EP0894127A2 - Procede permettant d'accelerer la maturation des cellules - Google Patents

Procede permettant d'accelerer la maturation des cellules

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Publication number
EP0894127A2
EP0894127A2 EP97915222A EP97915222A EP0894127A2 EP 0894127 A2 EP0894127 A2 EP 0894127A2 EP 97915222 A EP97915222 A EP 97915222A EP 97915222 A EP97915222 A EP 97915222A EP 0894127 A2 EP0894127 A2 EP 0894127A2
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EP
European Patent Office
Prior art keywords
cells
cell
insulin
transplantation
islet
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP97915222A
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German (de)
English (en)
Inventor
Gregory Korbutt
Ray Rajotte
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University of Alberta
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University of Alberta
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Publication date
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Publication of EP0894127A2 publication Critical patent/EP0894127A2/fr
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1652—Polysaccharides, e.g. alginate, cellulose derivatives; Cyclodextrin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0676—Pancreatic cells
    • C12N5/0677—Three-dimensional culture, tissue culture or organ culture; Encapsulated cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/126—Immunoprotecting barriers, e.g. jackets, diffusion chambers
    • A61K2035/128—Immunoprotecting barriers, e.g. jackets, diffusion chambers capsules, e.g. microcapsules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/24—Genital tract cells, non-germinal cells from gonads
    • C12N2502/246—Cells of the male genital tract, non-germinal testis cells

Definitions

  • the present invention relates generally to maturating cells, in particular, neonatal islet cells. These cells are useful for transplantation, for example, into a subject suffering from a diabetic condition. These transplanted cells supplement or replace the diabetic subject's deficient pancreatic islet cells, allowing the patient to produce insulin in response to glucose without insulin injections.
  • Insulin-dependent diabetes mellitus (Type I) is caused by the progressive destruction of the insulin-producing pancreatic islet cells, which eventually leads to life-long dependence by the diabetic subject or patient on insulin therapy.
  • a major focus of diabetes research has been to develop a better treatment to correct or alleviate the symptoms of diabetes, thereby preventing the disabling complications of the disease.
  • One approach is to transplant isolated insulin-producing pancreatic islet cells into the diabetic subject.
  • Clinical trials using certain types of islet transplantation have corrected the abnormally-high blood glucose levels in Type I diabetics, rendering the transplant recipients partially or totally independent of exogenous insulin therapy.
  • the transplantation has restored euglycemia.
  • human islet allotransplantation has corrected basal hyperglycemia, rendering the recipients insulin-independent for varying periods of time (1-6). This recent success is attributable to the development of reproducible methods for isolating and purifying human islets (7-9) and the production of an adequate cell mass to achieve insulin-independence.
  • islet transplantation is to become a widespread treatment for Type I diabetics, however, the supply of donor organs must be increased.
  • islet tissue from abundant and accessible animal sources is being considered for xenotransplantation (10-17).
  • Pigs meet the necessary requirements for a xenogeneic source of insulin-producing tissue as they breed rapidly, have large litters, and exhibit morphological and physiological characteristics comparable to humans.
  • Porcine insulin is structurally similar to human insulin and has been safely used for treating Type I human diabetics via exogenous insulin treatment.
  • rat (22-25), porcine (1 1, 12), and human (26) fetal pancreatic cells exhibit a poor insulin secretory response to glucose (22-26), and the onset and maturation of glucose-induced insulin secretion is more evident in the postnatal period (22-25).
  • the present invention provides a method for increasing the rate of proliferation of a cell comprising encapsulating the cell within a stabilizing matrix and culturing the resulting cell in vitro. Also, the rate of maturation of an undifferentiated cell can be increased via the encapsulation of that cell.
  • the cell can be encapsulated within a microcapsule to form a microencapsulated cell.
  • the microcapsule comprises alginate or agarose, which forms a thin layer of material around the encapsulated cell.
  • more than one cell type is encapsulated within the microcapsule.
  • Figure 1 shows an electron micrograph of porcine NIC aggregates after nine days culture. Beta cells appear well granulated, structurally intact and contain secretory granules which conform to adult granule morphology. The aggregate also contains non— granulated cells (*) and numerous duct-like structures (arrows) (x 130).
  • Figure 2 shows light micrographs of native neonatal (1-3 day old) porcine pancreas (A,B x250), 9 day cultured NIC aggregates (C,D x250), and porcine NIC aggregates 14 weeks after transplantation beneath the kidney capsule of alloxan diabetic nude mice (E,F xl25; g,h x250). Sections were counterstained with Harris's hemotoxylin then im unohisochmically stained for insulin (A,C,E,G) or glucagon (B,D,F,H).
  • A; OGTT oral
  • B; IPGTT intraperitoneal
  • Figure 4 shows electron microscopy of porcine NIC aggregates 14 weeks after transplantation beneath the kidney capsule of alloxan diabetic nude mice, ⁇ cells are structurally intact, highly granulated and contain well developed endoplasmic reticulum; some cells are shown to express mitotic figures (arrows, x 6200).
  • the present invention provides an in vitro method for enriching a population of preferred cells from a tissue source including treating the tissue source to form a preparation and then culturing the preparation in vitro with a serum-free basal media supplemented with a serum substitute, an agent to stimulate DNA synthesis, and an agent to promote survival of the selected cell type, thereby resulting in an enriched population of desired cells.
  • the selected cell type can include, but is not limited to, differentiated endocrine cells, such as neuroendocrine cells and adrenal cells; pancreatic endrocrine cells, endocrine precursor cells, such as stem cells and duct cells; hepatocytes, and the like.
  • the cells enriched are differentiated endocrine cells or endocrine precursor cells, with insulin-secreting neonatal islet cells most preferred.
  • the source of tissue can be any type so long as it contains the selected or desired cells for enrichment, and/or contains precursor cells capable of becoming the desired cells during the culture process.
  • tissue can include all or part of the liver, pancreas, thyroid gland, reproductive glands, myocardial tissue, renal tissue, blood, and the like, or a preselected group of cells from within these organs.
  • Most preferred as a tissue source is the pancreas, whereby it is isolated from a pig or human.
  • the source of the pancreatic tissue can be pre-adolescent, fetal or neonatal, with neonatal as the most preferred source.
  • the enriched population of preferred cells is substantially enriched as described herein.
  • substantially enriched means a population of selected cells wherein the majority of or at least about 90% of the cells are the selected cell type.
  • enriched aggregates of neonatal islet cells contain about 5% or less fibroblasts or pancreatic exocrine cells and most preferably contain about 2-5% or less of such cells.
  • the tissue is typically prepared by partial or full digestion, as necessary, using an agent or mixture of agents, such as proteases, collagenases, other enzymes, such as disphase, and the like to form a digest.
  • Collagenase such as Type V. is a preferred agent and can be obtained commercially from various vendors, such as Sigma, Serva and Boehringer Mannheim.
  • Other collagenases include liberase, Type P, and Type XI, which also can be purchased commercially.
  • a serum-free basal media supplemented with various factors or agents.
  • the agents include, but are not limited to, a serum substitute, an agent to stimulate DNA synthesis, and an agent to promote cell survival of the desired cell type.
  • the media must be serum free; serum is omitted from the media due to its ability to promote the survival of contaminating or undesired cells, such as fibroblasts, pancreatic exocrine cells, and the like. Therefore, the serum substitute must not promote, enhance, or proliferate a contaminating cell type.
  • albumin is utilized as the serum substitute, with bovine serum albumin being the most common.
  • An agent or agents capable of stimulating DNA synthesis in the selected cell type or precursor is added to the media as a supplement.
  • IBMX isobutylmethylxanthene
  • Another DNA-synthesis stimulating agent is nicotinamide, wherein it has the ability to stimulate islet cell DNA replication and to positively affect the metabolic function of fetal or neonatal islet cells.
  • Elevated glucose concentrations i.e., greater than or equal to 1 Ommol/liter, also stimulate DNA synthesis.
  • an agent capable of promoting cell survival of the cell desired for enrichment or capable of protecting the desired cell from destruction during the culture process is also included as a supplement in the media.
  • glucose and/or IBMX can be used as the cell survival promoting agent due to its cytoprotective effect of islet cells during culture and its ability to enhance islet cell replication.
  • the preferred supplements are present at about the following concentrations in the media: albumin from about 0.1% to about 1.0% weight/volume; IMBX from about 5 to about 100 ⁇ mol/liter; nicotinamide from about 0.5 to about 20 mmol/liter, and glucose from about 6 to about 30 mmol/liter.
  • the digest is typically cultured at a temperature from about 20° to about 39°C for about 7 to about 20 days in humidified air. Most preferred, the digest is cultured for about 9 days at a temperature of about 37°C.
  • the serum-free tissue culture or basal media is a commonly used liquid tissue culture media that is free of serum.
  • the media of the invention utilizes these media in combination with selected supplements or components to create a novel media to culture, enrich and allow the proliferation of, for example, the islet cells in vitro.
  • Basal media useful in the culture method of the invention is any serum-free tissue culture media known in the art, including Media 199 (Gibco), CMRL 1066 (Gibco) media, Ham's F10 (Gibco) tissue culture media, and the like. These media also contain various ingredients, for example, amino acids, vitamins, inorganic salts, buffering agents, and energy sources. Purified molecules, such as hormones, growth factors, transport proteins, trace elements, antibiotics, and substratum-modifying ingredients optionally can be included in the media.
  • the invention comprises a serum-free basal media supplemented with albumin, IBMX, nicotinamide, and glucose.
  • the pancreatic digest from one or more neonatal pig pancreas is preferably cultured with Ham's F10 tissue culture media, supplemented with about 10 m mol/liter of glucose, about 50 ⁇ mol/liter of IBMX, about 0.5% weight/volume of bovine serum albumin (BSA), and about 10 mmol/liter of nicotinamide.
  • the media can optionally be supplemented with amino acids, such as L-glutamine, and antibiotics, such as penicillin and streptomycin.
  • the amount of L-glutamine is about 2 mmol/liter, whereas the amount of penicillin and of streptomycin is about 100 U/ml and 100 ⁇ g/ml respectively.
  • the substantially enriched population of cells is produced by the culture, it can be utilized as described below or the cells can be further isolated, purified, or matured.
  • the present invention provides methods for culturing a source of islet cells for use in transplantation.
  • the resulting neonatal porcine islets can be transplanted into diabetic subjects; thereby improving their long-term prognosis.
  • a large-scale method of isolating neonatal islet cell (NIC) aggregates from a neonatal pancreas comprises digesting the pancreas with collagenase; and culturing the digest with a serum-free tissue culture or basal media supplemented with glucose, IBMX, albumin, and nicotinamide at about 37°C in humidified air for about nine days; resulting in neonatal islet cell aggregates.
  • the source of the pancreas can be from any animal, including a human, but porcine pancreases are preferred.
  • the islet cells as described herein are useful for gene therapy methods as well.
  • An exogenous or foreign gene of choice can be tranferred to the islet cells prior to transplantation.
  • An "exogenous” or “foreign” gene refers to genetic material from outside the islet cell which is introduced into the cell.
  • the term also includes a gene that has been modified from the native or natural form of a gene found in the cell to be transfected.
  • various cytokine genes or the like can be transfected into the cells by methods of transfection as calcium phosphate co-precipitation, conventional mechanical procedures such as microinjection. biolistics, insertion of a plasmid encased in liposomes, or by use of viral vectors.
  • one method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform the neuroblast (Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).
  • a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus
  • SV40 simian virus 40
  • bovine papilloma virus bovine papilloma virus
  • RNA virus such as a retrovirus.
  • retroviruses are useful particularly in the case of dividing cells. Therefore, the method of the invention, which provides a means for producing dividing and/or differentiated neonatal islet cells, provides cells that are susceptible to retrovirus infection.
  • the retroviral vector is a derivative of a murine or avian retrovirus.
  • retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
  • MoMuLV Moloney murine leukemia virus
  • HaMuSV Harvey murine sarcoma virus
  • MuMTV murine mammary tumor virus
  • RSV Rous Sarcoma Virus
  • GaLV gibbon ape leukemia virus
  • retroviral vectors can incorporate multiple genes. All of these vectors can transfer or incorporate a gene for a selectable marker so that transduced cells can be identified and generated.
  • helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus ⁇ gag, env, andpol genes) under the control of regulatory sequences within the long terminal repeat (LTR). These plasmids are missing a nucleotide sequence which enables the packaging mechanism to recognize an RNA transcript for encapsidation.
  • Helper cell lines which have deletions of the packaging signal include, but are not limited to ⁇ 2, PA317, PA 12, CRIP and CRE, for example. These cell lines produce empty virions, since no genome is packaged.
  • a retroviral vector is introduced into such cells in which the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virion produced.
  • the vector virions produced by this method can then be used to infect a tissue cell line, such as NIH 3T3 cells, to produce large quantities of chimeric retroviral virions.
  • tissue cell line such as NIH 3T3 cells
  • NIH 3T3 or other tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol and env, by conventional calcium phosphate or lipofection transfection. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.
  • the invention also envisions production of transgenic animals, e.g., pigs, which would produce an unlimited supply of islet cells for transplantation. Cells removed from such pigs are cultured and isolated prior to transplantation (allotransplants) by the methods described herein. (See US Patent No. 4,736,866; EP 247,494)
  • the invention also describes a method of treating a subject having diabetes by administering to the subject a therapeutically-effective amount of islet cells, such as transplanting the in vitro insulin secreting neonatal islets described herein.
  • a therapeutically-effective amount of islet cells such as transplanting the in vitro insulin secreting neonatal islets described herein.
  • “Therapeutically-effective” as used herein refers to that amount of islet cells that is of sufficient quantity to alleviate a symptom of the disease or to ameliorate the diabetic disorder.
  • “Ameliorate” refers to lessening or lowering the disease's or disorder's detrimental effect in the patient receiving the therapy. In the case of diabetics, the treatment can lower or eliminate the dependency on exogenous insulin.
  • the diabetic subjects or patients to be treated include animals, such as sheep, pigs, cats, rodents, cattle, non-human primates, and most preferably dogs and humans.
  • the culture methods of the invention provide a source of neonatal islet cells from humans or other species, such as bovine, sheep, pigs, or non-human primates, with pigs and humans as preferred sources.
  • the invention also describes methods for transplantation, typically of xenografts, but also of allografts. Such methods include culturing neonatal islet cells to enrich islet endocrine cells (differentiated and/or undifferentiated), and then transplanting the resulting cells into a suitable recipient. Glucose tolerance testing, as described herein, can be used to monitor the effectiveness of the islet transplant.
  • the selected cells or cells of interest can be surgically implanted into the recipient or subject. Further, the cells can be administered in an encapsulated form or non-encapsulated form.
  • Transplantation or implantation is typically by simple injection through a hypodermic needle having a bore diameter sufficient to permit passage of a suspension of cells therethrough without damaging the cells or tissue coating.
  • the typically encapsulated or coated cells are formulated as pharmaceutical compositions together with a pharmaceutically-acceptable carrier.
  • Such compositions contain a sufficient number of coated transplant cells which can be injected into, or administered through a laparoscope to, an animal, usually into the peritoneal cavity if islet cells are utilized.
  • other transplantation sites can be selected depending upon the cells and desired biological effect; these sites include the liver, spleen, kidney capsule, omental pouch, and the like.
  • the number of transplanted islets is from about 5 to about 10 thousand per kilogram of body weight.
  • the number of other cells, tissues, and the like will be calculated depending on their function.
  • an immunosuppressive agent may be desirable to administer an immunosuppressive agent to a recipient of the islet cells, prior to, simultaneous with, and/or after transplantation.
  • An agent such as Cyclosporine A (CsA) is preferable, however other immune suppressive agents can be used, such as rapamycin, desoxyspergualine, and like.
  • CsA Cyclosporine A
  • these agents are administered to cause an immunosuppressive effect in the subject, such that the transplanted islet cells are not rejected by that subject's immune system.
  • the immunosuppressive agent is administered continuously throughout the transplant treatment typically over a period of days or weeks; for example, CsA treatment ranges from about 2 to about 20 days at a dosage range of about 5 to 40 mg per kilogram of body weight per day.
  • the agent can be administered by a variety of means, including parenteral, subcutaneous, intrapulmonary, oral, intranasal administration and the like. Preferably, dosing is given by oral administration.
  • the enriched cells also can be encapsulated prior to transplantation.
  • the cells are typically microencapsulated, they can be encased in various types of hollow fibers or in a block of encapsulating material. Encapsulation provides an effective protective barrier to isolate the transplanted cells or tissues from the host or recipient's immune system.
  • Typical pharmaceutical encapsulation compositions include, e.g., liposomes, gelatin, polyvinyl alcohol, ethylcellulose, cellulose acetatephthalate and styrene maleic anhydride. See Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton PA (1990).
  • a variety cf microencapsulation methods and compositions are known in the art.
  • a number of microencapsulation methods for use in transplant therapy have focused on the use of alginate polymers or agarose to supply the encapsulation compositions.
  • Alginates are linear polymers of mannuronic and guluronic acid residues which are arranged in blocks of several adjacent guluronic acid residues forming guluronate blocks and block of adjacent mannuronic acid residues forming mannuronate blocks, interspersed with mixed, or heterogenous blocks of alternating guluronic and mannuronic acid residues.
  • monovalent cation alginate salts are soluble, e.g., Na-alginate.
  • Divalent cations such as Ca ++ , Ba ++ or Sf + , tend to interact with guluronate, and the cooperative binding of these cations within the guluronate blocks provides the primary intramolecular crosslinking responsible for formation of stable ion-paired alginate gels.
  • Alginate encapsulation methods generally take advantage of the gelling of alginate in the presence of these divalent cation solutions. In particular, these methods involve the suspension of the material to be encapsulated, in a solution of monovalent cation alginate salt, e.g., sodium. Droplets of the solution are then generated in air and collected in a solution of divalent cations, e.g., CaCI 2 .
  • 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.
  • Generation of alginate droplets has previously been carried out by a number of methods. For example, droplets have been generated by extrusion of alginate through a tube by gravitational flow, into a solution of divalent cations.
  • electrostatic droplet generators which rely on the generation of an electrostatic differential between the alginate solution and the divalent cation solution have been described. The electrostatic differential results in the alginate solution being drawn through a tube, into the solution of divalent cations.
  • this device comprises a capillary tube within an outer sleeve. Air is driven through the outer sleeve and the polymer solution is flow-regulated through the inner tube. The air flow from the outer sleeve breaks up the fluid flowing from the capillary tube into small droplets. See U.S. Patent No. 5,286,495.
  • Microencapsulation also has been applied in the treatment of diseases by transplant therapy. While traditional medical treatments for functional deficiencies of secretory and other biological organs have focused on replacing identified normal products of the deficient organ with natural or synthetic pharmaceutical agents, transplant therapy focuses on replacement of that function with cell or organ transplants. For example, the treatment of insulin-dependent diabetes mellitus, where the pancreatic islets of Langerhans are nonfunctional, can be carried out by replacing the normal secretion of insulin by the islets in the pancreas. Insulin may be supplied either by daily administra- tion of synthetic or substitute animal insulin, or by transplantation of functional human or animal islets.
  • U.S. Patent 4,744,933 describes encapsulating solutions containing biologically active materials in a membrane of inter-reacted alginate and polyamino acid.
  • U.S. Patent 4,696,286 reports a method for coating transplants suitable for transplanta ⁇ tion into genetically dissimilar individuals.
  • the method involves coating the transplant with a surface conforming bonding bridge of a multi-functional material that binds chemically to a surface component of the transplant, which is enveloped in a semiperme- able, biologically compatible layer of a polymer that binds chemically to the bonding bridge layer.
  • Encapsulation methods applied to make these materials have comprised a procedure for forming droplets of the encapsulating medium and the biological material and a procedure for solidifying the encapsulating medium.
  • Agarose encapsulated materials have been formed by chilling an emulsion of agarose droplets containing biological materials as shown by Nilsson, et al., Nature 302:629-630 (1983) and Nilsson, et al, Eur. J. Appl. Microbiol. Biotechnol. 17:319-326 (1983).
  • Injection of droplets of polymer containing biological materials into a body of coolant such as concurrently liquid stream has been reported by Gin, et al.,J. Microencapsulation 4:329-242 (1987).
  • an undifferentiated cell that is microencapsulated or encapsulated with a stabilizing matrix, i.e., block or hollow fibers will mature at a rate faster than that of an unencapsulated cell.
  • “Maturation” as used herein, means the ability of a cell to differentiate or to achieve a specific biological function or metabolic activity.
  • an encapsulated cell will mature at an increased rate over a cell not so encapsulated.
  • the maturation rate will be at least about a 2-fold increase.
  • the rate will be at least about a 2-fold increase.
  • microencapsulation prevents cell aggregation thereby eliminating central necrosis of the aggregates, and allows the enclosed cells to grow and mature during culture.
  • Another aspect of this invention is that encapsulation of a cell also increases the cell's rate of growth or proliferation in culture.
  • enclosing the neonatal islet cells within a microcapsule or stabilizing matrix allows them to mature, grow, and differentiate into insulin-secreting cells during the in vitro culture stage as well as after implantation. Further, once the cell is encapsulated it can be cultured in vitro in either a serum- containing media or serum-free media.
  • more than one cell type can be co-encapsulated in the capsule.
  • Cell types may include distinct stages of a single lineage of cells (e.g., both precursor and differentiated endocrine cells) or distinct cell lineage (e.g. , endocrine and blood)
  • any cell type including neonatal, fetal, and/or pre-adult cells, in such a protective growth-enhancing environment results in a increased rate of maturation or proliferation.
  • Cell types include endocrine cells, such as neuroendocrine and adrenal cells; pancreatic endrocine cells, endocrine precursor cells, such as stem cells and duct cells; hepatocytes, cholinergic neurons, hematopoietic cells, hippocampal cells and the like.
  • Other cell types that can be cultured within the microcapsule or matrix include, but are not limited to, any neonatal, fetal, pre-adolescent, adult, or other cell type capable of maturation and/or proliferation.
  • the aims of the present investigation were to develop a standardized method for the large scale isolation of porcine neonatal islet cell (NIC) aggregates, to define the cellular composition of these aggregates, and to assess their growth potential and viability both in vitro and in vivo.
  • the present invention shows the feasibility of using the neonatal porcine pancreas as a source of insulin-producing tissue for xenotransplantation into human Type I diabetics. Based upon existing methods of isolating fetal porcine islet tissue, a simple, reliable procedure was developed for the preparation of porcine neonatal islet cell aggregates with a reproducible and defined cellular composition.
  • tissue from one neonatal pig pancreas yielded approximately 50,000 islet cell aggregates, consisting of primarily epithelial cells (57%) and pancreatic endocrine cells (35%).
  • the total cell mass decreased initially, but subsequently increased 1.5-fold between days 3 and 9.
  • Transplantation of grafts consisting of 3 x 10 5 cells (1000 aggregates) under the kidney capsule of alloxan-diabetic nude mice corrected hyperglycemia in 75% (10/13) of the animals, whereas, 100% (20/20) of recipients implanted with 6 x 10 5 cells (2000 aggregates) achieved euglycemia within 8 weeks post ⁇ transplantation.
  • Nephrectomy of the graft bearing kidney at 14 weeks posttransplantation resulted in hyperglycemia in all recipients, and examination of the grafts revealed the presence of numerous well-granulated insulin- and glucagon-containing cells.
  • the cellular insulin content of these grafts was 20 to 30-fold higher than at the time of transplantation.
  • aggregates cultured in alginate microcapsules and then transplanted into diabetic recipients corrected the diabetes within 1-7 days post ⁇ transplantation.
  • HBSS Hank's balanced salt solution
  • BSA bovine serum albumin
  • ICN Biomedicals, Inc. Costa Mesa, CA
  • penicillin 100 U/ml penicillin
  • streptomycin 100 U/ml streptomycin.
  • Ham's F10 tissue culture medium was purchased from Gibco, isobutylmethylxanthine (IBMX) from ICN Biomedicals, nicotinamide from BDH Biochemical (Poole, England) and theophylline from Aldrich (Milwaukee, WIS).
  • Insulin release experiments were carried out in RPMI tissue culture medium (Gibco).
  • Collagenase Type V was obtained from Sigma, and crystalline trypsin, bovine pancreatic DNase, Proteinase K, and RNase A from Boehringer Mannheim (Laval, Quebec).
  • Picogreen P-7581
  • a fluorescent nucleic acid stain for quantification of double-stranded DNA was purchased from Molecular Probes (Eugene, OR).
  • Donor pancreases were obtained from 1 -3 day old Landrace- Yorkshire neonatal pigs (1.5-2.0 kg body weight) of either sex. Piglets were anesthetized with Halothane and subjected to laparotomy and complete exsanginuation. The pancreas was then carefully dissected from surrounding tissue and placed in cooled (4°C) HBSS (supplemented as above). Warm and cold ischemia was kept to ⁇ 10 and ⁇ 5 min., respectively. Eight glands were initially used to standardize the procurement and isolation procedure. Data were then obtained from 10 consecutive independent experiments, with islet cells prepared from 3 neonatal pig pancreases for each experiment.
  • mice Male, inbred, athymic nude Balb/c mice (age 6-8 weeks) were used as recipients of the NIC aggregates (Jackson Laboratories, Bar Harbour, Ma). Mice were rendered diabetic by intravenous injection of 90 mg/kg body wt alloxan (Sigma; freshly dissolved in 1 mmol/1 hydrochloric acid) 4 to 5 days before transplantation. Normoglycemic, a- ge-matched mice served as normal controls. All recipients entering this study exhibited blood glucose levels above 20 mmol/1. Blood samples were obtained from the tail vein for glucose assay (Medisense glucose meter, Medisense Canada, Mississauga. Ontario). Animals were maintained under Virus Antibody Free conditions in climatized rooms with free access to sterilized tap water and pelleted food.
  • Each of the glands were cut into fragments of approximately 1-2 mm 3 , then transferred to sterile tubes containing HBSS (supplemented as above) with 2.5 mg/ml collagenase, and gently agitated for 16-18 min. in a shaking water bath at 37 °C.
  • the digest was filtered through a nylon screen (500 um), washed four times in HBSS then placed into bacteriological petri dishes containing HAM's F10 tissue culture medium (10 mmol/1 glucose, 50 umol/1 IBMX, 0.5%> BSA, 2 mmol/1 L-glutamine, 10 ⁇ nmol/1 nicotinamide, 100 U/ml penicillin and 100 ug/ml streptomycin).
  • Culture dishes were maintained at 37°C (5% CO2, 95% air) in humidified air for 9 days, with the medium and dishes changed the first day after isolation and the medium every second day thereafter.
  • NIC aggregates from each preparation were microencapsulated with highly purified alginate (Metabolex, Inc.) producing uniform capsules of 250 to 350 ⁇ m in diameter. Encapsu ⁇ lated and non-encapsulated aggregates were then further cultured for 8 days (HAM's F10; 37 °C) in the presence and absence of 5% (v/v) heat-activated autologous neonatal pig serum. Controls consisted of non-encapsulated aggregates cultured for 16 days in serum free HAM's F10.
  • NIC aggregates were determined on the basis of cellular hormone, DNA, and amylase content. All measurements were assessed from duplicate aliquots of the islet cell suspensions. Hormone content was measured after extraction in 2 mmol/1 acetic acid containing 0.25% BSA. Samples were sonicated in acetic acid, centrifuged (800 x g, 15 min.), then supematants were collected and stored at -20 °C until assayed for insulin content by ELISA (Boehringer Mannheim) and for glucagon content by means of radioimmunoassay (Diagnostic Products Corp.; Los Angeles, CA).
  • Amylase content was determined in supernatant fractions collected from cell suspensions which were sonicated in supplemented HBSS, stored at -20 °C, then measured by an enzymatic amylase assay (Beckman, Carlsbad, CA). For DNA content, aliquots were washed in citrate buffer (150 mmol/1 NaCl, 15 mmol/1 citrate, 3 mmol/1 EDTA, pH 7.4) and stored as cell pellets at -20 °C.
  • cell pellets Prior to being assayed, cell pellets were placed in 450ul of lysis buffer (10 mmol/1 Tris, 1 mmol/1 EDTA, 0.5% Triton X-100, 4°C, pH 7.5), sonicated, supple- mented with 25 ul of Proteinase K solution (8 mg/ml), vortexed, and incubated at 65 and 70°C for 45 and 10 min., respectively. Lysates were supplemented with 25 ul of RNase A solution (10 mg/ml), vortexed, and incubated for 1 h at 37°C.
  • NIC aggregates were dissociated into single cell suspensions by gentle agitation in calcium-free medium containing trypsin (15 ug/ml) and DNase (4 ug/ml, ref. 27-29).
  • a Burker chamber was used for cell counts (27,28), and samples ranging from 2 - 5 x 10 4 cells were assayed for DNA content.
  • the cellular composition of each fraction was determined by electron microscopy and immunohistochemistry using methods similar to that previously described by Pipeleers et al (30-32). Aggregates were fixed in 2.5% (v/v) glutaraldehyde (Millonig's buffer, pH 7.2), post-fixed in 1.5% (w/v) OsO4, washed in distilled water, then dehydrated successively in 50, 70, 80, 90 and 100%) ethanol, prior to embedding in araldite. For electron microscopy, sections were stained with lead citrate and uranyl acetate then subsequently examined in a Hitachi H 7000 transmission electron microscope.
  • Primary antibodies (Dako; Carpinteria, CA) included, guinea pig anti-porcine insulin (1 : 1000) and rabbit anti-glucagon (1:100); biotinylated secondary antibodies and the ABC-enzyme complexes were purchased from Vector Laboratories (Burlingame, CA). Primary antibodies were incubated for 30 min. (room temperature), while secondary antibodies were applied for 20 min.
  • the NICs secretory response to glucose was determined following 9 days of tissue culture by using a static incubation assay (27). The cultured fractions were recovered from the Petri dishes, washed and aliquots of 50 to 100 aggregates were incubated for 120 min. in 1.5 ml of RPMI medium supplemented with 2 mmol/1 L-glutamine, 0.5% BSA and either 2.8 mmol/1 glucose, 20 mmol/1 glucose or 20 mmol/1 glucose plus 10 mmol/1 theophylline. Tissue and medium were then separated by centrifugation and assayed for their respective insulin contents. The insulin content of the medium was expressed as a percentage of the total content (i.e. tissue plus medium).
  • Stimulation indices were calculated by dividing the amount of insulin release at 20 mmol/1 glucose (+/- theophylline) by that released at 2.8 rnmol/1 glucose.
  • a portion of the freshly isolated NIC preparation was cultured for 9 days in the supplemented HAM's F10 medium, but without the addition of 10 mmol/1 nicotinamide, in order to assess whether nicotinamide influenced the insulin secretory capacity of porcine neonatal ⁇ cells. Transplantation and metabolic follow-up.
  • NIC aggregates were transplanted under the left kidney capsule of Halothane-anesthetized nude mice.
  • the cellular composition of the graft was characterized as outlined above, and in order to standard- ized the mass of islet cells transplanted in each experiment, representative aliquots of each preparation were counted, sized, and the final quantity of aggregates was converted to the number equivalent to a diameter of 150 um (33).
  • Aliquots consisting of 1000 or 2000 aggregate equivalents were aspirated into polyethylene tubing (PE-50), pelleted by centrifugation, and gently placed under the kidney capsule with the aid of a micromanip- ulator syringe. Once the tubing was removed, the capsulotomy was cauterized with a disposable high-temperature cautery pen (Aaron Medical Industries, St. Moscow, FL.).
  • mice and normal controls were monitored for blood glucose levels once a week between 8:00 and 11 :00 a.m. When the blood glucose level was ⁇ 8.4 numol/1, the graft was deemed a success.
  • an oral (OGTT) and then an intraperitoneal (IPGTT) glucose tolerance test 48 h later was performed on NIC recipients with normalized basal glycemia and in normal controls.
  • IPGTT intraperitoneal glucose tolerance test
  • the alginate microcapsule was removed from some of the aggregates by incubating the encapsulated aggregates in calcium-free HBSS containing 0.5% BSA and 1 mmol/1 ethylene glycol-bis-( ⁇ -amino ethyl ether) N'N'-tetra acetic acid for 30 min.
  • Non-encapsulated aggregates were also treated in the same manner.
  • Prior to implantation in order to standardized the mass of islet cells transplanted in each experiment, representative aliquots of each preparation were counted, sized, and the final quantity of aggregates was converted to the number equivalent to a diameter of 150 ⁇ m.
  • NIC recipients underwent a nephrectomy of the graft-bearing kidney for morphological analysis or to determine insulin and glucagon contents of the harvested grafts.
  • the grafts in four recipients receiving 2000 aggregates were however, not removed at this time, and these animals were monitored for an additional 7 months .
  • Nephrectomized animals were subsequently monitored to confirm a return of hyperglycemia.
  • the graft-bearing kidneys were immersed in Bouin's solution overnight and embedded in paraffin. Sections, 5 um thick, were then stained for the presence of insulin and glucagon containing cells, as described above. Pieces of native neonatal pig pancreas were also processed and analyzed according to this procedure.
  • the graft and adjacent kidney tissue was fixed in glutaraldehyde then processed for electron microscopy.
  • organs were homogenized and then sonicated at 4°C in 10 ml of 2 mmol/1 acetic acid (0.25%) BSA).
  • tissue homogenates were re-sonicated, centrifuged (8000 x g, 20 min.), then supernatants were collected and the pellets further extracted by sonication in an additional 8 ml of acetic acid.
  • the second supernatant was collected after centrifugation, combined with the first supernatant, total volume was measured, and samples were assayed for insulin and glucagon content.
  • the same procedure was also used to extract hormones from pancreases obtained from NIC recipients, normal control mice, and 1-3 day old neonatal pigs.
  • cultured preparations exhibited amylase values lower than 1% of the digests (Table I).
  • the insulin content per microgram of DNA or per amylase content (U) of the porcine NICs significantly increased during culture (Table I).
  • 9 day cultured NIC preparations were more than 6-fold (p ⁇ 0.0001) enriched in endocrine tissue compared to the freshly isolated material (Table I).
  • enrichment in endocrine tissue was more than 900-fold (p ⁇ 0.0001) after the 9 day culture period (Table I).
  • NIC aggregates developed into spherical structures by the third day of culture and began to exhibit a translucent appearance similar to adult pancreatic islets.
  • Tissue culture markedly reduced the percentage of exocrine cells, so that at 9 days post-isolation, ⁇ 5% of the cells were identified as exocrine (Table II). These morphological findings are therefore consistent with the observed reduction in amylase content following culture.
  • the nine day cultured preparations consisted of 35% structurally intact endocrine cells containing well-developed endoplasmic reticulum (Fig. 1). This percentage of endocrine cells was significantly higher than at the start of culture (p ⁇ 0.001 ; Table II).
  • 9 day cultured aggregates contained numerous n- ongranulated epithelial cells (57%) , as well as many duct-like structures were found in the aggregates (Fig. 1 ).
  • the quantity of aggregates recovered in each experiment was estimated using the method previously described for determining human islet equivalents (33).
  • NIC aggregates cultured for 9 days in the presence or absence of 10 mmol/1 nicotinamide was tested by comparing the percentages of cellular insulin that was released at low glucose (2.8 mmol/1), high glucose (20 mmol/1) and high glucose plus theophylline (10 mmol/1). No statistically significant differences were noticed in the amounts of insulin secreted at the low glucose concentration (Table III). Incubation in 20 mmol/l glucose significantly (p ⁇ 0.001) increased the secretory rate of both nicotinamide treated and nontreated NICs; this effect was further potentiated when the NICs were exposed to high glucose plus theophylline (Table III).
  • the amount of insulin released and the calculated stimulation indices after incubation with either 20 mmol/l glucose or 20 mmol/l glucose plus 10 mmol/l theophylline were significantly higher when the NICs were previously cultured with nicotinamide. Nicotinamide treated aggregates exhibited stimulation indices >5-fold when comparing insulin release at high glucose versus that at low glucose. When exposed to 20 mmol/l glucose in combination with 10 mmol/l theophylline, stimulation indices were >39-fold (Table III). Analysis of the total cellular insulin and DNA content recovered per NIC aggregate, and the percentage of insulin-positive cells, revealed no significant differences between nicotinamide treated and untreated preparations.
  • the composition of 9-day cultured NIC grafts was determined by electron microscopy, immunohistochemistry, hormone content, and DNA assay (Tables II and IV). As previously described, the NIC grafts were composed of 57% n- ongranulated and 35% endocrine cells; the remaining cell types were identified as either exocrine (3%) or damaged cells (5%; Table II). Immunohistochemical staining for insulin and glucagon was positive for 24 and 8% of the total cell population, respectively (Table II).
  • Glucose tolerance tests were performed on normoglycemic mice 12 weeks post ⁇ transplantation, and when compared to normal control mice, recipients of 2- 000-aggregates exhibited significantly lower glycemic values at all time points (Fig. 3).
  • the 1000-aggregate recipients were compared to normal controls, their blood glucose levels were not statistically different throughout both tests. Comparison of the two transplant groups indicated that the 1000-aggregate recipients exhibited statistically higher values at 15 and 30 min. in the OGTT and at 15. 30, and 60 min. for the IPGTT (Fig. 3). In all groups, the lycemia at min. 120 after the bolus of glucose was not significantly different from the values at min. 0.
  • NIC grafts contained on average 1.9 ug of insulin/ 1000 aggregates or 4.0 ug of insulin 2000 aggregates (Table IV).
  • Table VI The insulin content of grafts obtained from normoglycemic recipients implanted with 1000 aggregates contained 30-fold (63.7 ug) more insulin than what was initially transplanted (Table VI).
  • grafts contained more than 20-fold (88 ug) more insulin than at the time of implantation (Table VI).
  • the amount of insulin extracted from grafts obtained from recipients of 1000 and 2000 aggregates corresponds to, respectively, more than 74 and 141% of the pancreatic insulin content in aged-matched normal control mice (Table VI).
  • Glucagon content was similar to that found in pancreases of normal controls.
  • the recipients pancreatic insulin content was less than 1 % of that contained in normal control animals, whereas their glucagon content was similar to that in normal controls (Table VI).
  • NIC grafts Macroscopically, considerable growth of the NIC grafts was evident following 14 weeks posttransplantation. Immunohistological examination of the grafts revealed a highly vascularized tissue, consisting predominantly of well-granulated insulin- and gluca- gon-containing cells (Fig. 2 e,g and 2 f,h respectively). Epithelial cells were not frequently seen in the grafts. The ⁇ cells, which composed the major volume of the graft. were arranged in ductal-/tubular-like structures and the endocrine non- ⁇ cells were scattered randomly amongst the ⁇ cells. No marked differences in morphology were observed between the two transplant groups. In electron micrographs, donor endocrine cells were shown to be structurally intact, highly granulated, and mitotic activity was detected within some of the grafts ⁇ -cells (Fig. 4).
  • EXAMPLE 7 MICROENCAPSULATION OF ISLET CELLS Eight-day cultured aggregates were microencapsulated with purified alginate. then recultured for 8 days in the presence or absence of 5% (v/v) non-heat inactivated autologous serum, as described in EXAMPLE 1. Controls included nonencapsulated islets cultured in the same manner. In serum free medium, the number of cells and the amount of insulin recovered from nonencapsulated islets decreased by 38 ⁇ 3 and 69 ⁇ 4%, respectively. Moreover, when nonencapsulated islets were cultured with serum, extensive clumping and central necrosis occurred, causing significant loss (>90%) of islet cell mass and insulin content.
  • Table IV Composition of porcine neonatal islet cell grafls prior to transportation.
  • Values are means ⁇ SR of n iccipients.
  • ⁇ Normoglycemia defined as blood glucose values ⁇ 84 mmol/l.
  • Values are means i SR for n animals. All recipients exhibited normal glycemia and were analyzed at 1 weeks posttransplantation
  • Table VII Metabolic follow-up of microencapsulated porcine neonatal islet cell aggregate recipients.
  • Table IX Survival of Lewis rat islet allografts coencapsulated with Lewis Sertoli cells transplanted IP into diabetic Wistar-Furth recipients.
  • the present data indicate that viable porcine neonatal islet cells can be successfully isolated in large numbers by culturing collagenase digested pancreas for 9 days. Since we and others (34) observed that the endocrine portion of the neonatal pig pancreas does not contain intact and mature islets, but rather exhibits a random distribution of endocrine cells, no attempts were made to process these organs using methods conventionally used for isolating adult mammalian islets. Neonatal pig pancreases were therefore, digested according to a modification of the method of Korsgren et al (10) for preparing fetal pig islet cell clusters.
  • Tissue culture was then used to enrich the preparations in endocrine cells prior to assessing their viability through an in vitro stimulation assay and by transplantation into diabetic nude mice.
  • Selection of the media supplements was based on the following considerations: 1 ) serum was omitted due to its ability to promote the survival of contaminating fibroblasts and pancreatic exocrine cells, and based on previous reports (35,36), albumin was selected as a serum substitute; 2) IBMX was used for its capacity to stimulate DNA synthesis in rat islets (37).
  • nicotinamide was included due to its ability to stimulate islet cell DNA replication (40), and its beneficial effect on the metabolic function of porcine fetal islet cells (12).
  • NIC aggregates were predominantly composed of epithelial cells, exhibited a purity of 35% endocrine cells, and contained approximately 25% insulin-positive cells. With the technique used in this study, generally 50,000 NIC aggregates were recovered from one pancreas.
  • the number of ⁇ cells recovered from freshly isolated, 3-day. and 9-day cultured preparations is calculated to be 29.2, 8.5, 13.3 million cells per pancreas, respectively .
  • the calculated number of alpha cells is 1 1.7, 3.2, and 4.4 million cells per pancreas, respectively.
  • the decrease in endocrine cell mass between the isolation and 3 days culture is likely the result of deleterious effects of the collagenase digestion and the presence of potentially cytotoxic proteases released from degenerating exocrine cells during culture.
  • the increase in both ⁇ and ⁇ cell mass i.e.
  • nicotinamide may act as a protective agent, thereby preserving the insulin secretory activity of the neonatal ⁇ cell throughout the culture period. Nicotinamide has been shown to protect against the cytotoxic action of streptozotocin on ⁇ cells (41 , 42) and to slow or arrest the development of diabetes in the non-obese diabetic mice (43). The agent may therefore enhance survival of porcine NICs after collagenase digestion and protect them from chemical disruption due to the proteolytic activity of enzymes leaking from degenerating exocrine cells during culture. Interestingly, culture in nicotinamide did not increase the frequency of insulin-positive cells in the NIC aggregates. This observation is in contrast to that observed when fetal porcine (12) and human (44) islet cell clusters were cultured with nicotinamide.
  • Diabetic nude mice were transplanted with 1000 or 2000 NIC aggregates, containing either 3 or 6 x 10 5 ⁇ cells. At posttransplantation week 14, 75% of the mice receiving 1000 aggregates exhibited blood glucose values ⁇ 8.4 mmol/l, whereas, 2000 aggregates cured diabetes in 100% of the animals within 8 weeks after implantation. In contrast, Davilli and associates demonstrated that 2000 adult porcine islet equivalents infrequently produced normoglycemia in diabetic nude mice, and that 4000 islet equivalents with an average cellular insulin mass of 38.4 ug required >5 weeks to normalize diabetic nude mice (15).
  • NICs immature NICs to differentiate and proliferate may potentially allow them to replace any ⁇ cell mass lost to ischemic damage in the immediate posttransplantation period.
  • NIC grafts grown in the absence of culture in microcapsules were unable to correct diabetes immediately after transplantation, they eventually developed the capacity to establish and maintain euglycemia, likely because the relatively few ⁇ -cells implanted initially were subsequently supplemented by the growth and/or differentiation of additional new ⁇ cells.
  • NIC grafts produced sufficient insulin to keep recipients alive, although not euglycemia.
  • a hyperglycemic environment may be essential to inducing the growth and differentiation of new ⁇ -cells in our experimental model, and perhaps also in the clinical setting.
  • the insulin content of NIC grafts corresponded to only 5 - 10% of the pancreatic insulin content found in aged matched normal controls. Following transplanta ⁇ tion, the grafts insulin mass increased by >20-fold.
  • ⁇ cell proliferation did not contribute to at least some of the increased insulin content of the grafts, as electron micrographs indicated mitotic activity within some of the engrafted ⁇ cells.
  • semi-quantitative morphometric techniques such as bromodeoxyuridine labeling and simultaneous immunostaining for islet hormones (15) should provide further insight into the growth kinetics of porcine NIC grafts. It is worthy to note that the grafts glucagon content did not increase following transplantation, suggesting that no additional growth of a-cells occurred.
  • pancreas of normal control mice was shown to contain about 40 ug insulin and grafts obtained from recipients of 2000 NIC aggregates contained 88 ug insulin.
  • the apparent excess ⁇ cell mass in these animals could explain their lower blood glucose levels when compared to age-matched normal controls.
  • this phenomenon has also been observed in nude mice transplanted with adult porcine islets in which the grafts ⁇ cell mass did not differ from that found in the pancreas of normal control mice (15).
  • an alternative explanation for this observation is that the donors ⁇ cells eventually regulated the recipient's glucose homeostasis to that found in pigs, rather than in mice. Therefore, since plasma glucose levels in pigs and humans are similar, these results indicate that transplantation of porcine NICs into humans should theoretically maintain blood sugars within the recipients physiological range.
  • the neonatal porcine pancreas can be used for the isolation of a large number of functionally viable islet cells. Furthermore, due to their ready availability and inherent capacity to proliferate and differentiate both in vitro and in vivo, they constitute an attractive source of insulin-producing tissue for studies of islet cell neogenesis or as a source of xenogeneic islet cells for clinical transplantation.
  • Metabolism 5 (Suppl. l):29-32. 33. Ricordi C. 1991. Quantitative and qualitative standards for islet isolation assessment in humans and laree mammals. Pancreas 6:242-244.
  • Nicotinamide is a potent inducer of endocrine differentiation in cultured human fetal pancreatic cells. J. Clin. Invest. 92:1459-1466.

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Abstract

La présente invention concerne un procédé permettant d'accélérer la vitesse de maturation ou la vitesse de prolifération d'une cellule au moyen de techniques de microencapsulation. On décrit également un procédé permettant de traiter un patient souffrant de diabète au moyen de cellules produites selon le procédé de culture de la présente invention.
EP97915222A 1996-04-12 1997-04-11 Procede permettant d'accelerer la maturation des cellules Withdrawn EP0894127A2 (fr)

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US6352707B1 (en) 1992-02-24 2002-03-05 Anton-Lewis Usala Transplant encapsulation in a hydrogel matrix to obscure immune recognition
US6231881B1 (en) 1992-02-24 2001-05-15 Anton-Lewis Usala Medium and matrix for long-term proliferation of cells
CA2410965C (fr) 2000-05-31 2010-09-14 Encelle, Inc. Methode permettant de stimuler la croissance des cheveux
AU1112202A (en) * 2000-10-17 2002-04-29 Diatranz Ltd Preparation and xenotransplantation or porcine islets
ATE535601T1 (de) * 2001-09-28 2011-12-15 Diabcell Pty Ltd Züchtung von fremdtransplantationsmaterial in kultur
BR112014007592A2 (pt) * 2011-09-28 2017-04-11 Islet Sciences Inc maturação ex vivo de células das ilhotas
KR101914837B1 (ko) * 2016-12-08 2018-11-02 서울대학교산학협력단 신생돼지의 췌도 분리 방법
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EP0363125A3 (fr) * 1988-10-03 1990-08-16 Hana Biologics Inc. Produit contenant des cellules pancréatiques endocrines aptes à proliférer et procédé
US5227298A (en) * 1990-08-17 1993-07-13 The Trustees Of Columbia University In The City Of New York Method for microencapuslation of cells or tissue
US5578314A (en) * 1992-05-29 1996-11-26 The Regents Of The University Of California Multiple layer alginate coatings of biological tissue for transplantation
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US5849285A (en) * 1994-04-13 1998-12-15 Research Corporation Technologies, Inc. Autoimmune disease treatment with sertoli cells and in vitro co-culture of mammal cells with sertoli cells
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