WO1997016536A1 - Procede permettant une proliferation et une differentiation ex vivo de cellules des ilots pancreatiques adultes, milieux utiles pour ce procede et leurs utilisations - Google Patents

Procede permettant une proliferation et une differentiation ex vivo de cellules des ilots pancreatiques adultes, milieux utiles pour ce procede et leurs utilisations Download PDF

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WO1997016536A1
WO1997016536A1 PCT/US1996/016396 US9616396W WO9716536A1 WO 1997016536 A1 WO1997016536 A1 WO 1997016536A1 US 9616396 W US9616396 W US 9616396W WO 9716536 A1 WO9716536 A1 WO 9716536A1
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cells
cell
islet
proliferation
contacting
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PCT/US1996/016396
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English (en)
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Patrick Soon-Shiong
Maria Varsanyi-Nagy
Kevin Ferreri
Molly Moloney
Roswitha Heintz
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Vivorx, Inc.
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Priority to AU74439/96A priority Critical patent/AU7443996A/en
Publication of WO1997016536A1 publication Critical patent/WO1997016536A1/fr

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    • 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
    • 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
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • C12N2500/12—Light metals, i.e. alkali, alkaline earth, Be, Al, Mg
    • C12N2500/14—Calcium; Ca chelators; Calcitonin
    • 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
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/12—Hepatocyte growth factor [HGF]

Definitions

  • the present invention relates to methods and compositions for the proliferation of adult human or non-human pancreatic islets of Langerhans.
  • Invention methods are useful, for example, to provide a therapeutic for treatment of type 1 diabetes mellitus, wherein such cells are optionally encapsulated within alginate microcapsules before delivery to a subject.
  • the invention employs a series of complex cell culture media containing various nutrients which are sufficient to promote long term cell growth or multiplication and to avoid senescence or loss of biological function.
  • the invention includes methods for the proliferation of islets in the form of high fidelity three dimensional tissue-like structures employing a microgravity culture vessel.
  • the invention utilizes a novel beta cell marker using molecular biology for specifically measuring the transcriptional activity of the insulin promoter in pancreatic beta cells.
  • the invention provides a combination of donor and recipient cell types to provide an organoid with reduced immunogenic potential.
  • Type 1 insulin dependent diabetes mellitus is characterized by the loss of insulin producing beta cells from the pancreatic islets of Langerhans.
  • Standard therapy has included parenteral administration of insulin (either bovine or porcine or recombinant human) by means of multiple daily injections or an indwelling catheter and pump, but this treatment can only temporarily delay the pathological complications of the disease.
  • Adult human pancreatic islets have been transplanted into patients to achieve independence from insulin injections. Such transplantations require the use of immunosuppressive treatment (e.g., with cyclosporine A) to prevent rejection of the transplanted cells.
  • immunosuppressive treatment has been limited, however, by toxic side effects and by increased potential for infection.
  • inadequate supplies of human islets and the complications of graft rejection have necessitated the search for an improved source of islet cells.
  • Fetal pancreatic islets contain many undifferentiated beta cells which can mature after transplantation and which are less subject to rejection by the recipient, but they cannot be obtained in large enough quantities to serve as a practical therapeutic approach. Transplantation of individual cells or cellular communities (including human or porcine pancreatic islets, hepatocytes, keratinocytes, chondrocytes, acinar cells, or chromaffin cells) will require a virtually inexhaustible supply of functional living cells for use in human therapy.
  • Each mature islet of Langherhans is a cellular community comprising four distinct cell types arranged in the typical topographical distribution.
  • beta cells which secrete insulin in response to elevated glucose.
  • alpha cells which secrete glucagon
  • PP cells which secrete pancreatic polypeptide
  • Delta cells which secrete somatostain
  • mammalian cells in contrast, are more difficult to grow: they are easily damaged by the shear stresses of turbulent fluid flow, they require complex nutrient media to support cell growth, and they often grow better in the presence of an appropriate substrate surface which promotes cell attachment.
  • Free floating tumor cell spheroid aggregates both with and without attachment substrates such as microcarriers, have provided material for experimental analysis of embryological development and chemotherapeutic cytotoxicity.
  • Collagen coated cellulose sponges have allowed carcinoma cells to adhere, migrate, and proliferate on a solid substrate in the presence of fibrin, although degenerative changes were detected after ten days of culture.
  • Microcarrier beads provide increased surface area for cellular attachment, allowing them to assemble into tissue-like three dimensional structures which mimic the natural relationships. Agitation in a conventional impeller driven bioreactor vessel suspends the cells in the medium, delivers fresh nutrients, and removes metabolic waste products, but it also subjects them to high levels of shear stresses which can damage cells and inhibit cellular tissue assembly.
  • a specifically defined media as well as a specifically defined environment in which the cells are cultured, allow proliferation of adult differentiated islet cells, with ongoing insulin secretion in the cells thus proliferated.
  • the invention also describes a method of co-culturing proliferated islets from a donor pancreas, together with cell types optionally obtained from the recipient. These cell types include fibroblast, endothelial neural cells, and the like, which together may reduce the immunogenicity of the hybrid co-cultured organoid, as well as enhance the functional activity of the resulting pseudo islet.
  • co-culturing freshly isolated, non- proliferated islets with islet cells which have undergone proliferation, and encapsulating these co-cultured organoids results in cells having a longer viability, stability and insulin secretory activity than do either of the components of the co-cultured organoid alone.
  • encapsulating, together with these proliferated islets other cell types (including acinar cells, hepatocytes, and the like) which may provide enhanced activity of these co-cultured hybrid organoids.
  • the somatostatin transcription factor has been used as an identifying marker to detect specific functional activity of the insulin promoter concurrent with insulin expression in the beta cells of pancreatic islets.
  • This STF-l factor is used as a probe, not only to optimize the cell culture media in terms of developing a media which provides the highest STF-l activity, but also provides a probe for identifying STF-l cells, and thus insulin secreting cells.
  • Immunohistochemical and molecular biological techniques involving antibodies to STF-l make it possible to monitor and analyze insulin expression at the level of transcription of DNA into mRNA within the cell nucleus.
  • Figure 1 presents a flow chart outline of the invention method whereby adult human islet cells are produced by initiation, expansion and termination of adult human islet cell proliferation and pseudo islet formation in vitro.
  • Figure 2 presents a growth curve for proliferating human adult islet cells.
  • Figure 3 presents a diagram of the population doubling time of human adult islet cells generated by cell counting. About 20 doublings are required to produce 1 million cells.
  • Figure 4 presents a comparative bargraph representing the glucose + theophylline stimulated insulin secretion from 12,000-fold expanded islet cells after 24 h aggregation and encapsulation.
  • Figure 5A represents the perifusion curve of normal adult islets.
  • Figure 5B is a graphic presentation of the insulin secretion rate of 12,000-fold expanded adult human pseudo islets after encapsulation.
  • Figure 6A is a graphic presentation of insulin secretion from co-encapsulated adult islets with 1000-fold expanded pseudo islets.
  • Figure 6B is a graphic presentation of insulin secretion from encapsulated proliferated islets alone. Comparison of Figures 6A and 6B demonstrate enhanced viability and function of co-cultured islets.
  • a method for stimulating the ex vivo proliferation and differentiation of neonatal and/or adult pancreatic islet beta cells comprising: (a) contacting a primary culture of neonatal and/or adult pancreatic cells under conditions suitable to induce beta cell proliferation; and
  • step (b) contacting the differentiated cells produced in step (a) under conditions suitable to induce prolonged proliferation of said cells.
  • primary culture refers to a mixed cell population of neonatal and/or adult pancreatic cells which permits interaction of epithelial and mesenchymal cells within islet-like cell clusters.
  • ex vivo refers to cells which have been taken from a body, temporarily cultured in vivo , and then returned to a body.
  • proliferation refers to an increase in cell number.
  • differentiation refers to increased numbers of islet-like cell clusters containing an increased proportion of beta epithelial cells which produce increased amounts of insulin per cell.
  • Pancreatic tissue source suitable for use in the practice of the present invention include adult human pancreases (which can be obtained from cadaver organ donors, a ⁇ well as living donors) , neonatal human pancreases, neonatal and/or adult porcine pancreases, and the like.
  • Non-human adult pancreata are obtainable from porcine or bovine sources.
  • Pancreata are typically shipped on ice in standard medium (e.g., RPMI 1640, Irvine Scientific, Irvine, CA) supplemented with 10% normal human serum and antibiotics (penicillin 100 U/ml, streptomycin 0.1 mg/ml, and amphotericin B 1 mg/ml), and are processed within 6 to 12 hours of retrieval.
  • standard medium e.g., RPMI 1640, Irvine Scientific, Irvine, CA
  • antibiotics penicillin 100 U/ml, streptomycin 0.1 mg/ml, and amphotericin B 1 mg/ml
  • Pancreatic islet isolation can be carried out as known in the art, for example, by digesting human and non ⁇ human adult pancreata using collagenase (e.g., collagenase P, Boehringer, Indianapolis, IN) under aseptic conditions (see, for example, Soon-Shiong et al., in Transpl . 54:769- 774 (1992)). Islets are purified by gradient technical separation, viability tested by acridine orange/propidium iodide uptake, and beta cell concentration estimated via a specific vital dye stain (dithazone) i.e., DTZ uptake.
  • collagenase e.g., collagenase P, Boehringer, Indianapolis, IN
  • Islets are purified by gradient technical separation, viability tested by acridine orange/propidium iodide uptake, and beta cell concentration estimated via a specific vital dye stain (dithazone) i.e., DTZ uptake.
  • Explants subjected to tissue culture in accordance with the present invention may consist of highly purified adult islets (human or porcine) , or of adult islets mixed with exocrine or duct tissue, or of disaggregated single cells of highly purified adult islets.
  • Islet cell proliferation is initiated from highly purified whole islets, instead of monodispersed islet cells.
  • the advantage of not starting with single cells can be explained with reference to both the physiology and anatomy of the islet microorgan ⁇ (see, for example, Pipeleers et al., in Proc. Natl . Acad . Sci . USA 79:7322- 7325 (1982) , Cell Biology Section) .
  • Glucose-induced insulin release depends on functional cooperation between islet cells. Exposure to glucose caused release of 30-fold more insulin from beta cells lodged within intact islets as from purified single beta cells. Structurally coupled beta cells and single beta cells isolated with alpha cells responded 4-fold more effectively to glucose than single beta cells.
  • Glucose responsiveness is dependent not only the number and integrity of insulin containing beta cells, but also their interactions with their neighboring beta and non-beta cells. Insulin secretion is seen to depend on the micro anatomy and functional organization of the islets. (Pipeleers et al., supra) .
  • the solid growth substrate may be a surface- treated polystyrene petri dish (FALCON 3003) , a tissue culture flask, or the like, coated with a variety of agents (e.g., Matrigel, laminin, fibronectin, collagen, hyaluronic acid, and the like) for selective attachment. These solid growth substrates may be re-used after trypsinization. Islet cells may be co-cultured with fibroblast cells. The differentiated state is induced by extracellular matrix, by growth factors, or by contact with neighboring cells. The differentiated state is stabilized by cell-cell adhesion, cell-cell communication, cell substrate adhesion, cell substrate interaction, and the like.
  • FALCON 3003 tissue culture flask, or the like
  • Suitable culture medium is prepared using a standard commercially available cell culture medium (e.g., RPMI DMEM, Ham's F12) as a base, at a pH of about 7.4, containing an effective cell growth promoting concentration of water, calcium ions, sodium ions, glucose, insulin, transferrin, all essential amino acids, water soluble vitamins, coenzymes, and glucose.
  • the culture medium should contain a source of an aqueous mixture of lipoprotein, cholesterol, phospholipids, and fatty acids with low endotoxin.
  • a broad spectrum antibiotic e.g., gentamicin
  • Hepatocyte growth factor is added to the culture medium to stimulate the proliferation of adult pancreatic beta cells from primary cultures of adult pancreatic cells, to increase insulin production in primary and secondary cultures of adult pancreatic islet cells, and to prepare large quantities of functional adult pancreatic beta containing islets for transplantation into diabetic patients.
  • Hepatocyte growth factor (HGF or scatter factor) is an 87 kDa two chain glycoprotein cytokine, a potent hepatocyte mitogen, and a fibroblast secretory protein which increases the motility of epithelial cells. It has been purified to homogeneity, sequenced, and genetically cloned. It was identified immunohistochemically in pancreatic glucagon secreting A cells (but not exocrine cells) of adult humans or rats, and also in developing pancreatic acinar cells of fetal rats.
  • nicotinamide is added to cell growth media at the appropriate concentrations and at the appropriate stages of the proliferation, thereby inducing specific beta cell differentiation. While nicotinamide has been discovered to be toxic to cells when present at too high a concentration, it has also been discovered that the presence of nicotinamide at the appropriate stages of the culture period serves not only to desirably inhibit fibroblast overgrowth, but also to desirably induce beta cell differentiation and increase insulin content and output.
  • hormones which mimic the pregnant state.
  • hormones include human placental lactogen, hormones of the pituitary (including corticotropin, somatotropin, oxytocin, vasopressin, and the like) , as well as hormones provided from hypothalamic extracts (e.g., growth hormone releasing hormone, thyrotropin releasing hormone, corticotrophin releasing hormone, gonadotropin releasing hormone, luteinizing releasing hormone, prolactin releasing hormone, adrenocorticotropic hormone, thyrotropin stimulating hormone, follicle stimulating hormone, luteinizing hormone, and the like) .
  • hypothalamic extracts e.g., growth hormone releasing hormone, thyrotropin releasing hormone, corticotrophin releasing hormone, gonadotropin releasing hormone, luteinizing releasing hormone, prolactin releasing hormone, adrenocorticotropic hormone, thyrotropin stimulating hormone, follicle
  • growth media contemplated for use in the practice of the present invention are established at a pH of 7.4, an osmolarity between 270 and 320 mOsmol, a temperature of 37°C, and surface tension sufficiently low to prevent formation of air bubbles.
  • the media contain an effective cell growth promoting concentration of water, sodium ions (Na + ) , potassium ions (K + , 0.23 g/L), calcium ions (Ca ++ , between 0.37 and 1.1 mM) , magnesium ions (Mg ++ ) , zinc ions (Zn ++ ) , chloride ions (Cl ) , sulfate ions (S0 4 ) , bicarbonate ions (HC0 3 ) , glucose (1500 mg/L) , all essential amino acids, cysteine, tyrosine, glutamine (between 2 and 7 mM) , water soluble vitamins, nicotinamide, coenzymes, and inorganic trace elements.
  • Glucose is preferably present at 0.8 to 1.2 mg/mL.
  • the culture medium contains a source of an aqueous mixture of lipoprotein, cholesterol, phospholipids, and fatty acids with low endotoxin.
  • a broad spectrum antibiotic e.g., gentamicin
  • the media described in the present invention are of various types for use at different stages of the proliferation and differentiation process. They include:
  • VRX-E Establishing media for endocrine cell selection (see Example 1) .
  • VRX-S Beta cell specific differentiation media containing various concentrations of nicotinamide (see Example 2) .
  • VRX-P Extended proliferation media for extended propagation. This medium contains a reduced dose of nicotinamide, and optionally no scatter factor.
  • VRX-C Media to bring about cessation of proliferation whereby growth factors are removed from the base medium.
  • V. VRX-A Media utilized for the aggregation of pseudo islets.
  • Growth factors, hormones and differentiation factors contemplated for use in the above-described media include pregnancy hormones (e.g., lactogen) , gastrointestinal hormones (e.g., gastrin or CCK) , pituitary hormones (e.g., prolactin or growth hormone), steroid hormones, thyroid hormones (e.g. T 3 or T 4 ) , insulin (as Na insulin monomer) , epidermal growth factor (EGF) , hepatocyte growth factor (HGF) , fetal bovine serum (FBS) 4%, attachment factors, spreading factors, binding proteins, and the like.
  • pregnancy hormones e.g., lactogen
  • gastrointestinal hormones e.g., gastrin or CCK
  • pituitary hormones e.g., prolactin or growth hormone
  • steroid hormones e.g. T 3 or T 4
  • thyroid hormones e.g. T 3 or T 4
  • insulin as Na insulin monomer
  • Gas phase employed for the above-described culturing is introduced as follows: The culture is perfused with a gas mixture comprising either 5% C0 2 plus 95% air plus 2.5 ng/mL selenous acid in a C0 2 incubator, or 95% 0 2 plus 5% N 2 .
  • the gas temperature is maintained at 37°C and the relative humidity is maintained at 90%.
  • the above-described method further comprises:
  • step (c) contacting the proliferated cells produced in step (b) under conditions suitable to arrest the growth thereof, and thereafter, optionally
  • step (d) culturing the cells produced in step (c) under conditions suitable to promote the formation of three dimensional tissue-like structures.
  • Conditions suitable to arrest the growth of said cells comprise culturing the differentiated/proliferated cells in growth cessation media, VRX-C.
  • Conditions suitable to promote the formation of three dimensional tissue-like structures comprise culturing cells in a microgravity device in the presence of aggregation media, VRX-A.
  • a rotational wall vessel such as, for example, the device disclosed by Schwarz et al. , in U.S. Patent No. 4,988,623, or the device disclosed by Schwarz et al., in U.S. Patent No.
  • the low gravity process simultaneously minimizes the fluid shear stress, provides three dimensional freedom for islet cell cluster and substrate spatial orientation, and increases localization of the various cell types of the islet (i.e. Delta, Beta and PP cells) in a similar spatial region for significant periods during the cell culture, thereby increasing the pseudo islet formation.
  • Cells and substrate rotate about an axis nearly perpendicular to gravity. Cells of greatly different sedimentation rates orbit in particular paths and remain spatially localized for many minutes or hours. This allows individual islet cells sufficient interaction time to form multicellular structures and to associate with each other.
  • a vessel diameter is chosen which has the appropriate volume for the intended quantity of cultured material and which will allow a sufficient seeding density of cells, tissues, and substrates.
  • the outward particle drift due to centrifugal force is exaggerated at higher vessel radii and for rapidly sedimenting particles.
  • Selected levels of shear stress may be introduced into the culture environment by differential rotation of the vessel components, as a means for controlling the rate and size of tissue formation and for maintaining optimal particle sizes and associated sedimentation rates.
  • Individual pancreatic islet cells cultured under microgravity conditions lead to the formation and maintenance of three dimensional aggregates possessing similar morphology and anatomical structure to that normally found in natural tissue. Thus, islet cells (after several fold expansion) were introduced into a microgravity vessel containing culture medium, growth factors, and an attachment matrix.
  • Simulated microgravity was created (in ordinary unit gravity) by modulating the horizontal rotation of a culture vessel completely filled with culture medium containing the matrix. These conditions cause cells to co-locate in one spatial region and encourage the maintenance of aggregates because shear stresses arising from the relative motion of the medium with respect to the walls of the vessel are minimized.
  • a method for treating a subject with type 1 diabetes mellitus comprising:
  • step (a) contacting a primary culture of neonatal and/or adult pancreatic cells under conditions suitable to induce beta cell proliferation; (b) contacting the differentiated cells produced in step (a) under conditions suitable to induce prolonged proliferation of said cells;
  • step (c) contacting the proliferated cells produced in step (b) under conditions suitable to arrest the growth thereof;
  • step (d) culturing the cells produced in step (c) under conditions suitable to promote the formation of three dimensional tissue-like structures containing increased numbers of insulin producing islet-like cell clusters containing beta epithelial cells;
  • a method to proliferate or differentiate neonatal and/or adult pancreatic islet cells in clinically useful quantities comprising:
  • a composition for transplanting functional neonatal and/or adult pancreatic tissue into patients comprising: a pharmaceutically acceptable vehicle, containing primary cultures of neonatal and/or adult pancreatic islet cells which have been contacted ex vivo with a differentiation and proliferation inducing amount of a cytokine (hepatocyte growth factor) sufficient to induce an increase in cell number, an increase in the formation of islet-like cell clusters containing beta epithelial cells, and retain the ability to produce insulin in response to stimulus.
  • a cytokine hepatocyte growth factor
  • Exemplary pharmaceutically acceptable vehicles include alginate microcapsules, as well as sterile aqueous or non-aqueous solutions, suspensions, or emulsions.
  • non-aqueous vehicles examples include propylene glycol, polyethylene glycol, vegetable oils, such as olive oil and corn oil, gelatin, and injectable organic esters such as ethyl oleate.
  • Such dosage forms may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. They may be sterilized, for example, by incorporating sterilizing agents into the compositions, by irradiating the compositions, or by heating the compositions. They can also be manufactured in the form of sterile water, or some other sterile injectable medium immediately before use.
  • compositions comprising a combination of freshly isolated islet cells and proliferated neonatal and/or adult islets.
  • Such compositions are optionally encapsulated to facilitate administration to a patient.
  • such compositions can be employed for treating a subject with type 1 diabetes mellitus. This is accomplished by parenterally administering an effective amount of the above-described optionally encapsulated combination of cells to said subject.
  • methods to identify proliferated and/or differentiated cells as beta cells comprising monitoring said cells for the expression of STF-l.
  • Such method can also be employed to optimize culture media for the differentiation of epithelial cells for selection for insulin-secreting beta cells.
  • a cell population is monitored for the presence of STF-l as a function of variations in the culture media, and the media then modified so as to maximize expression of STF-l.
  • Phenotyping of proliferating cells is accomplished using immunochemical techniques directed against insulin, glucagon, somatostatin, SF receptor, STF-l, vimentin, and Factor VIII.
  • PCR polymerase chain reaction
  • Function in vitro is assessed by sterile static glucose stimulation of adherent cells and by dynamic glucose stimulation to modulate the insulin secretion rate.
  • Cell aggregation and pseudo islet formation is performed in the microgravitation system.
  • Function in vivo is assessed by transplantation of encapsulated islet cell aggregates into mice with autoimmune diabetes.
  • Somatostatin transcription factor regulates insulin expression in beta cells of pancreatic islets. It stimulates the insulin gene by recognizing two islet specifying elements on the insulin promoter. Specification of the four islet cell types (secreting either insulin, glucagon, somatostatin, or pancreatic polypeptide) during development may be partially determined by the expression of STF-l relative to other islet cell factors, since the development of endocrine cell types in the pancreas is believed to involve the progressive restriction of pluripotent stem cells.
  • STF-l is a member of the homeobox class of transcription factors and is required for pancreatic organogenesis (Jonsson et al., Nature 371:606-609 (1994)). It is expressed during early development by the epithelial cells of the gut and most of the cells that will eventually form the pancreas. However, in the adult, STF-l expression is lost in pancreatic ductal, exocrine, and alpha cells, and is restricted to the duodenal epithelium, beta cells, and a subset of delta cells (Guz et al., Development 121:11-18 (1995)).
  • STF-l In adult beta and delta cells, STF-l is required for the hallmark phenotype of these cells, the expression of insulin in beta cells (Peers et al., Mol Endocrinol 8:1798-1806 (1994)) and somatostatin in delta cells (Leonard et al., Mol Endocrinol 7:1275-1283 (1993)). STF-l binds to the CT2 box in the human insulin promoter (Petersen et al., Proc Nat Acad Sci USA 91:10465-10469 (1994)) resulting in increased transcription of the insulin gene. In accordance with the present invention, STF-l is utilized as both a marker for mature islet cells and as a requirement for insulin expression. Further in accordance with the present invention, culture conditions are optimized based upon STF-l expression and activity in conjunction with measurements of glucose responsive insulin release.
  • a method for the preparation of cell clusters from proliferated, differentiated, growth arrested cells comprising subjecting said cells to aggregation conditions in a microgravity vessel, such as, for example, the device disclosed by Schwarz et al., in U.S. Patent No. 4,988,623, or the device disclosed by Schwarz et al., in U.S. Patent No. 5,026,650, both of which are hereby incorporated by reference herein in their entirety.
  • a microgravity vessel such as, for example, the device disclosed by Schwarz et al., in U.S. Patent No. 4,988,623, or the device disclosed by Schwarz et al., in U.S. Patent No. 5,026,650, both of which are hereby incorporated by reference herein in their entirety.
  • This example describes the process of establishing a primary culture from highly purified adult islets and a method to induce endocrine cell selection through the use of specific defined media (VRX-E: "primary establi ⁇ hing media”) in a cell culture environment which allows for adherence of endocrine cells and negative selection for fibroblasts.
  • VRX-E specific defined media
  • the media which is described below, reflects the discovery that growth factors mimicking that of the pregnant state, such as human placental lactogen, growth factors from extracts of the pituitary and hypothala us, and factors specific for beta cell of selection (hepatocyte growth factor) , are critical.
  • Establishing media is the base media set forth in Table 1, containing components 1-58 thereof. Derivatives or variants of the base media are then prepared by adjusting (either by addition or subtraction or a combination thereof) the base media, for example, by adding one or more of the optional components 59-61 and/or by deletion of such additives as (13) , (56) , (57) and/or (58) .
  • Hypothalamus extract 0.03-0.10 g/L (protein)
  • Vitamin B-12 1.4 mg/L
  • Serum (fetal, bovine 10 - 50 ml/L or human)
  • Islets were isolated from a pancreas retrieved from a 22 year old cadaver male donor. Following collagenase digestion, the islets were purified by density gradient centrifugation, and cultured overnight in standard cell culture media containing RPMI 1640 (Biowhittakes, Inc.) supplemented with 10% fetal bovine serum. Following 24 hours culture, the islets were collected, washed and then placed in cell culture vessels which allowed attachment, optionally including tissue culture flasks coated with either matrogel, la inan fibrinogen or standard petri dishes. The cells were fed every three to four days with 5-15 mM of VRX-E establishing growth media for a period of 14 days. Endocrine cells rapidly attached to the surface of the tissue culture vessel within 24-48 hours.
  • islet cell proliferation was confirmed in 57 different HLA typed, HIV negative, pancreatic donors between the ages of 15 to 65 yrs. Normal Chromosomal analysis of the islet cells proliferated at various stages of doublings demonstrated that, indeed, these cells were primary cell cultures capable of propagation without transformation.
  • the endocrine cells thus selected were frozen by cryopreservation, and stored in liquid nitrogen as a master cell bank, and are used for further expansion in subcultures.
  • nicotinamide is critical to successful proliferation and differentiation of islets.
  • high doses of nicotinamide are needed during the early phases of the culture period to induce differentiation, but if such high doses are maintained during the extended proliferation phase (described below in Example 3) , nicotinamide is observed to be toxic to the cells and is, therefore, highly undesirable at this specific stage of cell growth.
  • the timing of nicotinamide application is also seen to be very important during the growth phase of induced differentiation. This observation has not been previously reported in the art, and gives rise to the beta cell specific differentiation media defined below, utilizing different concentrations of nicotinamide.
  • VRX-s (specific beta cell proliferation medium) is formulated by the addition of the following amounts (g/L) of nicotinamide to 1000 ml of VRX-E (to produce the concentrations shown in parenthesis below) :
  • Glucose stimulated insulin secretion in adult human islet cells cultured in consecutive subcultures was examined, with and without nicotinamide. After 14 days of primary culture utilizing the VXR-E establishing media described above, the cells were reseeded in VRX-S media, in the presence and absence of nicotinamide (10 mM) , and subcultured for five weeks with weekly passaging.
  • a sterile static glucose stimulation (SGS) test was performed.
  • the sterile static glucose stimulation test was carried out as follows: First the cells were washed with glucose free Krebs Ringer bicarbonate (KRB) buffer (containing 100 mg/ml human serum albumin) , then incubated with basal 60 mM glucose (KRB 60) buffer at 37°C in a C0 2 incubator. After 60 minutes the supernatants were saved and replaced with 350 mg/ml glucose (KRB 350) buffer for the next 60 minutes. Incubation was completed with a final 60 minutes KRB 60 exposure. Supernatants were tested for insulin content by radio immunoassay (RIA) .
  • RIA radio immunoassay
  • This example illustrates the importance of scatter factor for the maintenance of beta cell phenotype in proliferating adult islet cell culture, especially during the phase of specific beta cell selection (i.e., employing VRX-S media) .
  • Islet cell cultures were initiated and established in VRX-E media as described in Example 1. Cells were then subcultured in VRX-S media as described in Example 2. Control cells were submitted to subculture in VRX-S medium, but without the addition of scatter factor. Adherent cells at 50% confluency were fixed and processed for insulin with immunocytochemistry.
  • Immunocytochemical staining involved a two stage peroxidase procedure with peroxidase-conjugated secondary antibodies.
  • First antibodies were Guinea Pig anti-insulin, rabbit anti-glucagon, and rabbit anti-somatostatin.
  • Peroxidase reaction was developed with 3,3-diaminobenzidine (DAB) substrate and 0.01% hydrogen peroxide (Vector Lab).
  • Counterstaining was performed with Gill's Hematoxyline (blue nuclei) .
  • beta cells which are stained positively for insulin are also seen to enter the mitotic cycle. This demonstrates clearly that adult differentiated insulin secreting cells have the capacity, under the appropriate conditions, to divide. This is the first clear demonstration that, with the use of scatter factor, beta cell specific selection is induced.
  • islets were isolated from seven different donors, cultured for 14 days in establishing medium then trypsinized and reseeded in VRX-S beta cell specific proliferation medium. Control cultures were set up without scatter factor. Sterile SGS was performed with Glucose (350 mg/dl) plus Theophylline
  • Glucose + theophylline stimulated insulin secretion of proliferating islet cells cultured with and without scatter factor
  • Insulin ⁇ IU/1.0 M cells/60 min
  • This example illustrates the islet cell growth pattern and extended cell proliferation utilizing a media designed specifically for this purpose (VRX-P media) .
  • VRX-P media a media designed specifically for this purpose.
  • islets were cultured for 14 days in VRX-E establishing medium then cells were trypsinized and reseeded in VRX-S beta cell specific proliferation medium for the next 14 days.
  • On day 28 cells were passaged into VRX-P medium (i.e., a medium with lower concentrations of nicotinamide, and optionally without the addition of scatter factor) and grown in ⁇ erial culture for cell expansion.
  • VRX-P medium i.e., a medium with lower concentrations of nicotinamide, and optionally without the addition of scatter factor
  • VRX-P medium is a modification of the beta cell specific proliferation media (VRX-S medium) in that scatter factor can optionally, at this stage, be removed and the nicotinamide concentration is reduced to a range of about 0.0003 mM to 5.0 mM.
  • the distinction between primary cell cultures and transformed tumor cell lines is important in that, to date, substantial efforts have been invested in the possibility of developing tumor cell lines in attempt ⁇ to develop an unlimited supply of insulin producing cells.
  • the final product of the invention process is significantly different.
  • the proliferated cells prepared according to the invention are not tumor cell line ⁇ , but instead are normal non-transformed cells which have undergone mitosis.
  • Figure 2 graphs the growth pattern of pancreatic cells up to 88 million fold expansion.
  • the Figure also show ⁇ that cell propagation ⁇ topped after growth factor withdrawal (i.e., conver ⁇ ion to VRX-C media).
  • the growth curve was generated by cell counting after trypsinization and re ⁇ eeding 0.5M cell ⁇ per di ⁇ h. Cell viability wa ⁇ checked at each pas ⁇ with trypan blue exlusion and was always >90%.
  • pancreatic cell growth was carried on for a total of 70 days. Population doubling time was calculated at each pas ⁇ age and plotted on the Y axis (See Figure 3) .
  • RT PCR Reverse transcriptase polymerase chain reaction
  • This example illustrates the successful production of islet cell clusters (pseudo islet ⁇ ) by aggregation of proliferated and growth ceased adult islet cells in a three dimensional, rotational wall vessel with low shear stress.
  • a ves ⁇ el with an integrated rotating wall (RWV) and a slow turning, lateral vessel solve ⁇ thi ⁇ problem.
  • both ve ⁇ sels are ba ⁇ ed on two de ⁇ ign principles: the vessel is rotating horizontally when it is filled completely with culture media and the cells are oxygenated by a silicon rubber membrane. As the vessel rotates, the liquid inside quickly accelerates and the fluid ma ⁇ rotates at the same angular rate. This environment eliminates destructive shear gradients. Cells obey simple kinematics and uniformly suspend in the fluid (Prewett et al., J. Tissue Cult . Meth . 15:29-36 (1993)).
  • the three dimensional aggregation of islet cells was then monitored and determined beginning at 6 hr and continued for a 42 hr period.
  • the number of aggregates was determined in 35 mm petri dishes in islet size component ⁇ (>300, 200-300, 100-200 and 50-100 ⁇ m) .
  • the size distribution of aggregates can readily be determined by photomicrograph, which reveal ⁇ aggregate ⁇ of several cells up to large aggregates approaching hundreds of cells.
  • VRX-A The media u ⁇ ed during thi ⁇ phase of aggregation is defined a ⁇ VRX-A.
  • VRX-A aggregation medium
  • I ⁇ olated i ⁇ let function i ⁇ characterized by the insulin secretion response to gluco ⁇ e challenge, where glucose stimulation can be carried out in dynamic and static circumstances.
  • Dynamic glucose stimulation (Figure 5) was performed in a perifusion (PF) system.
  • the sy ⁇ tem consisted of two reservoirs containing KRB-60 and KRB-350 + theophylline (lOmM) , a cassette pump, tubing set with a Swinnex filter holder (13mm) and 3 way stopcock and fraction collector. The solutions and the filter holder were immersed into a 37°C waterbath.
  • 50-100 islet equivalent pseudo islet ⁇ (150 mm) were placed on a 40 mm ⁇ terile filter in 6 well plates. Islets were incubated in KRB 60 solution to determine basal insulin secretion. Following incubation for 60 min at 37°C, the filters holding the islets were transferred into KRB 350 solution with 10 mM theophylline. The as ⁇ ay was finished in a second KRB 60 incubation. Supernatants were collected for insulin determination.
  • Figure 5B shows the rate of insulin release from pseudo i ⁇ let ⁇ aggregated for 48 hour ⁇ .
  • Figure 5A shows the normal insulin secretion rate of nonproliferated islets.
  • STF-l is only present in beta cells and delta cells.
  • the presence of STF-l protein was used to characterize the proliferating cells. Cultures of proliferated i ⁇ lets (10 cm plates) at passage 1 and pas ⁇ age
  • the blot was probed with a STF-l specific antibody (Peers et al., Mol Endocrinol 8:1798-1806 (1994)) (1:1000 in 1% gelatin, 0.02% Tween 20, 500 mM NaCl, 20 mM Tris HCl pH 7.5) and detected with [1251] protein A (Amer ⁇ ham IM 144, 1:1000 in 20 mM Tris HCl pH 7.5, 500 mM NaCl, 0.5% bovine serum albumin, 0.5% Triton X 100, 0.2% SDS) followed by autoradiography. There was no observed change in the relative level ⁇ of STF-l protein from passage 1 to passage 4 indicating that the proliferated cells are islet cell ⁇ .
  • Proliferated beta cells from representative islet cultures are extracted with TriReagent (Sigma #T 9424) and both RNA and protein samples are prepared.
  • the mRNA levels of STF-l are determined by quantitative Reverse Transcriptase Polymerase Chain Reaction (RT PCR) utilizing primer ⁇ that di ⁇ tinguish the correctly spliced message.
  • RT PCR quantitative Reverse Transcriptase Polymerase Chain Reaction
  • Northern blot analyses of selected samples can be used to confirm the RT PCR results.
  • High level STF-l expression is seen in cultures containing predominantly pancreatic endocrine cells but not in cultures of acinar cells or fibrobla ⁇ ts.
  • the proliferative capacity of the beta cells in the culture can be determined and optimized for each experiment.
  • the culture conditions for each donor islet population can be tailored based upon these re ⁇ ult ⁇ .
  • This example demonstrates the use of the level and activity of STF-l protein to optimize beta cell culture medium.
  • the interaction of STF-l with the CT2 element in the insulin promoter is augmented in insulinoma cells following exposure to glucose, implying that both the level and state (phosphorylation, glycosylation, etc.) of the STF-l protein is important for insulin expression.
  • Islet cell culture conditions were optimized in part based on the expression level and glucose dependent modification of the STF-l protein.
  • the levels of STF-l were determined in the protein extracts by We ⁇ tern blot analy ⁇ e ⁇ using specific antibodies (Peers et al., Mol Endocrinol 8:1798-1806 (1994)) and compared with control proteins and with other markers.
  • STF-l protein levels remain constant.
  • Glucose dependent modifications of STF-l were examined in cell extracts by electrophoretic mobility shift assays, DNA footprint experiments, and by isoelectric focusing. The results of the assays were compared with SGS and perfusion experiment data to correlate observed changes in STF-l with glucose responsive in ⁇ ulin release. Culture conditions were then changed as appropriate to achieve optimal levels.
  • islet cells are identified in representative culture ⁇ by in situ hybridization with a STF-l probe.
  • Co-localization of STF-l and other beta cell marker ⁇ in cultured cells allow the morphological identification of in ⁇ ulin producing cells, leading to the sub-culturing of homogeneous beta cell clones. Physical isolation of these clones may be possible by robotic instrumentation, including the use of laser directed splicing of the cell and retrieval of these clone ⁇ from the cell culture vessel for further subculture.
  • STF-l as a probe to confirm specific beta cell ⁇ election and to optimize media for beta cell selection has not previously been de ⁇ cribed.
  • Thi ⁇ example demonstrates regulation of the STF-l promoter. It i ⁇ pre ⁇ ently believed that high level expre ⁇ ion of STF-l is required for expres ⁇ ion of the beta cell phenotype.
  • a STF-l promoter fragment ha ⁇ been shown to direct the expression of a beta-galactosidase reporter gene predominantly to pancreatic beta cells. It is thus possible to introduce a similar reporter construct into proliferating beta cell populations and monitor the effect of media components in order to identify factors that maximize transcription for the STF-l promoter. Reporter gene expression data can be correlated with the other data and conditions optimized for glucose dependent insulin release.
  • This example demonstrate ⁇ the increased efficacy of insulin secretion following long term culture using freshly isolated islet ⁇ co-cultured with proliferated i ⁇ lets.
  • freshly isolated islet ⁇ , together with proliferated islets were encapsulated in an alginate-based membrane and placed in an incubator for an extended culture period of 30 days.
  • the performance of this combination of islet ⁇ wa ⁇ compared to the performance of proliferated islets encapsulated alone.
  • i ⁇ lets which are co-encapsulated demon ⁇ trate an ability to maintain insulin output for an extended period of time when so cultured.
  • a ⁇ can be ⁇ een in Figure 6, when proliferated islets from donor 186 were co-encapsulated with freshly isolated islets from donor 180, excellent insulin secretion was noted at day 150 and even day 180 of culture. In contrast, when islets from donor 186 were encapsulated and cultured alone, a significant drop in insulin output was noted in day 123 (even though good insulin output was noted in response to glucose stimulation at day 89) .
  • endothelial cells from the recipient may one day be retrieved and co-cultured with proliferated islet ⁇ from the donor, and in thi ⁇ way provide a tolerant hybrid organ which may or may not require immuno ⁇ uppre ⁇ ion.
  • This example demonstrates the use of encapsulated proliferated human islets for transplant into a type 1 diabetic patient.
  • the first patient to receive encapsulated proliferated human islets was a 41- year-old male with insulin dependent diabetes for 33 years requiring a mean +10 era of 0.6 +0.01 units of insulin per kg per day.
  • encap ⁇ ulated human i ⁇ let ⁇ (60% of which were derived from proliferated source) were injected into the peritoneal cavity.
  • this patient received a dose of co-encapsulated, freshly isolated and proliferated islet cell aggregate ⁇ .
  • the patient demon ⁇ trated immediate islet function from the interperitoneally transplanted cells.
  • insulin secretion wa ⁇ noted within 24 hour ⁇ .
  • the mean ⁇ erum gluco ⁇ e levels fell from a pre transplant level of 232 +9.5 mg per dl to 116 +18.6 mg per dl on the first post operative day, with all exogenous insulin being essentially discontinued.
  • the patient thereafter maintained a stable daily mean blood glucose level ranging from 116 +18.6 to 155 +12 mg per dl with less lability relative to hi ⁇ pre-tran ⁇ plant levels, while on a significantly (P ⁇ 0.001) reduced dose of insulin of approximately 0.05 to 0.2 units per kg per day for a period of three weeks. Fasting pro insulin levels and c peptide levels were significantly increased.
  • This example demonstrates the proliferation of non-human adult on neonatal porcine islet cells.
  • Islets were isolated from adult porcine pancreata, as well as from neonatal pancreata, by standard collagenase digestion techniques and purified u ⁇ ing a density grade inseparation. Following purification of these islet cell masses, they were subjected to the same culture techniques a ⁇ de ⁇ cribed in Example 1, u ⁇ ing the VRX-E e ⁇ tablishment media, followed by the VRX-S beta cell specific media. Using these non ⁇ human islet cell populations, similar evidence of proliferation and mitosis as demonstrated in examples de ⁇ cribed above u ⁇ ing human i ⁇ let ⁇ was noted.

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Abstract

L'invention concerne un procédé pour induire la prolifération et la différentiation de cellules d'îlots pancréatiques de foetus et /ou d'individus adultes humains ou non humains, pour obtenir un produit utile, par exemple, comme agent thérapeutique pour le traitement du diabète. Le procédé de l'invention fait appel à une série de milieux complexes pour la culture de cellules contenant les éléments nutritifs et les éléments de croissance nécessaires, une cytokine humaine (facteur de croissance ou facteur de dispersion des hépatocytes), un récipient de culture à microgravité pour favoriser une croissance tridimensionnelle et des dosages de biologie moléculaire pour mesurer l'activité du promoteur de l'insuline. L'invention concerne également un procédé pour réaliser un organoïde hybride comprenant une combinaison de cellules du donneur et de cellules du receveur.
PCT/US1996/016396 1995-10-30 1996-10-11 Procede permettant une proliferation et une differentiation ex vivo de cellules des ilots pancreatiques adultes, milieux utiles pour ce procede et leurs utilisations WO1997016536A1 (fr)

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997039107A2 (fr) * 1996-04-12 1997-10-23 The Governors Of The University Of Alberta Procede permettant d'accelerer la maturation des cellules
WO1999061586A1 (fr) * 1998-05-29 1999-12-02 Cythera, Inc. Promotion de la differenciation cellulaire par des cellules ayant subi initialement plusieurs passages
EP0994185A2 (fr) * 1998-10-17 2000-04-19 Tai-Wook Yoon Procédé de culture de cellules d'ilot de Langerhans
WO2000046351A2 (fr) * 1999-02-04 2000-08-10 Mcgill University Plate-forme de differenciation cellulaire
WO2001032839A2 (fr) * 1999-10-29 2001-05-10 Mcgill University Milieu servant a la preparation de cellules dedifferenciees
WO2001068108A1 (fr) * 2000-03-10 2001-09-20 The Regents Of The University Of California Induction de la differenciation de cellules beta dans des cellules humaines par stimulation du recepteur glp-1
EP1297841A1 (fr) * 2000-06-02 2003-04-02 Hiroshi Okamoto Promoteur de la proliferation des cellules de langerhans beta pancreatiques et inhibiteur d'apoptose, ainsi que criblage de composes candidats pour ces medicaments
US6562620B2 (en) * 1997-09-19 2003-05-13 Mcgill University Medium to promote islet cell survival
EP1438395A1 (fr) * 2001-09-28 2004-07-21 Diabcell Pty Limited Croissance d'une matiere pour xenotransplant dans une culture
US6911324B2 (en) 2001-10-18 2005-06-28 The Regents Of The University Of California Induction of beta cell differentiation in human cells
EP1572949A2 (fr) * 2002-06-07 2005-09-14 The Regents Of The University Of California Entretien des ilots de langerhans
WO2009090424A1 (fr) * 2008-01-14 2009-07-23 University Of Brighton Système de culture cellulaire pour les îlots pancréatiques
WO2012022351A1 (fr) * 2010-08-18 2012-02-23 Drugmode Aps Système de bioréacteur
US9850458B2 (en) 2010-12-15 2017-12-26 Drugmode Aps Bioreactor with lid for easy access to incubation cavity
WO2018216021A1 (fr) * 2017-05-24 2018-11-29 Ramot At Tel-Aviv University Ltd. Procédé pour favoriser la prolifération in vitro de cellules bêta

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Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997039107A3 (fr) * 1996-04-12 1997-12-11 Univ Alberta Procede permettant d'accelerer la maturation des cellules
WO1997039107A2 (fr) * 1996-04-12 1997-10-23 The Governors Of The University Of Alberta Procede permettant d'accelerer la maturation des cellules
US6562620B2 (en) * 1997-09-19 2003-05-13 Mcgill University Medium to promote islet cell survival
WO1999061586A1 (fr) * 1998-05-29 1999-12-02 Cythera, Inc. Promotion de la differenciation cellulaire par des cellules ayant subi initialement plusieurs passages
EP0994185A3 (fr) * 1998-10-17 2001-07-25 Tai-Wook Yoon Procédé de culture de cellules d'ilot de Langerhans
EP0994185A2 (fr) * 1998-10-17 2000-04-19 Tai-Wook Yoon Procédé de culture de cellules d'ilot de Langerhans
AU775691B2 (en) * 1998-10-17 2004-08-12 Korea Islet Transplantation Institute Inc. Method for culturing langerhans islets and islet autotransplantation islet regeneration
WO2000046351A2 (fr) * 1999-02-04 2000-08-10 Mcgill University Plate-forme de differenciation cellulaire
WO2000046351A3 (fr) * 1999-02-04 2001-07-19 Univ Mcgill Plate-forme de differenciation cellulaire
US6638765B1 (en) 1999-02-04 2003-10-28 Mcgill University Platform for the differentiation of cells
AU781750B2 (en) * 1999-02-04 2005-06-09 Mcgill University Platform for the differentiation of cells
WO2001032839A3 (fr) * 1999-10-29 2001-11-01 Univ Mcgill Milieu servant a la preparation de cellules dedifferenciees
WO2001032839A2 (fr) * 1999-10-29 2001-05-10 Mcgill University Milieu servant a la preparation de cellules dedifferenciees
WO2001068108A1 (fr) * 2000-03-10 2001-09-20 The Regents Of The University Of California Induction de la differenciation de cellules beta dans des cellules humaines par stimulation du recepteur glp-1
US6448045B1 (en) * 2000-03-10 2002-09-10 The Regents Of The University Of California Inducing insulin gene expression in pancreas cells expressing recombinant PDX-1
EP1297841A1 (fr) * 2000-06-02 2003-04-02 Hiroshi Okamoto Promoteur de la proliferation des cellules de langerhans beta pancreatiques et inhibiteur d'apoptose, ainsi que criblage de composes candidats pour ces medicaments
EP1297841A4 (fr) * 2000-06-02 2006-06-28 Hiroshi Okamoto Promoteur de la proliferation des cellules de langerhans beta pancreatiques et inhibiteur d'apoptose, ainsi que criblage de composes candidats pour ces medicaments
EP1438395A1 (fr) * 2001-09-28 2004-07-21 Diabcell Pty Limited Croissance d'une matiere pour xenotransplant dans une culture
EP1438395A4 (fr) * 2001-09-28 2006-04-12 Diabcell Pty Ltd Croissance d'une matiere pour xenotransplant dans une culture
US6911324B2 (en) 2001-10-18 2005-06-28 The Regents Of The University Of California Induction of beta cell differentiation in human cells
EP1572949A2 (fr) * 2002-06-07 2005-09-14 The Regents Of The University Of California Entretien des ilots de langerhans
EP1572949A4 (fr) * 2002-06-07 2007-01-03 Univ California Entretien des ilots de langerhans
WO2009090424A1 (fr) * 2008-01-14 2009-07-23 University Of Brighton Système de culture cellulaire pour les îlots pancréatiques
GB2468624A (en) * 2008-01-14 2010-09-15 Univ Brighton Cell culture system for pancreatic islands
WO2012022351A1 (fr) * 2010-08-18 2012-02-23 Drugmode Aps Système de bioréacteur
US9574166B2 (en) 2010-08-18 2017-02-21 Drugmode Aps Bioreactor system
US9850458B2 (en) 2010-12-15 2017-12-26 Drugmode Aps Bioreactor with lid for easy access to incubation cavity
WO2018216021A1 (fr) * 2017-05-24 2018-11-29 Ramot At Tel-Aviv University Ltd. Procédé pour favoriser la prolifération in vitro de cellules bêta

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