulfate, tetramethyl ethylenediamine, lysine, omithine, histidine or arginine.

    [0224] In one embodiment, the cocatalyst is triethanolamine.

    [0225] In one embodiment, the photoactive polymer solution also includes an accelerator which is N-vinyl pyrrolidinone, 2-vinyl pyridine, 1-vinyl imidazole, 9-vinyl carbazone, 9-vinyl carbozol, acrylic acid, n-vinylcarpolactam, 2-allyl-2-methyl-1,3-cyclopentane dione, or 2-hydroxyethyl acrylate.

    [0226] In one embodiment, the accelerator is N-vinyl pyrrolidinone.

    [0227] In one embodiment, the photoactive polymer solution also includes a viscosity enhancer which is selected from the group including natural and synthetic polymers. In a one embodiment, the viscosity enhancer is 3.5 kD PEG-triol or 4 kD PEG-diol.

    [0228] In one embodiment, the biological material for the encapsulation method is neurologic, cardiovascular, hepatic, endocrine, skin, hematopoietic, immune, neurosecretory, metabolic, systemic, or genetic. In one embodiment, the biological material is from an animal of Subclass Theria of Class Mammalia. In a one embodiment, the animal is from an Order of Subclass Theria which is Artiodactyla, Carnivora, Cetacea, Perissodactyla, Primate, Proboscides, or Lagomorpha. In one embodiment, the animal is a Human.

    [0229] In another embodiment, the invention is directed to a composition for encapsulating biological material which includes a polymerizable high density ethylenically unsaturated PEG having a molecular weight between 900 and 20,000 Daltons, and a sulfonated comonomer.

    [0230] In one embodiment, the composition for encapsulating biological material has the quality of permselectivity. In one embodiment, the permselectivity can be engineered by manipulating the composition.

    [0231] In one embodiment, the composition for encapsulating biological material further is biodegradable. In one embodiment, the composition is biodegradable in a mammal. In one embodiment, the composition is biodegradable in a sub-human primate. In one embodiment, the composition is biodegradable in a human.

    [0232] Further aspects, features and advantages of this invention will become apparent from the detailed description of the one embodiments which follow.

    [0233] One embodiment, is related to compositions and methods of treating one or more diseases or disorders, such as neurologic (e.g., Parkinson's disease, Alzheimer's disease, Huntington's disease, Multiple Sclerosis, blindness, peripheral nerve injury, spinal cord injury, pain and addiction), cardiovascular (e.g., coronary artery, angiogenesis grafts, valves and small vessels), hepatic (e.g., acute liver failure, chronic liver failure, and genetic diseases effecting the liver), endocrine (e.g., diabetes, obesity, stress and adrenal, parathyroid, testicular and ovarian diseases), skin (e.g., chronic ulcers and diseases of the dermal and hair stem cells), hematopoietic (e.g., Factor VIII and erythropoietin), or immune (e.g., immune intolerance or auto-immune disease), in a subject in need of treatment comprising: providing cells or tissue, such as pancreatic islets, hepatic tissue, endocrine tissues, skin cells, hematopoietic cells, bone marrow stem cells, renal tissues, muscle cells, neural cells, stem cells, embryonic stem cells, or organ specific progenitor cells, or genetically engineered cells to produce specific factors, or cells or tissue derived from such; enclosing said cells or tissue within at least one encapsulating material, such as a hydrogel, made of physically or chemically crosslinkable polymers, including polysaccharides such as alginate, agarose, chitosan, poly(amino acids), hyaluronic acid, chondroitin sulfate, dextran, dextran sulfate, heparin, heparin sulfate, heparan sulfate, gellan gum, xanthan gum, guar gum, water soluble cellulose derivatives, carrageenan, or proteins, such as gelatin, collagen, albumin, or water soluble synthetic polymers with ethylenically unsaturated groups or their derivatives, such as poly(methyl methacrylate) (PMMA), or poly(2-hydroxyethyl methacrylate) (PHEMA), poly(ethylene glycol) (PEG), poly(ethylene oxide) (PEO), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(thyloxazoline) (PEOX); or a combination of the above, such as alginate mixed with PEG, or more hydrophobic or water insoluble polymers, such as poly(glycolic acid) (PGA), poly(lactic acid) (PLA), or their copolymers (PLA-GA), or polytetrafluoroethylene (PTFE) and administering a therapeutically effective amount of said encapsulated cells or tissue to the subject in need of treatment via subcutaneous injection or implant, or directly into organs via either direct injection into the substance of the organ or injection through the vascular system of those organs.

    [0234] Organs maybe selected from, but not limited to, liver, spleen, kidney, lung, heart, brain, spinal cord, muscle, and bone marrow.

    [0235] The subject in need of treatment may be selected from, but not limited to, mammals, such as humans, sub-human primates, cows, sheep, horses, swine, dogs, cats, and rabbits as well as other animals such as chickens, turkeys, or fish.

    [0236] In a further embodiment, the encapsulated cell or tissue may be administered to a subject in need of treatment in combination with an immunosuppressant and/or an anti-inflammatory agent. The immunosuppressant may be selected from, but not limited to cyclosporine, sirolimus, rapamycin, or tacrolimus. The anti-inflammatory agent may be selected from, but not limited to, aspirin, ibuprofen, steroids, and non-steroidal anti-inflammatory agents.

    [0237] The immunosuppressant and/or an anti-inflammatory agent is administered for six months following implantation or injection of the encapsulated cells or tissue. The immunosuppressant and/or an anti-inflammatory agent is administered for one month following implantation or injection of the encapsulated cells or tissue

    [0238] In a embodiment, encapsulated islets are implanted or injected subcutaneously or into liver or spleen. In one aspect, micro-bulk coated islets are administered subcutaneously.

    [0239] In some embodiments, the concentration of ingredients and composition of encapsulating solution may vary. Concentration ranges are as follows.

    [0240] For Buffer solution a concentration is 1 to 200 mM, 5 to 100 mM, and 10 to 50 mM.

    [0241] For CaCl.sub.2 a concentration is 0.1 to 40 mM, 0.5 to 20 mM, and 1 to 5 mM. For Manitol a concentration is 10 mM to 6M, yet more is 50 mM to 3M, yet more is 100 mM to 1M, and yet more is 200 to 300 mM.

    [0242] For pH of CaCl.sub.2/Manitol solution a value is 6 to 8, 6.4 to 7.6, and 6.6 to 7.4.

    [0243] For DEN-EY a concentration is 0.005 to 8 mg/ml, 0.01 to 4 mg/ml, and 0.05 to 2 mg/ml.

    [0244] For DEN-EY conjunction level a level is 0.15 to 68, 1 to 34, and 1.5 to 15.

    [0245] For pH of macromer solution a value is 6.5 to 9.5, 7 to 9, and 7.5 to 8.5.

    [0246] For PEG TA a concentration is 0.1 to 100%, 0.2 to 50%, and 1 to 25%.

    [0247] For PEG TA a density is 0.05 to 20 K, 0.1 to 10 K, 0.5 to 5 K, and 0.8 to 2.5 K.

    [0248] For PEG-triol a concentration is 0.1 to 100%, 1 to 75%, and 2 to 50%.

    [0249] For PEG-triol a density is 0.15 to 70 K, 0.3 to 35 K, 1.5 to 15 K, and 2.3 to 7.5 K.

    [0250] For PEG-diol a concentration is 0.1 to 100% 1 to 75%, and 2 to 50%.

    [0251] For PEG-diol a density is 0.2 to 80 K, 0.5 to 40 K, 1 to 20 K, and 2 to 10 K.

    [0252] For TEoA a concentration is 5 mM to 2 M, 10 mM to 1M, 50 to 500 mM, and 75 to 125 mM.

    [0253] For AMPS a concentration is 2 to 640 mg/ml, 5 to 300 mg/ml, and 10 to 150 mg/ml.

    [0254] For NVP a concentration is 0.01 to 40 μl/ml, 0.1 to 20 μl/ml, and 0.5 to 10 μl/ml.

    [0255] For Nycodenz a concentration is 0.1 to 100%, 1 to 50%, and 5 to 25%.

    [0256] For the Laser a strength is 10 mW/cm.sup.2 to 4 W/cm.sup.2, 25 mW/cm.sup.2 to 2 W/cm.sup.2, and 75 mW/cm.sup.2 to 1 W/cm.sup.2.

    [0257] For the light source a time is 3 seconds to 20 minutes, 6 seconds to 10 minutes, and 12 seconds to 3 minutes.

    [0258] In an embodiment, the encapsulating material comprises a hydrogel that forms a sphere around at least one cell or tissue.

    [0259] In one embodiment, a cell or tissue may be encapsulated in a biocompatible alginate microcapsule, wherein the alginate is made biocompatible by coating the alginate in a biocompatible material, such as PEG or hyaluronic acid, purifying the alginate and/or removing the poly-lysine and replacing it with PEG.

    [0260] The disease to be treated is diabetes, the cells or tissue comprise insulin producing cells or tissue, or cells or tissue derived from pancreatic cells or tissue, or cells derived from progenitor or stem cells that are converted into insulin producing cells, and the encapsulated cells or tissue are administered to the subject in need of treatment via subcutaneous or liver injection or implant.

    [0261] According to an embodiment the microcapsules of encapsulated insulin-producing cells or tissue may have an average diameter of 10 micron to 1000 micron, 100 micron to 600 micron, 150 micron to 500 micron, and 200 micron to 300 micron.

    [0262] In another embodiment, the invention relates to an insulin-producing cell or tissue encapsulated in microcapsules having a concentration of at least 2,000 IEQ (islet equivalents)/ml, at least 9,000 IEQ/ml, and at least 200,000 IEQ/ml.

    [0263] In another embodiment, the volume of insulin-producing cells or tissue encapsulated in microcapsules administered per kilogram body mass of a subject may be 0.001 ml to 10 ml, 0.01 ml to 7 ml, 0.05 ml to 2 ml.

    [0264] In one embodiment, the ratio of microcapsule volume to insulin producing cell or tissue volume is less than 300 to 1, less than 100 to 1, less than 50 to 1, and less than 20 to 1.

    [0265] In one embodiment, micro-bulk coated insulin-producing cells or tissue may have an average membrane thickness of 1 to 400 micron, 10 to 200 micron, and 10 to 100 micron. In one embodiment the invention relates to a micro-bulk coated insulin-producing cell or tissue having a concentration of at least 10,000 IEQ/ml, at least 70,000 IEQ/ml, at least 125,000 IEQ/ml, and at least 200,000 IEQ/ml.

    [0266] In one embodiment, the volume of the micro-bulk coated insulin producing cell or tissue administered per kilogram body mass of a subject may be 0.01 to 7 ml, 0.01 to 2 ml, and 0.04 to 0.5 ml.

    [0267] In one embodiment, the ratio of micro-bulk coating volume to insulin-producing cell or tissue volume is less than 13 to 1, less than 8 to 1, less than 5 to 1, and less than 2.5 to 1.

    [0268] In one embodiment, the microcapsules of encapsulated cells or tissue may have an average diameter of 10 micron to 1000 micron, 100 micron to 600 micron, 150 micron to 500 micron, and 200 micron to 300 micron.

    [0269] In one embodiment, the ratio of microcapsule volume to insulin producing cell or tissue volume is less than 300 to 1, less than 100 to 1, less than 50 to 1, and less than 20 to 1.

    [0270] In one embodiment, micro-bulk coated cells or tissue may have an average membrane thickness of 1 to 400 micron, 10 to 200 micron, and 10 to 100 micron.

    [0271] In one embodiment, the ratio of micro-bulk coating volume to cell or tissue volume is less than 13 to 1, less than 8 to 1, less than 5 to 1, and less than 2.5 to 1.

    [0272] In one embodiment, relates encapsulated cells or tissue where the cell density is at least about 100,000 cells/ml. The encapsulated cell is micro-bulk coated. The cell is micro-bulk coated with an encapsulating material comprising acrylated PEG.

    [0273] In one embodiment, a method of treating diabetes in a subject comprising administering encapsulated islets where the cell density is at least about 6,000,000 cells/ml, where the curative dose is less than about 2 ml per kilogram body mass of the subject.

    [0274] In one embodiment, related to agricultural animals or pets, such as cows, sheep, horses, swine, chickens, turkeys, rabbits, fish, or dogs and cats; to change the growth rate, or alter the condition of the animal (e.g., increase meat or dairy production), or protect them from or treat them for different diseases.

    [0275] In one embodiment, a method of providing cells or tissue to an agriculturally relevant animal comprises: a) providing a cell or tissue; b) enclosing said cell or tissue within at least one encapsulating material, such as a hydrogel, made of physically or chemically crosslinkable polymers, including polysaccharides such as alginate, agarose, chitosan, poly(amino acids), hyaluronic acid, chondroitin sulfate, dextran, dextran sulfate, heparin, heparin sulfate, heparan sulfate, gellan gum, xanthan gum, guar gum, water soluble cellulose derivatives, carrageenan, or proteins, such as gelatin, collagen, albumin, or water soluble synthetic polymers or their derivatives, such as methyl methacrylate (MMA), or 2-hydroxyethyl methacrylate (HEMA), polyethylene glycol (PEG), poly(ethylene oxide) (PEO), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(thyloxazoline) (PEOX); or a combination of the above, such as alginate mixed with PEG, or more hydrophobic or water insoluble polymers, such as poly(glycolic acid) (PGA), poly(lactic acid) (PLA), or their copolymers (PLA-GA), or polytetrafluoroethylene (PTFE); and c) administering said encapsulated cell or tissue to the subject in need of treatment via subcutaneous injection or implant, or directly into organs via either direct injection into the substance of the organ or injection through the vascular system of those organs.

    [0276] In one embodiment, a method for encapsulation of at least one islet cell encapsulated in a microcapsule, comprising the steps of: a) coating at least one islet cell encapsulated in a microcapsule with photoinitiator; b) suspending the at least one coated islet cell encapsulated in a microcapsule in a macromer solution comprised of macromer; and c) irradiating the suspension with light.

    [0277] In one embodiment, the macromer is a water soluble, ethylenically unsaturated, polymer susceptible to polymerization into water insoluble polymer through interaction of at least two carbon-carbon double bonds.

    [0278] In one embodiment, the macromer is selected from the group consisting of ethylenically unsaturated derivatives of poly(ethylene oxide) (PEO), poly(ethlyene glycol) (PEG), poly(vinyl alcohol) (PVA), poly(vinylpyrrolidone) (PVP), poly(ethyloxazoline) (PEOX), poly(amino acids), polysaccharides, and proteins.

    [0279] In one embodiment, the polysaccharides are selected from the group consisting of alginate, hyaluronic acid, chondroitin sulfate, dextran, dextran sulfate, heparin, heparin sulfate, heparan sulfate, chitosan, gellan gum, xanthan gum, guar gum, water soluble cellulose derivatives and carrageenan.

    [0280] In one embodiment, the proteins are selected from the group consisting of gelatin, collagen, and albumin.

    [0281] In one embodiment, the photoinitiator is any dye that absorbs light having a frequency between 320 nm and 900 nm, can form free radicals, is at least partially water soluble, and is non-toxic to the at least one islet cell at the concentration used for polymerization.

    [0282] In one embodiment, the macromer solution further comprises a primary, secondary, tertiary, or quaternary amine cocatalyst and the photoinitiator is selected from the group of ethyl eosin, eosin Y, fluorescein, 2,2-dimethoxy, 2-phenylacetophenone, 2-methyl, 2-phenylacetonphenone, camphorquinone, rose bengal, methylene blue, erythosin, phloxime, thionine, riboflavin, and methyl green.

    [0283] In one embodiment, the microcapsule is comprised of material selected from the group of alginate, chitosan, agarose, and gelatin.

    [0284] In one embodiment, the macromer solution further comprises an accelerator to increase the rate of polymerization.

    [0285] Additional embodiments are described in the following paragraphs.

    [0286] Paragraph 1. A composition comprising: encapsulating devices comprising a micro-bulk coating, and cell aggregates, wherein said composition has a cell density of at least about 100,000 cells/ml, wherein the micro-bulk coating for the encapsulating devices comprises a polymerizable high density ethylenically unsaturated polyethylene glycol (PEG) having a molecular weight between 900 and 20,000 Daltons, and a sulfonated comonomer, and wherein the micro-bulk coating comprises salt, MOPS (3-(N-morpholino)propanesulfonic) acid, co-monomer, a diol containing compound, an x-ray contrast agent and a photo-initiator.

    [0287] Paragraph 2. The composition of paragraph 1, wherein the encapsulating devices are micro-bulk capsules.

    [0288] Paragraph 3. The composition of paragraph 1 where the PEG is selected from the group consisting of a diacrylate of PEG with a molecular weight in the range of 2 kD to 16 kD, a triacrylate of PEG with a molecular weight in the range of 3 kD to 16 kD, a tetra-acrylate of PEG with a molecular weight in the range of 4 kD to 20 kD, and combinations thereof.

    [0289] Paragraph 4. The composition of paragraph 1 where the co-monomer is selected from the group consisting of AMPS (2-Acrylamido-2-methylpropane sulfonic acid), ammonium AMPS, 2-methyl-2-((1-oxo-2-propenyl)amino)-monoammomium salt, nVP (N-Vinylpyrrolidone), polyvidone, polyvinylpolypyrrolidone and similar types of co-polymers.

    [0290] Paragraph 5. The composition of paragraph 1 where the x-ray contrast agent is selected from the group consisting of nycodenz, iohexol, omnipaque and similar low-osmolality agents.

    [0291] Paragraph 6. The composition of paragraph 1 where the salt has the formula of XCl.sub.2(H.sub.2O).sub.a; where X=calcium, magnesium, barium or strontium; and a=0, 1, 2, 4 or 6.

    [0292] Paragraph 7. The composition of paragraph 1 where the photo-initiator is selected from the group consisting of eosin Y, tetrabromo derivative of fluorescein, methylated eosin Y, ethylated eosin Y, eosin yellowish, bromofluoresceic acid, acid red 87, bromoeosine, eosin B, dibromo dinitro derivative of fluorescein, and similar compounds.

    [0293] Paragraph 8. The composition of paragraph 1 where the diol containing compound is selected from the group consisting of PEG-diol, beta propylene glycol, propylene-1,3,diol, bisphenol A, 1,4-butanediol and similar compounds.

    [0294] Paragraph 9. The composition of paragraph 2, wherein the micro-bulk capsule envelopes the cell aggregate.

    [0295] Paragraph 10. The composition of paragraph 9, wherein the cell aggregate is pancreatic islets.

    [0296] Paragraph 11. The composition of paragraph 9, wherein the cell density is at least about 6,000,000 cells/ml.

    [0297] Paragraph 12. The composition of paragraph 1, where the cell is selected from the group consisting of neurologic, cardiovascular, hepatic, endocrine, skin, hematopoietic, immune, neurosecretory, metabolic, systemic, and genetic.

    [0298] Paragraph 13. The composition of paragraph 12, where the cell is selected from the group consisting of autologous, allogeneic, xenogeneic and genetically-modified.

    [0299] Paragraph 14. The composition of paragraph 12, where the endocrine cell is an insulin producing cell.

    [0300] Paragraph 15. A composition comprising a plurality of encapsulating devices having an average diameter of less than 500 μm, said encapsulating devices comprising encapsulated cell aggregates within a micro-bulk coating of an encapsulation material, wherein the composition comprises at least about 500,000 cells/ml and wherein the encapsulation material comprises a polymerizable high density ethylenically unsaturated PEG having a molecular weight of between 900 and 20,000 Daltons, and a sulfonated comonomer, wherein the micro-bulk coating contains the encapsulated cell aggregates.

    [0301] Paragraph 16. The composition of paragraph 15, wherein the average diameter of the encapsulating device is less than 400 micron.

    [0302] Paragraph 17. The composition of paragraph 15, wherein the average diameter of the encapsulating device is less than 300 micron.

    [0303] Paragraph 18. The composition of paragraph 15, wherein the average diameter of the encapsulating device is less than 200 microns.

    [0304] Paragraph 19. The composition of paragraph 15, wherein the average diameter of the encapsulating device is less than 100 micron.

    [0305] Paragraph 20. A composition comprising a plurality of micro-bulk encapsulating devices having an average diameter of less than −500 μm, said encapsulating devices comprising encapsulated cells aggregates micro-bulk coated in an encapsulation material, wherein the composition comprises a ratio of volume of encapsulating device to volume of cells of less than about 20:1 and wherein the encapsulation material comprises a polymerizable high density ethylenically unsaturated PEG having a molecular weight between 900 and 20,000 Daltons, and a sulfonated comonomer, and wherein the encapsulation material comprises salt, MOPS (3-(N-morpholino)propanesulfonic) acid, co-monomer, a diol containing compound, an x-ray contrast agent and a photo-initiator.

    [0306] Paragraph 21. The composition of paragraph 20, wherein the composition comprises a ratio of volume of encapsulating device to volume of cells of less than about 10:1.

    [0307] Paragraph 22. The composition of any one of paragraphs 1, 15, or 20, where the polymerizable high density ethylenically unsaturated PEG is a high density acrylated PEG.

    [0308] Paragraph 23. The composition of paragraph 22, where the polymerizable high density acrylated PEG has a molecular weight of 2 kD to 20 kD.

    [0309] Paragraph 24. The composition of any one of paragraphs 1, 15, or 20, where the sulfonated comonomer is selected from the group consisting of 2-acrylamido-2-methyl-1-propanesulfonic acid, vinylsulfonic acid, 4-styrenesulfonic acid, 3-sulfopropyl acrylate, 3-sulfopropyl methacrylate, and n-vinyl maleimide sulfonate.

    [0310] Paragraph 25. The composition of paragraph 24, where the sulfonated comonomer is 2-acrylamido-2-methyl-1-propanesulfonic acid.

    [0311] Paragraph 26. The composition of any one of paragraphs 1, 15, or 20, further comprising a cocatalyst selected from the group consisting of triethanolamine, triethylamine, ethanolamine, N-methyl diethanolamine, N,N-dimethyl benzylamine, dibenzyl amino, N-benzyl ethanolamine, N-isopropyl benzylamine, tetramethyl ethylenediamine, potassium persulfate, tetramethyl ethylenediamine, lysine, omithine, histidine and arginine.

    [0312] Paragraph 27. The composition of paragraph 26, where the cocatalyst is triethanolamine.

    [0313] Paragraph 28. The composition of any one of paragraphs 1, 15, or 20, further comprising an accelerator selected from the group consisting of N-vinyl pyrrolidinone, 2-vinyl pyridine, 1-vinyl imidazole, 9-vinyl carbazone, 9-vinyl carbozol, acrylic acid, n-vinylcarpolactam, 2-allyl-2-methyl-1,3-cyclopentane dione, and 2-hydroxyethyl acrylate.

    [0314] Paragraph 29. The composition of paragraph 28, where the accelerator is N-vinyl pyrrolidinone.

    [0315] Paragraph 30. A composition comprising encapsulating devices comprising encapsulating cells in an encapsulation material with a polyethylene glycol (PEG) coating having a molecular weight between 900 and 3,000 Daltons, wherein said composition has a cell density of at least about 6,000,000 cells/ml.

    [0316] Paragraph 31. The composition of paragraph 30, wherein the encapsulating devices are microcapsules.

    [0317] Paragraph 32. The composition of paragraph 31, wherein the microcapsules are micro-bulk coated cell aggregates.

    [0318] Paragraph 33. The composition of paragraph 32, wherein the cell aggregates are pancreatic islets.

    [0319] Paragraph 34. The composition of paragraph 30, where the cell is selected from the group consisting of neurologic, cardiovascular, hepatic, endocrine, skin, hematopoietic, immune, neurosecretory, metabolic, systemic, and genetic.

    [0320] Paragraph 35. The composition of paragraph 34, where the cell is selected from the group consisting of autologous, allogeneic, xenogeneic and genetically-modified.

    [0321] Paragraph 36. The composition of paragraph 35 where the endocrine cell is an insulin producing cell.

    [0322] Paragraph 37. A composition comprising a plurality of encapsulating devices having an average diameter of less than 400 μm, said encapsulating devices comprising encapsulated cells in an encapsulation material, wherein a cell density is at least about 6,000,000 cells/ml.

    [0323] Paragraph 38. The composition of paragraph 37, wherein the average diameter of the encapsulating device is less than 300 micron.

    [0324] Paragraph 39. The composition of paragraph 38, wherein the average diameter of the encapsulating device is less than 200 micron.

    [0325] Paragraph 40. The composition of paragraph 39, wherein the average diameter of the encapsulating device is less than 100 micron.

    [0326] Paragraph 41. The composition of paragraph 40, wherein the average diameter of the encapsulating device is less than 50 micron.

    A. Background of PEG Interfacial Polymerization for Islets

    [0327] One of the inventors developed a PEG based islet encapsulation methods and patents that were utilized for their primate studies and their FDA approved clinical trials. This technology is defined in the 2008 U.S. Pat. No. 7,427,415. The clinical trial was closed with partial islet function for greater than one year. The uniqueness of this technology was that islets were not first encapsulated into a matrix that was then was treated for crosslinking and permeability parameters. So this technology was a clear departure from alginate and similar islet hydrogel encapsulation techniques. Instead, each islet was coated with a dye, Eosin Y, which was placed on the surface of the islets. These stained islets were then placed into solution with the PEG encapsulating components. Since the photoinitiator was located in the solution along with other encapsulating ingredients, the radical based crosslinking of the polymer was accomplished by exposure to a high intensity laser beam focused on the bottom of the dish containing the stained islets. A major problem with this approach was the interfacial polymerization on the Y ((polyethylenimine-3-(acrylamidopropyl) trimethyl ammonium chloride. Eosin-5-Isothiocyanate), The other components were combined into new islet capsules using PEG-acrylates NVP (N-vinyl-2-pyrrolidinone), a co-monomer, AMPS (Sodium 2-Acrylomido-2-methyl-1-propansesulfonic acid) solution and also a co-monomer along with other salt solutions using high intensity LED light to cross link the polymetric coating. The new process now permits the formation of a new, minimal volume coating of individual islets that can readily replace the interfacial coatings.

    B. PEG Micro-Bulk Phase, Minimal Volume Capsules Proposed Alternative for Human Islet/iPS/ESC Encapsulation

    [0328] A significant alternative has been developed to the interfacial polymerized PEG for a diabetes therapy product sourcing for human pancreatic islet cells (Islets)/Induced Pluripotent Stem cell (iPS cell)/Embryonic Stem Cell (ESC's) formed aggregates. PEG acrylate Micro-Bulk phase technology can encapsulate individual human Islets, iPS cell islet aggregates, and/or ESC islet aggregates by minimal volume capsules. Several technical improvements in islet encapsulation enable this new approach. Minimal Volume Capsules of alginate have been developed to reduce standard sizes of alginate capsules from 500+ micron sized capsules containing a single islet to 250-400 micron sized islets that can centralize them within the capsule. By applying the methods used to make these smaller sized alginate capsules to PEG encapsulation, one can now develop “Micro-Bulk” phase islet encapsulation. Micro-Bulk phase encapsulation techniques to encapsulate Islets/IPS cells/ESC's as minimal volume capsules within the very similar PEG coatings. This starts with islets/IPS cells/ESC's being encapsulated in the PEG reactants as small capsules that are then irradiated with LED light to crosslink the PEG surrounding the islets. This reduces the oxygen based radical islet destruction since the eosin Y is no longer on the islet surface in high concentration but is now in the Micro-Bulk phase encapsulation solution at lower concentrations. It also eliminates the requirement to contractually produced dendrimer eosin Y to keep it on the cell aggregate surface for interfacial polymerization. Finally, this approach eliminates the laser requirement due to recent advances in LED technology that can deliver the energy required to cross link the islet containing PEG coatings without the high energy required to cross link islets in a large petri dish. Now each islet can be crosslinked as it moves through the encapsulation device using far less energy as compared to crosslinking in large petri dish sized containers.

    1. Micro-Bulk Phase Encapsulation Systems for Islets/iPS Cells/ESC's

    [0329] a. Conversion of the Nisco Encapsulator for Islet Micro-Bulk Phase Encapsulation

    [0330] Nisco Encapsulator was developed for standard, large-scale alginate encapsulation of cell aggregates utilizing high voltage to reduce islet capsule sizes. This method can also encapsulate islets/iPS cells/ESC's in PEG small capsules that do not require calcium or barium crosslinking. Instead of forming the alginate encapsulated islets and collecting them in a calcium chloride bath for crosslinking, the Nisco Encapsulator can be modified so that the PEG Micro-Bulk phase encapsulation can permit LED illumination following the formation of the minimal volume capsules in PEG.

    [0331] Just like alginate capsules using this approach, the PEG encapsulated islets are not centralized leading to portions on the edge of capsules inadequately covered as well as high concentrations of empty capsules.

    b. Micro-Bulk Encapsulation Using a Tower Apparatus for Formation of the PEG Capsules Containing Islets/iPS Cells/ESC's

    [0332] This technique is a larger scale alternative to the other approaches as a system that would be more scalable to manufacturing levels. The development of this approach was done by the following three steps:

    i. Formation of Micro-Bulk Droplets

    [0333] The first step was to develop a method in which the droplet formation of the PEG polymer that would include islets/iPC cells/ESC's could be controlled and optimized in terms of a micro-drop size. A small system was built which controlled the flow rate of the fluid containing the PEG polymer exiting a small bore inner needle while simultaneously providing controlled nitrogen gas to flow through a larger outer needle to reduce droplet size. Collection of these PEG droplets initially was into open petri dishes. Flow rates were readily available to slow the formation of polymer droplets that fell into the petri dish containing HBSS. These droplet sizes could be controlled well and when captured underneath focused LED lights of the proper wavelength would readily crosslink.

    ii. Optimization of PEG Encapsulation Components

    [0334] Once this was demonstrated, a series of PEG capsules were produced at a small size as a platform used to optimize the PEG ratios of components required to readily optimize the crosslinking of the capsules. The following components were optimized for capsule size, speed of polymerization, and optimal capsule morphology:

    [0335] a. PEG acrylate size—from 1.1 kD diacrylate to 10 kD diacrylate and 10 kD tetra-acrylate at different concentrations

    [0336] b. Optimization of nVP concentrations

    [0337] c. Optimization of AMPS concentrations

    [0338] d. Optimization of Nycodenz concentrations

    [0339] e. Optimization of PEG diol concentrations

    [0340] Once these optimizations were accomplished, they were combined into a final formulation and method that was tested under a variety of conditions. These results demonstrate the empty Micro-Bulk PEG capsules can clearly and uniformly be crosslinked in a very short time, can lose their surface stickiness, can maintain their shape and size over many days time, and are stable in tissue culture solutions for a few weeks.

    [0341] Once this was accomplished, we turned to the final optimization of the encapsulation and collection steps. The first step in the tower application optimization was to replace the nitrogen gas with a liquid to drive the PEG polymer solution containing the islets/iPC cells/ESC's down the inner cannula to their exit maintain very small capsule size. With this completed, we turned to developing the collection system after capsule formation since dropping them into the bottom of a petri dish was not an optimizable step as the PEG Micro-Drop capsules readily coalesced prior to their being adequately crosslinked. Instead a collection column was developed that became the site of the LED irradiation step to crosslink the PEG polymer. By controlling the density of the liquid in this reaction chamber, the time to complete crosslinking of the PEG micro-bulk capsules could be completed while still slowly falling through this density-controlled liquid. Thus, the recovery step appeared to be more simple by collecting the Micro-Bulk crosslinked PEG polymer encapsulated islets into HBSS off the bottom of the density gradient in the reaction chamber. Unfortunately, testing with this method proved to not be simple with the PEG-diacrylate capsules continuing to coalesce prior to their being completely crossed-linked by the LED light. So this method of forming and crosslinking Micro-Bulk PEG-diacrylate capsules was also placed on hold until other methods could be evaluated.

    c. Micro-Bulk Encapsulation Using Micro-Fluidics Formation of PEG Capsules Containing Islets/IPS Cells/ESC's

    [0342] The use of micro-fluidics has readily been demonstrated to form small beads that contain cells including islets. Also explored was the potential to cross link PEG-acrylate capsules containing islets/iPS cells/ESC's that can be formed into Micro-Bulk capsules using this micro-fluidics approach of capsule formation. Initially, the capsules well formed with the micro-fluidics that then exited the device to drop into a collection system. But, this exit approach essentially returned us to the Micro-Bulk droplet formation then falling into a collection mode of either a petri dish or a tower in which the time to complete the encapsulation of the PEG-diacrylate was also too long to prevent coalescence of the Micro-Bulk PEG capsules into larger and larger blobs of PEG-diacrylate macro-capsules containing multiple islets. In order to perform the LED induced crosslinking completely within a micro-fluidic may be possible but is beyond our current capabilities. So this approach was also placed on hold.

    d. Micro-Bulk Encapsulation Utilizing Emulsion Techniques

    [0343] An emulsion technique was developed that was required for the encapsulation of the TRGel thermally crosslinked capsules. This technique had to be modified to replace the thermal crosslinking with LED illumination crosslinking of the emulsion produced PEG capsules that can contain islets.

    [0344] Previous research has demonstrated the feasibility of encapsulating islets by conformal coatings laid down on the surface of islets by binding the photoinitiator to the surface of the islets, followed by laser exposure of light into dish containing the stained islets and all the encapsulation constituents in the media. The crosslinking took place with light activation at the site of the bound eosin Y forming a conformal coating of islets that was self-limiting in thickness of capsule with decreasing crosslinking from the surface to the outer edge of the formed capsules.

    [0345] The application discloses micro-bulk coating, which is an improved method of encapsulation compared to conformal coating. Emulsion technology was developed to form a micro-bulk coating on the surface of islets. A light box with LED's focused inside achieve high levels of crosslinking which keeps the capsules from fragmenting in vivo. A method was developed to measure fragmentation of capsules. An in vitro capsule testing system was developed to measure the capsule strength in terms of stress and strain prior to implantation. Fragmentation was reduced by increasing concentrations of the micro-bulk components.

    [0346] Capsule size is an important feature for a successful encapsulation procedure. Reduce Capsule size was reduced by manipulating the encapsulation component conditions.

    [0347] The following examples are provided merely for illustrative purposes of the present invention and are not to be read as limiting the scope of protection of the present invention.

    EXAMPLES

    Example 1

    PEG Micro-Bulk Encapsulation for Islets

    [0348] Alginate capsules have been the standard method of isolating islets for over 30 years (FIG. 1A). Their primary problem has been the large size of these capsules relative to the size of the islets that produces implant problems of very large quantities of alginate required for clinical application. While we and others can reduce the size of alginate capsules now and can also increase the numbers of islets per capsule (FIG. 1B), this problem of capsule size remains a critical impediment for considering alginate encapsulation as a way forward to a clinical product. In addition, while highly purified alginates are being formulated, the basic lot-to-lot variations of the seaweed required to produce a standard alginate remains a problem. Conformal coating of islets by PEG tri-acrylate coatings was the best candidate produced in terms of the smallest coating (50-100 microns thick) as well as excellent biocompatibility and functional results (FIG. 1C). It also produced the optimal coatings for long term implant success in diabetic non-human primate implants without insulin or immunosuppression requirements. However, it was lost to further development for human clinical trials due to investor fatigue in supporting the development of a clinical trials and future products that resulted in abandoning its strong patent position.

    [0349] Volume of PEG conformal coating=20-50 micron coating layer over 150 micron sized islet, then the volume of the conformal coated islet=8×10.sup.6 um.sup.3

    [0350] If volume of 300 micron capsule of PEG Micro-bulk capsule=14×10.sup.6 um.sup.3 If volume of earlier PEG conformal coating=1, then PEG micro-bulk=1.7 times its volume.

    [0351] This compares with alginate capsules that usually are 27 times larger if volume of 750 micron diameter alginate capsule is measured with volume=220×10.sup.6

    Example 2

    [0352] Initial Implant Results from 1% 1 kDA PEG-Diacrylate Empty Capsules

    [0353] FIGS. 2A & 2B show that the optimized alginate capsules formed with highly purified alginate when formed into empty capsules remain intact two weeks after implant in both Nu/Nu immune incompetent mice and C57Bl6 normal mice. FIGS. 2C & 2D for Nu/Nu implants of 1% 1 kDa PEG Diacrylate in the Nu/Nu mouse recipients demonstrate significant PEG-diacrylate capsule fragmentation after two weeks of implant (FIGS. 2E & 2F). FIGS. 2G & 2H show C57Bl6 implants of 1% 1 kDa PEG-diacrylate also demonstrate significant fragmentation of the empty capsules (FIGS. 2I & 2J).

    Example 3

    Unrestrained Compression Testing for Capsule Strength Testing

    [0354] With evidence of capsule fragmentation post-implant, it became obvious that some valid strength testing apparatus had to be developed to initially produce stronger capsules in development and then function as a quality control test post-production prior to implantation. FIG. 3 shows a small apparatus that was developed to place a drop of polymer onto a microscopic glass slide that was then covered by a second glass slide. Specific calibrated weights were sequentially added to document the weight that either broke the polymer bead or reached the maximal weight without breakage. In addition to the weights, Elasticity is actually reported in Pasquelles by calculating by actual measurements of Stress and Strain by scientific definitions. FIG. 4 demonstrates the results of this testing in actual quantification that has been used going forward for all micro-bulk capsules produced.

    Example 4

    Development of PEG Acrylate Micro-Bulk Capsule Size Distribution

    [0355] In terms of essential quality control testing, the development of accurate methods to measure islet capsule size distribution of each lot of produced micro-bulk capsules is paramount. To this end, we have developed an improved image analysis system over the one that has been in use for documenting isolated islet size. This new system quantifies islet size by actual calculations and plots the results of the scan in a few minutes as shown in FIG. 5.

    Example 5

    Higher Concentrations of PEG-Diacrylates Prevents In Vivo Capsule Fragmentation

    [0356] The implants of 5% 1 kDa PEG Diacrylate in both Nu/Nu mice (FIG. 6A) and C57Bl6 (FIGS. 6B & 6C) demonstrate intact capsules without evidence of fragmentation after two weeks of implant. Implants of 7.5% 1 kDa PEG Diacrylate capsules also demonstrate the lack of in vivo capsules at two weeks of implant (FIGS. 6D & 6E). Following implants of 7.5% 1 kDa PEG Diacrylate micro-bulk capsules (FIG. 6F) for longer periods of time demonstrated that these highest concentrations of the polymer became fragile over time eliminating their consideration as a final product. Histologic evidence of the 5% 1 kDa PEG Diacrylate in both the Nu/Nu mice and the C57Bl6 mice demonstrate excellent responses in vivo without evidence of fragmentation.

    Example 6

    Choice of PEG-Acrylates for Micro-Bulk Capsules

    [0357] Tetraethylene glycol tri-acrylate (PEG-acrylate) was utilized in the product as its primary encapsulation compound for the production of the confocal encapsulation products but had to be custom manufactured as it is not readily produced under standardized conditions. PEG tri-acrylate is also not listed in the National Center for Biotechnology Information (NCBI) PubChem listing of chemicals. We had determined that the size of the arms is critical for its use in cell encapsulation as the “middle” arm of the three arms is readily hindered by the other two arms in most sizes and applications so there is little room for lot to lot variations in a custom made product. Therefore, we chose to concentrate instead on the PEG-diacrylate and the PEG-tetra-acrylate product candidates to develop for this emulsion based, micro-bulk cell encapsulation product that would greatly reduce the component costs of a final product. FIG. 7 summarizes the results of in vitro testing for micro-bulk islet encapsulation. Starting with the PEG diacrylates, there are three that are readily available (3.5 kDa, 5 kDa, and 10 kDa) by standardized manufacturing and a fourth (1 kDa) that can be obtained by custom manufacturing. The emulsion based micro-bulk capsules produced by both the 5 kDa and the 10 kDa PEG diacrylates produced very soft micro-bulk capsules with a wide range of variable crosslinking from batch to batch and were not tested further. The emulsion produced 3.5 kDA PEG diacrylates were of consistency that could be taken forward as a possible candidate and were placed on hold to complete the testing of these different sized micro-bulk capsule candidates. The emulsion micro-bulk capsules produced by the 1 kDa diacrylate results were concentration dependent. At 1% 1 kDa polymer, the micro-bulk capsules were very soft with little strength. At 5% 1 kDa polymer, these micro-bulk capsules were strong and stable in vitro and appeared stable in the in vivo implants, becoming a potential candidate for final testing. However, as a custom made product, unacceptable lot to lot variations of this difficult to produce PEG diacrylate were encountered due to its very small size and eliminated it as a candidate going forward. At 7.5% 1 kDa PEG diacrylate, the micro-bulk capsules were very strong in vitro, but were found to be very brittle in mouse implants and thus not taken forward. Testing of the 10 kDA four armed PEG tetra-acrylate for in vitro and in vivo testing has been proven now to be the optimal combination of strength and pliability for micro-bulk islet encapsulation. With its 4 arms for reactivity, it has been shown to produce adequately crosslinked micro-bulk capsules with only 20 seconds of LED light exposure compared to the 120 seconds required to achieve adequate crosslinking with the optimized 3.5 kDa PEG diacrylate using the same light exposure. By choosing the 10 kDa PEG tetra-acrylate, we eliminated the problem of encapsulated islet reduced viability from the long, intense and required LED light exposure for the 3.5 kDa PEG diacrylate. Thus, this 10 kDA PEG tetra-acrylate is the final choice to move through the small and large animal pre-clinical studies and on to the clinical trials.

    Example 7

    Elasticity Measurements of Different PEG-Acrylates

    [0358] The actual elasticity measurements are shown for the 5% 1 kDa PEG diacrylate and the 7.5% 1 kDa PEG diacrylate showing the increase in measured elasticity parameters of the micro-bulk capsules produced with the higher concentration of PEG-diacrylate concentration (FIG. 8). Since reaching these levels of Elasticity, we have not observed any additional fragmentation in the implanted micro-bulk capsules.

    Example 8

    Improved Encapsulated Islet Viability Using 10 kDa PEG-Tetra-Acrylate Micro-Bulk Capsules

    [0359] Conversion to the 5% 10 kDa PEG-tetra Acrylate to form the PEG micro-bulk capsules reduced the encapsulation intense light exposure to 20 seconds maintaining excellent encapsulated islet viability. Use of the 5% 1 kDa PEG-Diacrylate (FIGS. 9A & 9B) required 150 seconds to achieve the same degree of crosslinking achieved by the use of the 5% 10 kDa PEG-tetra-Acrylate for only 20 seconds (FIGS. 9C & 9D). But the islet viability was remarkably reduced by the increased light exposure time. So the final polymer choice for the ongoing development of the micro-capsules is clearly the 5% 10 kDa PEG-tetra-Acrylate.

    Example 9

    Successful Implants of PEG Encapsulated Human Islets in Subcutaneous Site

    [0360] Histologic results from implanting empty micro-bulk 5% 10 kDa PEG-tetra-Acrylate capsules that had previously been tested for elasticity and strength in both the Nu/Nu mice (FIGS. 10A & 10B) and the C57Bl6 mice (FIGS. 10C & 10D) in the subcutaneous site show excellent implants without evidence of fragmentation. There is some evidence of these capsules tending to show some signs of crystallization that is observed as histology knife cutting fractures that are not fragmentation. Human islets were also encapsulated as 5% 10 kDa PEG tetra-acrylate micro-bulk capsules (FIGS. 10E & 10F). After two weeks in culture there is clear evidence of viable islets in both the Nu/Nu mice and C57Bl6. The C57Bl6 recipients show more of a non-specific cellular infiltrate surrounding the human islet containing capsules than was seen with the Nu/Nu mouse recipients that is an expected outcome. These results set the stage to start implanting curative dose of 5% 10 kDa PEG-tetra-acrylate micro-bulk capsules which is the next step.

    Example 10

    Description of Illumination for Photoencapsulation

    [0361] The encapsulation vessel is made of glass that is optically transmissive in the wavelength band for which photopolymerization is activated. The vessel is contained completely within a chamber made of highly reflective surfaces. Both specular (mirror-like) and diffusing surfaces can be used. The surfaces are arranged to nearly completely enclose the vessel and maximally contain light from sources emitting into the vessel. The surfaces are highly reflective for the emission wavelength band of the light sources which in turn are matched to the active, absorbing wavelengths of the photoactive component of the encapsulation monomer. In one form, these surfaces can be non-reflective for wavelengths that are not useful for stimulating the photoactive component in order to allow loss of light energy from the chamber that is not contributing to photopolymerization.

    [0362] The chamber has an array of apertures provided. The apertures are optimized in size to allow passage of light from the light source emitters while minimizing loss of light out of the chamber. These apertures can be physical holes or also windows through which the light source wavelengths are effectively transmitted.

    [0363] The sources are an array of light emitters. In our particular case, these are LEDs. These LEDs have a lens integrated onto the electronic emitter base to provide maximal gathering and directionality of the LED emitted light energy.

    [0364] The array is geometrically arranged around the outside perimeter of the encapsulation vessel. In our case, there are six emitters spaced at equal intervals around the perimeter. The emitters are placed in close proximity to the encapsulation vessel to maximize light transmission into the vessel. Optical elements such as lenses or wavelength filters may be introduced between the light sources and the vessel to optimize transmission into the encapsulation vessel.

    [0365] The light sources emit wavelengths selected or adjusted to match as closely as possible the absorption wavelengths of the photoactive component of the macromer. In our case, we have used eosin derivatives with an absorption activity peak near 532 nm and LEDs with an emission peak around 525 nm. This wavelength may be adjustable during the course of photopolymerization in order to optimize the effect of encapsulation.

    [0366] The positions and power out of the LEDs is adjusted to give a nearly uniform intensity of approximately 120 mW/cm.sup.2 within the photopolymerization chamber. This intensity may be programmatically adjusted to optimize the photopolymerization process during the time course of encapsulation.

    [0367] The duration of exposure is controlled to provide illumination for between 15 sec and approximately 250 sec.

    [0368] The amount of fluorescence emitted by photoactive components within the macromer may be measured during photopolymerization to monitor the progress of the process.

    Example 11

    Micro-Bulk Islet Encapsulation Methodology

    [0369] The apparatus requires a digital overhead stirrer with a speed range up to 2,000 rpm such as IKA RW 20 Digital Overhead Stirrer that includes the required stirring rods and mixing blades. Heavy glass walled round bottom flasks with a single neck of different sizes for scaling that are used to form the emulsion. The single neck is required in order to enable to spread the emulsion throughout most of the vertical distance of the flask. The Chem Glass CG-1506 fits this requirement along with glass stirring shafts CG-2078 (10 mm) and CG-2087 (19 mm) that attach with Teflon (PTFE) heavy duty stirring blades (CG-2089). One must drill a single 2 mm hole into the glass flask just below the neck for in process chemical additions. The second major apparatus is the LED light box unit that contains a mirrored box with 5 sides, each containing placement of an LED unit that is driven by the appropriate direct current power supplies. The entire apparatus fits into a standard vertical laminar hood equipped with an elevator to lower the base of the unit below the standard hood surface, permitting the apparatus height to all fit within laminar air flow.

    [0370] The required reagents include cyclonethicone as the water insoluble portion and water based liquids as the soluble portion to form the unstable emulsion by mixing all components contained within the round bottom flask. The water soluble components include the polyethylene glycol acrylate of choice as the primary polymer reactant with co-monomers, MOPS, n-Vinyl pyrrolidione, and AMPS, the photoinitiator eosin Y, Hanks balanced buffer solution, and others. To initiate the formation of the unstable emulsion, one 50 ml conical of cyclomethicone, one of HBSS, and one of water are set aside. The PEG polymer is thawed at 10° C. The round bottom flask is warmed to 37° C. Ambient oxygen is removed by sparging in argon gas from the MOPS and polymer solutions. The sterile flask is positioned beneath the mixer with the mixer shaft and propeller to the bottom of the flask and positioned with the flask and propeller with in the light box with appropriate clamps to secure the apparatus for the high spinning run. Place a few drops of the cyclomethicon within the flask to permit turning on the propeller without friction and set the propeller speed to 1850 rpm. When ready to perform the encapsulation run, ambient lights must be off. To start the run, remove the warm cyclomethicone and add to the flask through the small hole prepared at the top of the flask. With a sterile syringe and needle withdraw air to the 200 μl mark. Take 300 μl of polymer from refrigerator and mix with 20,000 IEQ of human islets within the syringe avoiding any ambient air. Place the needle of the syringe into the small hole in the flask and slowly deliver the islet polymer mixture into the flask. Turn the propeller on to the optimal speed for 30 seconds eliciting the emulsion of the cyclomethicone and the islet/polymer mixture. Activate the LED light bank on for a 20 second exposure of light. Turn on the ambient light and lower the light box below the flask. Remove the propeller from the flask. Decant the oil off the water based liquid containing the micro-bulk encapsulated islets. Slowly fill the flask with HBSS and gently agitate for two minutes and let the product settle. Replace the HBSS with islet culture medium, PIM(R), and rinse twice with additional culture medium. Divide the 20 k micro-bulk islet containing beads evenly into two portions and culture in two non-tissue culture treated T-150 culture flasks with PIM(R) culture media. Then separate small aliquots of the product for the required testing.

    Example 12

    Micro-Bulk PEG Islet Encapsulation

    [0371] PEG Conformal Coatings have demonstrated that allografts encapsulated islet implanted over two years into a subcutaneous site of diabetic baboons and having initiated a clinical trial of human islet allografts using the same capsules, the inventors developed a second generation of PEG micro-bulk encapsulated islets. FIGS. 11A, 11B & 11C shows the micro-bulk PEG encapsulated human islets along with their viability. These first results were designed to increase the number of human islets per capsule, initially in large capsules, and then reducing the size of these micro-bulk PEG capsules to increase the concentration of human islets per capsule. FIGS. 11A, 11B & 11C show these human islets encapsulated within the micro-bulk PEG capsules are predominantly viable. The in vitro results of glucose stimulation of these micro-bulk encapsulated human islets show their feasibility.

    [0372] These first large PEG capsules demonstrated that multiple human islets can be encapsulated per capsule to demonstrate the feasibility of this approach for clinical application. Although these first capsules showed the ability to encapsulate multiple human islets per capsule, they were far too large for clinical application. We ran viability tests and GSIR prior to reducing the size of the capsules.

    [0373] Ethidium bromide/fluorescene diacetate (EB/FDA) staining of the micro-bulk Phase PEG large capsules was performed after one day of culture in PIM(R)® culture medium supplemented with PIM(ABS)® and PIM(G)® (FIG. 11D). The majority of the encapsulated human islets within a single micro-bulk PEG capsule were staining positive (green) and were all completely encapsulated within the large capsule with a few of the smaller islets staining negative (red).

    [0374] The GSIR results showed the viability of the encapsulated human islets (red) (FIG. 11E). However, compared with the same batch of human islets that were not encapsulated (blue), there is a delay in the encapsulated islets insulin response to increasing glucose. This most likely is a diffusion delay due to these very large PEG capsules and confirm the plans to reduce the size of the micro-bulk PEG capsules for islets.

    [0375] For the size-reducing studies of the micro-bulk PEG capsules, islet dosing per capsule was reduced. Manipulation of the encapsulation parameters was fairly straight forward using the new device, designed and built specifically for the formation of the micro-bulk PEG capsule

    [0376] The first step in capsule size reduction was to reduce from >1000 microns to be in the range of 500-1000 microns (FIGS. 11F & 11G). Encapsulated human islets are clearly represented in the photomicrographs as encapsulated within the Micro-Bulk PEG capsules (FIGS. 11H, 11I & 11J).

    [0377] The reduction from >1000 micron-sized micro-bulk to <500 micron size is critical to providing a clinically relevant encapsulation technology for human islets (FIGS. 11K, 11L & 11M). We increased the number of human islets per capsule as we reduced the size, these early <500 micron sized demonstrated the ability to hold up to three human islets per capsule. Development is ongoing to produce capsules in this range that can routinely hold multiple numbers of islets. It is also critical to have the encapsulated human islets in <400 micron sized capsules since removable rods in implantable devices are around 500 microns in diameter.

    [0378] It is important to note that the mechanism of crosslinking micro-bulk PEG capsules relies on LED-induced radical polymerization to produce the polymer crosslinking. While this can be very toxic to islets, approaches we utilize during the encapsulation protect the islets from radical damage during the short crosslinking time as demonstrated in the GSIR results. In vivo results of micro-bulk PEG encapsulated human islets are very good.

    Example 13

    [0379] Implant micro-bulk human islets subcutaneous into diabetic mice with sufficient islets to cure the mice. Monitor their blood glucose levels and add long term insulin support for two weeks so human islets can survive in the subcutaneous site with the temporary insulin support.

    Example 14

    [0380] Implant micro-bulk human islets subcutaneous into 9 diabetic mice that get 3 implanted with micro-bulk islets in the subQ site, 3 implanted with micro-bulk islets in the IP site, and 3 implanted with free human islets into the IP site.