WO2009006600A2 - Isolement de gradient de densité de cellules d'îlot pancréatique - Google Patents

Isolement de gradient de densité de cellules d'îlot pancréatique Download PDF

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WO2009006600A2
WO2009006600A2 PCT/US2008/069215 US2008069215W WO2009006600A2 WO 2009006600 A2 WO2009006600 A2 WO 2009006600A2 US 2008069215 W US2008069215 W US 2008069215W WO 2009006600 A2 WO2009006600 A2 WO 2009006600A2
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islet cells
pancreatic islet
cells
solution
pancreatic
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PCT/US2008/069215
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WO2009006600A3 (fr
WO2009006600A9 (fr
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José OBERHOLZER
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The Board Of Trustees Of The University Of Illinois
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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
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/70—Enzymes
    • 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
    • C12N2501/998—Proteins not provided for elsewhere

Definitions

  • the application relates to the field of cell biology.
  • the application relates to solutions, cells, methods, kits, and processes useful for the isolation, culture and transplantation of cells and tissues.
  • pancreatic islet transplantation can reverse insulin-dependent diabetes
  • mesenchymal stem cells can improve heart function after an acute myocardial infarction (Scheuleri et al., 2007, Handb Exp Pharmacol 180:195-218)
  • BM-MNC bone marrow mononuclear cells
  • hematopoietic stem cells can resolve multiple sclerosis (Portaccio E et al., 2007, Mult Scler, 13(5): 676-8).
  • pancreatic islets transplantation With respect to pancreatic islets transplantation, unfortunately, the procedure is hampered by a short supply of islets and a gradual loss of islet function after transplantation. The inconsistency of islet isolation outcomes has been a major limitation to widespread clinical application of islet transplantation.
  • U.S. Patents describe known methods of isolating, culturing, and/or transplanting pancreatic islets: 6,506,599, 6,562,620, 6,783,964, and 6,815,203.
  • This invention provides methods and reagents for density gradient isolation and purification of cells liberated by enzymatic treatment.
  • the invention provides methods for isolating pancreatic islet cells from enzyme-treated pancreata.
  • the methods of the invention provides Biocoll density gradient and methods for using such gradients in centrifugation to purify pancreatic islet cells derived from enzyme -treated pancreata away from stromal components.
  • the gradient is a narrow gradient prepared using a mixture of Biocoll and University of Wisconsin (UW) solution or equivalent solution for preserving cells and tissue for transplantation.
  • UW Biocoll and University of Wisconsin
  • the invention provides methods of isolating pancreatic islet cells from a mammal, comprising (a) treating pancreatic tissue from the mammal with one or a plurality of enzymes for producing unpurified pancreatic islet cells therefrom; (b) collecting the unpurified pancreatic islet cells from the tissue treated with the enzyme; (c) incubating the unpurified pancreatic islet cells in a tissue preservation solution; and (d) subjecting the unpurified pancreatic islet cells to a continuous density gradient to isolate pancreatic islet cells, wherein the density gradient is formed in a mixture of Biocoll and the tissue preservation solution, and wherein the density gradient formed has a density range of from about 1.06 g/mL to about 1.08 g/mL.
  • the mammal is a human
  • the density gradient has a density range from about 1.068 g/mL to about 1.079 g/mL.
  • the unpurified pancreatic islet cells are collected by spinning down the cells and resuspending the cells in the tissue preservation solution.
  • the tissue preservation solution is UW solution.
  • the unpurified pancreatic islet cells are isolated by centrifugation in the density gradient at 3000 rpm for 5 min.
  • pancreatic tissues are treated with an enzyme, most preferably a protease such as collagenase, to liberate islet cells from associating stromal components.
  • an enzyme most preferably a protease such as collagenase
  • said treatment is performed using cell culture media, such as RPMI or CMRL or other commercially-available or proprietary media.
  • pancreatic tissues are treated with an enzyme in the presence of UW solution.
  • included in the pancreas treatment and isolation solutions are hemoglobin-based O 2 carriers (HBOCs) that are useful in delaying or reducing ischemia in the isolated islets, as set forth in more detail in International Application No.
  • HBOCs hemoglobin-based O 2 carriers
  • the pancreatic tissue from the mammal is incubated in the presence of polymerized hemoglobin at 4 0 C prior to enzymatic treatment and at 37 0 C during enzymatic treatment.
  • the polymerized hemoglobin is derived from human blood.
  • the polymerized hemoglobin is pyridoxylated.
  • the methods further comprise the step of spinning down the unpurified pancreatic islet cells after enzymatic treatment, resuspending the cells in a tissue preservation solution, such as UW solution, before incubating the unpurified pancreatic islet cells in the tissue preservation solution.
  • a tissue preservation solution such as UW solution
  • the unpurified pancreatic islets are incubated in UW solution for at least 30 minutes.
  • the methods further comprise the step of separating the unpurified pancreatic islet cells by gentle physical dissociation.
  • the step of gentle physical dissociation occurs after the enzymatic treatment step, before, during or after the incubation with the tissue preservation solution step, or before the step of subjecting the unpurified pancreatic islet cells to the density gradient.
  • the tissue preservation solution is UW solution.
  • the invention provides purified pancreatic islet cells produced according to the methods of the invention.
  • the invention provides methods for treating a mammal that suffers from a defect, disorder, disease or deficiency of pancreatic islet cells, comprising a step of administering to the mammal pancreatic islet cells purified according to the method of the invention.
  • the defect, disorder, disease or deficiency of pancreatic islet cells is diabetes mellitus.
  • the mammal is a human.
  • the invention provides methods for treating a mammal that suffers from a defect, disorder, disease or deficiency of pancreatic islet cells, comprising administering to the animal in need of such treatment a pharmaceutical composition or a cellular preparation of a therapeutically effective amount of the purified pancreatic islet cells of the invention.
  • the methods of the invention for treating a mammal having a defect or deficiency of pancreatic islet cells comprise administering to the animal in need of such treatment a pharmaceutical composition of a therapeutically effective amount of purified pancreatic islet cells and at least one pharmaceutically-acceptable carrier, excipient or diluent.
  • the mammal is a human.
  • the invention provides purified pancreatic islet cells for use in therapy in treating a mammal having a defect, disorder, disease or deficiency of pancreatic islet cells.
  • the invention provides methods for using the purified pancreatic islet cells to prepare a pharmaceutical composition or formulation for treating a defect or deficit of pancreatic islet cells in a mammal in need thereof.
  • the mammal is a human.
  • the invention provides pharmaceutical compositions comprising a therapeutic effective amount of purified pancreatic islet cells of the invention and at least one pharmaceutically acceptable excipient, diluent or carrier.
  • the invention provides compositions comprising pancreatic islet cells purified according to the methods of the invention and at least one pharmaceutically acceptable excipient, diluent or carrier.
  • kits for isolating pancreatic islet cells comprising (a) a first Biocoll and a tissue preservation solution gradient mixture having a density of about 1.06 g/mL; and (b) a second Biocoll and a tissue preservation solution gradient mixture having a density of about 1.08 g/mL.
  • the first Biocoll and UW solution gradient mixture has a density of about 1.068 g/mL and the second Biocoll and UW solution gradient mixture has a density of about 1.079 g/mL.
  • the tissue preservation solution is UW solution.
  • the kit further comprises at least one enzyme that is capable of digesting pancreatic tissue and liberating pancreatic islet cells from the associating stromal components.
  • the enzyme is a protease, preferably a collagenase.
  • the kit further comprises polymerized hemoglobin.
  • Figure 1 shows the viability of islets from both groups expressed in percentages
  • Figure 3 A shows changes in ratio-metric values (Fura 2/AM) as a measurement of intracellular calcium levels in two representative islets under basal (2 mM) and stimulated
  • Figure 4 A shows changes in ratio-metric values (Fura 2/AM) as a measurement of intracellular calcium levels in two representative islets under basal glucose (2 mM) conditions after the addition of Tolbutamide (100 ⁇ M).
  • Figure 4B shows the area under the curve (AUC) for intracellular calcium levels under basal glucose concentration (2 mM) in islets from both groups after the addition of
  • FIG. 5 shows insulin secretion of islets in response to glucose challenge, expressed as a stimulation index (SI), represented as mean ⁇ SEM.
  • SI stimulation index
  • FIG. 6A shows levels of Rhodamine 123 (Rhl23)-fluorescence outside the mitochondrial inner membrane in two representative islets under basal (2 mM) and glucose- stimulated conditions (14 niM). A gradual decrease in fluorescence represents the incorporation of Rh 123 into the membrane as an indirect measurement of membrane potentials.
  • Figure 6B shows the percentage change in mitochondrial potentials in islets from
  • FIG. 7 shows mitochondrial morphology. Mitochondria were stained with
  • Rh 123 dye Two representative images (confocal reconstructions) from individual islets from PoIySFH-P and control groups are shown. Images are maximum intensity projections, l ⁇ m slice thickness. Cell nuclei in the islets are identified with the letter "n”. Mitochondrial morphology and distribution around the nuclei appear superior in the PoIySFH-P group than in the control. Contrast has been balanced to reveal details of mitochondrial morphology.
  • Scale bar is 5 ⁇ m.
  • FIG 8 shows the number of days (lag time) to reach normoglycemia after islet transplantation in mice.
  • *p 0.02.
  • Figure 9 shows the results of an Intraperitoneal Glucose/ Arginine Tolerance Test
  • Figure 10 is a graph showing viability staining specific for beta and non-beta cells from isolated islet cell populations. Cells were assayed for cell membrane stability
  • Figures HA through HD are graphs showing purity and tissue volume distribution in each fraction after purification using conventional methods (SM) ( Figure
  • Figures 11C and 1 ID show the purity distribution based on the density of each fraction for both SM and UIC-UB gradient groups, respectively. Power lines in Figures 11C and 1 ID are theoretical lines fitted to the density lines (the least squares fit through the experimental data points). DETAILED DESCRIPTION OF THE INVENTION
  • This invention provides methods for isolating cells or collections of cells that have been liberated from stromal components of organs or tissues.
  • the cells or collections of cells can be liberated from stromal components using physical or more preferably chemical methods, including but not limited to enzymatic treatment.
  • the invention provides methods for isolating pancreatic islet cells from enzyme-treated pancreata. Preferred enzymes include without limitation proteases.
  • the invention provides methods for isolating pancreatic islet cells by using modified and improved Biocoll density gradient centrifugation, particularly with regard to the composition and density range of the gradient and the specifics concerning the handling of enzyme-treated tissues as set forth herein.
  • inventive methods are illustrated by improved separation of islet cells from stromal components of enzyme -treated pancreata as compared to the isolated islets from methods known in the art, wherein the isolated islets show superior yields, purity, and viability. Additional advantages include higher digested tissue volume capacity of the gradient, which reduces the processing time and ischemic insult to the cells as well as resulting in higher yield.
  • tissue preservation solution preferably UW solution
  • incubation in a tissue preservation solution prevents cell swelling of the exocrine tissue, thus preserves the distinction in density between pancreatic islet cells and other exocrine tissue.
  • pancreatic islets As used herein, the term “pancreatic islets”, “islets”, “islet cells”, and “pancreatic islet cells” are used interchangeably to refer to endocrine cells of the pancreas located and grouped in the islets of Langerhans.
  • the term "unpurified pancreatic islet cells” refers to the pancreatic islets cells liberated from pancreatic tissue by enzymatic treatment before purification by density gradient centrifugation.
  • the unpurified pancreatic islet cells are spun down and resuspended in UW solution before loading onto the density gradient.
  • the unpurified pancreatic islet cells may further comprise polymerized hemoglobin and/or UW solution.
  • pancreatic islet cells refers to isolated or enriched pancreatic islet cells that are substantially free of contaminating cells or tissues such as stromal components.
  • a tissue preservation solution refers to a solution used for preserving the viability of organs, tissues, or cells for transplantation, the use of such solution allows prolonged tolerable ischemic time.
  • transplantation preservation solutions are known in the art. See, e.g., P. Michel et al, 2002, J. Heart Lung Transplant. 21(9): 1030-39.
  • a tissue preservation solution is UW solution.
  • the invention provides a modified density gradients for isolating pancreatic islet cells, methods for preparing and using the gradients, and kits for isolating pancreatic islet cells comprising the solutions for preparing the gradient.
  • the invention provides purified pancreatic islet cells isolated using the inventive methods, and methods for using the purified pancreatic islet cells for transplantation.
  • the inventive methods provide Biocoll gradients having a different density range than methods known in the art, with improved yield, purity, and viability of the isolated islets.
  • the gradient is prepared using commercially- available Biocoll and a tissue preservation solution.
  • the inventive gradients are prepared using Biocoll and University of Wisconsin (UW) solution, the composition of which is known in the art (for example, see Belzer et al., 1988, Transplantation 45: 673-6).
  • the term "density range” as used herein refers to the density differential from the lightest to the heaviest density points in a density gradient.
  • the density gradient has a density range of from about 1.06 g/mL to about 1.08 g/mL, preferably, from about 1.068 g/mL to about 1.079 g/mL.
  • the density gradient has a density range of from about 1.062 g/mL to about 1.073 g/mL.
  • the composition of the gradient used produces a gradient density range that is "shallower” or “narrower” (i.e., does not change as greatly per unit length of the gradient) than previously-attempted gradients using Biocoll. It has been the understanding in the field that a density gradient with a wide or broad density range should be used for islet cells purification to recover cells with different density. It was unexpectedly discovered by the Applicant that a narrower or shallower gradient, in which each density fraction contains a larger volume provides better separation results.
  • the density range of the gradient has a density differential, i.e., the difference in density between the lightest and the heaviest density points in the gradient, of about 0.02 g/mL.
  • the density range of the gradient has a density differential of about 0.015 g/mL, more preferably 0.011 g/mL, most preferably not more than 0.011 g/mL.
  • the gradients of the invention have a lower viscosity. It is unexpectedly discovered that not only did the inventive density gradient with a narrower density range provide better separation of pancreatic islet cells from stromal components, the inventive gradients, because of the overall lower concentrations of Biocoll in the gradients, greatly reduced Biocoll- associated toxicity to the pancreatic islet cells.
  • the inventive gradients are advantageously prepared using UW solution, which serves to preserve isolated cells and tissues for transplantation.
  • UW solution also known as VIASP AN ®
  • VIASP AN ® was a tissue or organ preservation solution designed for use in organ transplantation.
  • UW solution is commercially available from, for example, DuPont Pharma, Bad Homburg, Germany and further described in Salehi et al. (Transplantation, 2006, 82: 983-985).
  • the composition of UW solution used in the invention is shown below: Potassium lactobionate: 100 mM, KH 2 PO 4 : 25 mM, MgSO 4 : 5 mM, Raff ⁇ nose: 30 mM, Adenosine: 5 mM, Glutathine: 3 mM, Allopurinol: 1 mM, Hydroxyethyl starch: 50 g/L.
  • the density gradient of the invention having a narrower density range provided markedly better separation of islets from exocrine tissue.
  • the density of an intact islet is approximately 1.070 g/cm
  • the density of surrounding acinar tissue is approximately 1.10 g/ cm (according to Eckhard et al, 2004, Transplantation Proc. 36: 2849-54).
  • a shallower gradient as provided herein was shown to increase discrimination between the islets and extraneous tissues co-digested from the organ during preparation. It is particularly advantageous to use a preservation solution such as UW solution in the gradient.
  • the methods disclosed herein are practiced by loading unpurified pancreatic islets (produced ⁇ or example by proteolytic digestion as set forth herein), preferably comprising UW solution, onto the gradient.
  • unpurified pancreatic islet cells are spun down, suspended in 150 ml of UW solution.
  • the unpurified pancreatic islet cells are incubated in the UW solution, preferably for at least 30 min before loading onto the density gradient of the invention.
  • the combination of Biocoll gradient separation using UW solution has been recently reported by Huang et al. (2004, Transplantation 77: 143-145). However, in this method conventional Biocoll gradient centrifugation was used rather than the modified gradient disclosed herein.
  • inventive gradients disclosed herein can be used to purify larger amounts of the unpurified islet cells. Packed tissue volume up to 50 mL or more can be purified in one gradient of the invention.
  • the methods of the instant invention are also advantageous because the higher sample volume capacity minimizes the volume of gradient required and shortens the isolation process time. In general only one gradient run will be necessary during one human islet isolation process, as compare to two to four with the conventional method. Consequently, the cold ischemia time is reduced using the inventive methods, which increases islet cell viability.
  • the invention provides methods for isolating cells from tissues wherein the enzymatic treatment solution further contains polymerized hemoglobin (such as PoIySFH-P) to reduce ischemia during isolation of cells, particularly pancreatic islet cells.
  • polymerized hemoglobin such as PoIySFH-P
  • Maintaining an appropriate O 2 level is important to prevent ischemic damage and reperfusion injury during organ preservation, pancreatic islet isolation, and cell culture.
  • artificial oxygen carriers such as perfluorocarbons (PFC)
  • PFC perfluorocarbons
  • TLM two layer method
  • Artificial oxygen carriers are synthetic solutions capable of binding, transporting and unloading O 2 .
  • Artificial oxygen carriers have been originally developed as blood substitutes, but none of the PFC based products have been approved for clinical use, and in clinical trials anaphylactic reactions were observed.
  • PFCs have the inconvenience of being hydrophobic and difficult to keep in aqueous solution.
  • Hemoglobin-based O 2 carriers such as PoIySFH-P (polymerized stroma-free hemoglobin pyridoxylated, also known as POLYHEME ® ), are water soluble. No anaphylactic reactions have been observed in phase I and II trials of POLYHEME ® .
  • U.S. Patent No. 6,498,141 which is hereby incorporated by reference in its entirety, describes the preparation of representative HBOCs. In contrast to PFC, PoIySFH-P gives an O 2 saturation curve similar to that of red blood cells.
  • PoIySFH-P is essentially tetramer-free (thereby eliminating certain biological responses to contaminating tetramer), substantially stroma- free, polymerized, and pyridoxylated hemoglobin derived from human blood.
  • hemoglobin refers to hemoglobin from mammals (preferably bovine, ovine, or human hemoglobin), synthetic hemoglobin, hemoglobin obtained by transgenic means, hemoglobin obtained from cell lines that naturally produce or have been manipulated to produce hemoglobin in vitro, hemoglobins obtained in mutant form, and chemically modified forms of hemoglobin.
  • the hemoglobin is human hemoglobin.
  • the hemoglobin of the invention comprises hemoglobin species including but not limited to Hemoglobin A, ( ⁇ 2 ⁇ 2 ,), Hemoglobin A2, ( ⁇ 2 ⁇ 2 ) and fetal hemoglobin ( ⁇ 2 ⁇ 2 ), as well as mixtures thereof.
  • polymerized hemoglobin refers to hemoglobin that has been polymerized so that it can serve as a physiologically competent oxygen carrier, wherein the placement of molecular bridges between molecules or tetrameric subunits of the hemoglobin results in the increased size and weight of the resulting polymerized molecule with respect to native or tetrameric hemoglobin.
  • Polymerized hemoglobin can absorb oxygen at the partial pressures of oxygen prevailing at the site of oxygenation of hemoglobin, for example, in the lungs of humans, and release the bound oxygen to the tissues of the same organisms in amounts that are life supporting.
  • Polymerized hemoglobins can be obtained, for example, by treatment with glutaraldehyde or raff ⁇ nose, as discussed in U.S. Patent No. 5,998,361, which is hereby incorporated by reference. Polymerized hemoglobins are also described, for example, in U.S. Patent No. 6,498,141, which is hereby incorporated by reference.
  • the polymerized hemoglobin derived from human blood is pyridoxylated. Pyridoxylation may be used to modulate the oxygen half-saturation pressure (P-50) of the polymerized hemoglobin to a desirable range.
  • P-50 oxygen half-saturation pressure
  • the hemoglobin derived from human blood is preferably pyridoxylated, as described in U.S. Patent No. 6,498,141.
  • solution containing polymerized hemoglobin to preserve and maintain viability of cells or tissues has been described in PCT application PCT/US2007/60987.
  • Preferred solutions containing polymerized hemoglobin are aqueous and are formulated to contain from about 5-15 g/dL of polymerized hemoglobin, more preferably from about 8-12 g/dL of polymerized hemoglobin, and most preferably from about 9-11 g/dL of polymerized hemoglobin.
  • Particularly preferred solutions contain about 10 g/dL of polymerized hemoglobin.
  • the solutions containing polymerized hemoglobin are formulated to have a pH of from about 7-8, more preferably from about 7.5-7.9, most preferably from about 13-1.6.
  • Polymerized hemoglobin containing solution described above preferably contains from about 0.5X to 2X of cell culture medium (where IX medium is a concentration equivalent to IX RPMI). More preferred polymerized hemoglobin/cell culture medium solutions contain about IX cell culture medium. In certain embodiments, the polymerized hemoglobin/cell culture medium further contains UW solution.
  • Cell culture medium refers to a medium suitable for the culture, maintenance, proliferation, and/or growth of cells in vitro. Examples of cell culture media that can be used are disclosed in U.S. Patent Nos. 6,670,180 and 6,730,315, which are incorporated by reference. One of skill in the art will recognize that the type of cell culture media useful in a solution of the invention can be selected based on the type of cell, tissue, and or organ for which the solution is to be used. For example, where the cells are pancreatic islets, the cell culture medium can be RPMI, as described herein.
  • Solutions of polymerized hemoglobin and an enzyme are formulated to contain the above amounts of hemoglobin and from about 0.1 - 10 mg/mL of the enzyme.
  • Preferred solutions are formulated to contain from about 0.5 - 5 mg/mL of enzyme, more preferably from about 0.75 - 1.25 mg/mL of enzyme.
  • Particularly preferred solutions contain about 1 mg/mL of enzyme.
  • Solutions of polymerized hemoglobin, cell culture medium, and enzyme are formulated to contain the amounts of these components described above and within the above-recited pH ranges.
  • the solutions and suspensions can be prepared by mixing the components thereof. Oxygenating the solutions and suspensions can be achieved, for example, by bubbling 100% O 2 gas through the solutions and suspensions for a sufficient period of time, or by otherwise contacting the solutions and suspensions with O 2 gas.
  • Buffer refers to a system, such as a solution, that acts to minimize the change in concentration of a specific chemical species in solution against addition or depletion of the species, particularly with regard to the hydrogen ion concentration (pH) of the solution. Examples of buffers are well-known to those of skill in the art.
  • protease a protease
  • Non-limiting examples of proteases suitable for use in the invention include trypsin, chymotrypsin, pepsin, furin, dispace, thermolysin, elastase, and mixtures thereof such as pancreatin and liberase (a purified enzyme blend of collagenase isoforms I and II from Clostridium histoliticum and thermolysin from Bacillus thermoproteolyticus).
  • Enzymatically produced refers to the action of an enzyme in the presence or absence of polymerized hemoglobin according to the invention, particularly proteolytic enzymes useful in digesting extracellular matrix proteins and other proteins involved in maintaining the integrity of a tissue or organ in vivo.
  • stromal component refers to the connective, nonfunctional supportive framework of a cell, tissue or organ.
  • the invention provides a composition, pharmaceutical composition or a cellular preparation comprising a therapeutic effective amount of the pancreatic islet cells prepared according to the inventive density gradient.
  • the cellular preparations and pharmaceutical compositions of the invention may contain formulation materials for modifying, maintaining, or preserving, in a manner that does not hinder the physiological function and viability of the pancreatic islet cells obtained according to the method of the invention, for example, pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition.
  • Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobial compounds, antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, betacyclodextrin, or hydroxypropyl-beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as serum albumin, gelatin, or immunoglobulins), coloring, flavoring and diluting agents, emulsifying
  • the primary vehicle or carrier in a pharmaceutical composition may be aqueous in nature.
  • a suitable vehicle or carrier for injection may be physiological saline solution.
  • Optimal pharmaceutical compositions can be determined by a skilled artisan depending upon, for example, the intended route of administration, delivery format, desired dosage and recipient tissue. See, e.g., REMINGTON'S PHARMACEUTICAL SCIENCES, supra. Such compositions may influence the physical state, stability, and effectiveness of the composition.
  • Polymerized hemoglobin derived from human blood was concentrated to about 7 g/dL and the pH of the solution was adjusted to between 7.30 and 7.60 with 0.1 M HCl. This solution was concentrated to 12 g/dL PoIySFH-P.
  • a sufficient amount of 1OX RPMI solution containing 2.5 g/L ascorbic acid and water for injection (“WFI") was added to produce a final PolySFH-P/RPMI solution containing 10 g/dL PoIySFH-P, IX RPMI, and 0.25 g/L ascorbic acid.
  • the pH of the PolySFH-P/RPMI solution was verified to be between 7.30 and 7.60.
  • PolySFH-P/RPMI solution was then sterile filtered and 250 mL were transferred aseptically into 500 mL bags. Bags were filled only half-full to allow for simplified oxygenation of the solution (within the bag) at the time of use. Filled bags were stored at 2-8 0 C.
  • 1OX RPMI solution containing 2.5 g/L ascorbic acid was prepared as follows. RPMI 1640 powder without NaHCO 3 , phenol red and L-Glutamine, obtained from Cellgro (Mediatech, Herndon, VA), was added to water for injection to obtain a concentration 10 times as concentrated as IX RPMI 1640 (see below). 7.5% NaHCO 3 , obtained from Invitrogen (Carlsbad, CA), was added to obtain a concentration of 267 mL/L. 200 mM L- Glutamine, received as a frozen solution from Invitrogen, was thawed and added to obtain a concentration of 102.5 mL/L. In addition, ascorbic acid was added to obtain a final concentration of 2.5 g/L.
  • the first method using compressed air, led to a higher conversion to Met Hb (17.6 %Met Hb) as compared to using compressed oxygen (9.3 %Met Hb).
  • the amount of MetHb formed was directly proportional to the amount of time taken to oxygenate PoIySFH-P polymerized hemoglobin or the time kept at 37 0 C, or both. Despite this conversion of a small amount of the oxygenated PoIySFH-P polymerized hemoglobin to the Met Hb form, a significant amount of Hb (79.7%) remained that was capable of carrying oxygen to the islet cells. [0069] 2B.
  • PoIySFH-P polymerized hemoglobin Once PoIySFH-P polymerized hemoglobin has been oxygenated, one of the enzymes to be tested (collagenase or liberase) was added to PoIySFH-P polymerized hemoglobin (lmg/lmL) at 4-8 0 C and kept at this temperature for 10 minutes. After the 10-minute at 4-8 0 C, Cooximetry and HPLC samples were evaluated for PoIySFH-P polymerized hemoglobin degradation and methemoglobin conversion. The PoIySFH-P polymerized hemoglobin/enzyme solution was heated to 37-39° and this temperature maintained for approximately 20 minutes. Cooximetry and HPLC samples were then tested for PoIySFH-P polymerized hemoglobin degradation and methemoglobin conversion. HPLC analysis was used to determine degradation of the PoIySFH-P polymers by analyzing for differences over time in the integrated areas of the peaks representing each polymeric species.
  • PoIySFH-P polymerized hemoglobin to evaluate the suitability of PoIySFH-P polymerized hemoglobin-supplemented RPMI for use in pancreatic islet cell harvesting.
  • a 10OmL sample of PoIySFH-P polymerized hemoglobin at 4-8 0 C was oxygenated to at least 85.0% O 2 Hb.
  • a Cooximetry sample was evaluated for the extent of oxygenation. Once the oxyhemoglobin level was at least 85.0%, an osmolality sample was evaluated as a control.
  • the RPMI 1640 was then added to PoIySFH-P polymerized hemoglobin (lg/lOOmL) at 4-8 0 C and thoroughly mixed to homogeneity prior to determining the osmolality of the mixture.
  • a buffer solution of RPMI (10.10g/1.0L) was formulated.
  • the buffer solution was used to carry out a four- volume wash (diafiltration) of the 20OmL PoIySFH-P polymerized hemoglobin. Upon completion of the diafiltration, the Cooximetry and osmolality of the sample was tested.
  • Pancreatic islets were isolated from experimental animals (rats) using in vitro culture media containing collagenase and with or without the addition of PoIySFH-P prepared as described in Example 1. All animal procedures involving animals were performed in accordance with the guidelines of the National Institutes of Health and the Animal Care Committee (ACC) at the University of Illinois Chicago. Male Lewis rats (Harlan Industries, Indianapolis, IN), weighing between 175-200 g were used as pancreas donors for islets. Animals were anesthetized by isoflurane inhalation using a vaporizer and masks (Viking Medical, Medford Lakes, NJ.).
  • Rat islet isolation was performed following a conventional technique previously described in Lacy & Kostanovsky (1967, Diabetes 16:35-39), modified by using the warm ischemia model described in Avila et al. (2003, Cell Transplant 12:877-881). Briefly, after the animal was anesthetized, a laparotomy incision was performed followed by incision into the thoracic cavity and section of the heart for euthanasia by exsanguination.
  • Collagenase type XI (Sigma Chemical Co., St. Louis, MO) was reconstituted to a final concentration of 1 mg/mL in either PolySFH-P/RPMI solution (Treatment) or RPMI 1640 medium (Control), and both Treatment and Control were oxygenated by bubbling the solutions with 100% O 2 for 15 minutes.
  • the effect of collagenase on the stability of polymerized hemoglobin was determined by HPLC analysis. PolySFH-P/RPMI solution was incubated with or without collagenase under different conditions, before and after oxygenation, at 4 0 C and 37 0 C.
  • the oxygenated enzyme solutions were injected via the bile duct and into the main pancreatic duct for distention of the pancreas.
  • the pancreas was then excised, and each pancreas placed in a 50 mL conical tube with 7.5 mL of its respective perfusion solution. This was followed by incubation in a 37 0 C water bath (digestion phase) for 18 minutes. After this step, each pancreas was gently shaken in the tubes, washed with cold RPMI 1640 medium, and transferred into a 500 mL beaker. Islets were purified from the exocrine tissue by discontinuous Ficoll density gradients (Mediatech Inc., Herndon, VA).
  • the islet/exocrine tissue mixtures were applied to the Ficoll density gradients and then centrifuged for 15 minutes at 1,500 rpm; the islet cell portion of the gradient was identified by visual inspection from the middle layer of the Ficoll gradient and handpicked. Isolated islets were then washed and cultured in RPMI 1640 medium containing 10% fetal calf serum (FBS), 10% Penicillin/Streptomycin (Invitrogen) and without glutamine, for 24 hours culture at 37 0 C.
  • FBS fetal calf serum
  • Penicillin/Streptomycin Invitrogen
  • pelleted cells were then resuspended using the wash buffer in the kit according to the manufacturer's instructions and washed twice in this buffer by centrifugation and resuspension.
  • the cells were then resuspended in 100 ⁇ L of the wash buffer and the contents of each tube transferred into individual wells of a black microtiter plate. Fluorescence intensity was measured using an excitation wavelength of 485 nm and emission wavelength of 535 nm in a fluorescent plate reader (GENios, Tecan US Inc., Durham, NC).
  • a fluorescent plate reader Genios, Tecan US Inc., Durham, NC.
  • isolated islets from PoIySFH-P perfused pancreata showed fewer apoptotic cells compared to the control as detected by lower caspase 3 activity.
  • islet cell function was assayed by incubation with varying amounts (5, 8 and 14 mM) of glucose.
  • Intracellular divalent calcium ion concentration during glucose stimulation was measured for functional evaluation in isolated islets, using standard wide- field fluorescence imaging with dual-wavelength excitation fluorescent microscopy.
  • islets were loaded with a calcium-specific dye (Fura-2/AM; Molecular Probes, Eugene OR) by incubating the islets for 25 min at 37 0 C in Krebs solution supplemented with 2 rnM glucose (KRB2), containing 5 ⁇ M Fura-2/AM.
  • KRB2 2 rnM glucose
  • the islets were placed into a temperature-controlled perfusion chamber (Medical Systems Inc, Paola, KS) mounted on an inverted epifluorescence microscope (TE-2000U, Nikon, Inc.) and perfused by a continuous flow (rate 2.5 mL/min) with 5% CO 2 -bubbled KRB2 buffer at 37 0 C (pH 7.4). Krebs buffers containing different glucose concentrations (5, 8, and 14 mM) were administered to the islets and resulting fluorescence followed for 15 min each, rinsing with KRB2 in between. Multiple islets were imaged with 10x-20x objectives for each sample.
  • Fura-2 dual-wavelength excitation was set at 340 nm and 380 nm (excitation wavelengths), and fluorescence detected at 510 nm (emission wavelength). Fluorescence was analyzed using Metafluor/Metamorph imaging acquisition and analysis software (Universal Imaging Corporation, West Chester, PA) and images collected using a high-speed, high-resolution charge-coupled device (Roper Cascade CCD, Arlington, AZ). Estimation of Ca 2+ levels was accomplished using an in vivo calibration method. The percentage change of intracellular Ca 2+ between both groups was calculated by the maximum increase after glucose stimulation, minus the basal (2 mM glucose) Ca 2+ level for each group.
  • Intracellular calcium ion concentration was also assessed in these islet cells in the presence of tolbutamide, an inhibitor of K + -ATP channels.
  • tolbutamide was added to the perfusion media at a final concentration of 100 ⁇ M in Krebs perfusion media containing 2mM glucose (basal levels) and used to perfuse islet cells in the absence of glucose stimulation. These measurements were performed on islets as described above.
  • Islet cell function was also assessed for glucose-induced insulin secretion. Static glucose incubation was used to compare glucose induced insulin secretion (stimulation index, SI) between islets isolated in the presence or absence of PoIy-SFH-P as described in Example 1. SI as used herein was defined by the ratio of stimulated versus basal insulin secretion. Briefly, for each experiment, groups of 5 handpicked islets with similar size (approximately 100 ⁇ m) were placed in five different wells of a 12 well-plate (5 replicates), then pre-incubated with 1 mL of Krebs buffer at low glucose concentration (1.6 mM glucose final concentration) for 30 min, after which the supernatant was collected and discarded.
  • SI stimulation index
  • Isolation in the presence of O 2 created the potential for reactive oxygen species (ROS) to have injured the functional integrity of islet cells, particularly at the mitochondrial and cell membranes, which could be disrupted inter alia by ROS-peroxidation.
  • Functional integrity of islet cells isolated in the presence or absence of PoIy-SFH-P as disclosed in Example 1 was further assessed by analyzing mitochondrial membrane integrity.
  • mitochondrial membrane potential were assessed using the fluorescent dye Rhodamine 123 (Rhl23), a lipophilic cation that integrates selectively into the negatively- charged mitochondrial membranes and can be used as a probe of mitochondrial transmembrane potential.
  • Rhl23 In cells pre-loaded with Rhl23, membrane potential increase (hyper-polarization) that occurs after glucose stimulation in functional islet cells causes more Rh 123 to be concentrated in the mitochondrial membrane, leading to aggregation of dye molecules and a decrease (quenching) of the fluorescence signal. Rhl23 was used as previously described.(Zhou et al., 2000, Am J Physiol Endocrinol Metab 278: E340-E351).
  • islets were incubated for 20 min at 37 0 C in Krebs solution containing 2mM glucose and supplemented with 10 ⁇ g/mL Rhl23 (Molecular Probes, Eugene, OR), then placed into a temperature-controlled perfusion chamber (Medical Systems Inc.) mounted on an inverted epifluorescence microscope (TE-2000U, Nikon Inc, Melville, NY.) The islets were perfused with a continuous flow (rate 2.5 mL/min) of 5% CO 2 -bubbled Krebs buffer at 37 0 C (pH 7.4). Islets were then stimulated with 14 mM glucose and the changes in fluorescence measured for 15 min after glucose stimulation.
  • Rhl23 fluorescence was determined using 540 nm as excitation wavelength and 590 nm as emission wavelength, and images collected with a charged coupled device camera (Roper Cascade CCD). Data were normalized to the average fluorescence intensity recorded during a five-minute period prior to glucose stimulation. The percentage change in fluorescence intensity between both islet isolation groups (i.e., isolated in the presence or absence of PoIy-SFH-P) was calculated as the maximum reduction in fluorescence intensity after 14 mM glucose stimulation, minus the basal fluorescence intensity for each group.
  • Rh 123 was used to assay islet cells for changes in mitochondrial morphology.
  • islets from PoIySFH-P and control groups were incubated for 15 minutes in Krebs buffer containing 2.5 ⁇ M Rhl23 and visualized using a Carl Zeiss LSM 510 confocal microscopy equipped with 60 X water immersion objective.
  • the 488 nm line from an argon-krypton laser used for excitation and Rh 123 emission was detected through an LP 505 filter. The intensity and the distribution of fluorescence were used to morphologically characterize mitochondrial integrity in these islet cells.
  • Islets were collected, washed with phosphate buffered saline (PBS) at pH 7.5, resuspended in 500 ⁇ L of 50 mM TRIS buffer containing 1 mM EDTA and then sonicated.
  • PBS phosphate buffered saline
  • the sonicated islet cell mixture was centrifuged to clear the supernatant of debris and the fluorescence from the cleared supernatant detected using a fluorescence plate reader (GENios, Tecan US Inc., Durham, NC) with an excitation wavelength of 380 nm and an emission wavelength of 470 nm.
  • MDA malondialdehyde
  • TBA thiobarbituric acid
  • 500 islets were sonicated in 700 ⁇ L PBS into a cell lysate. After centrifugation at 15,000 rpm to clear the lysate of debris, 500 ⁇ L of the supernatant were extracted and mixed with 875 ⁇ L of the reaction mixture, then boiled at 95-98°C for 1 hour. After this process, samples were cooled and mixed with 750 ⁇ L of n-butanol in order to extract MDA and avoid interference of other compounds. After a brief centrifugation, 100 ⁇ L of this supernatant were extracted and fluorescence assessed in duplicate on a 96 well plate with a fluorometer (GENios, Tecan US Inc. Durham, NC) at an excitation wavelength of 530/25 and an emission wavelength of 575/15. Samples were assayed in comparison with MDA standards (obtained from Sigma) prepared at different concentrations (2, 4, and 8 mM).
  • pancreatic islets isolated in the presence of oxygenated PoIySFH-P were structurally and functionally superior to islets isolated without oxygenated PoIySFH-P.
  • Islet function was assessed in vivo by transplantation under the kidney capsule of diabetic athymic nude mice (Harlan Industries), using animals treated as set forth in Example 1 with the exception that these animals were housed and surgeries performed under a laminar flow hood located in "barrier" rooms to prevent adventitious infection.
  • Diabetes was induced in these animals by a single intraperitoneal (IP) injection of streptozotocin (Sigma) at a dose of 220 mg/kg body weight. Diabetes was considered induced in treated animals after three or more non- fasting blood glucose levels of >300 mg/dL taken from the tail vein, which generally occurred after a maximum of 72 hours post injection.
  • IP intraperitoneal
  • streptozotocin Sigma
  • IPG/ ATT Intraperitoneal Glucose/ Arginine tolerance test
  • mice transplanted with islets treated with PoIySFH-P achieved normoglycemia and reversed diabetes in a significantly shorter time than the mice transplanted with islets from the Control group ( Figure 8).
  • the mice receiving PolySFH-P-treated islets showed better graft function with lower glucose levels during IPG/ATT ( Figure 9).
  • islets were dissociated and the cells staining with the following dyes: 7- aminoactinomycin D (7aad, specific for cell membrane stability), teramethylrhodamine ethyl ester (TMRE, mitochondrial membrane stability) and Newport Green (NG, wherein NG high populations were beta cells and NG low populations were non-beta cells).
  • 7- aminoactinomycin D 7aad, specific for cell membrane stability
  • TMRE teramethylrhodamine ethyl ester
  • NG Newport Green
  • Pancreata were removed from the sterile interior of its transport jar by a sterile gowned team member. The spleen and duodenum were dissected away from the pancreas and the pancreas decontaminated by immersion into three solutions: Betadine (300 cc, 5%) Kefzol/Fungizone (150 cc HBSS, lgr Kefzol, lOOmg Fungizone) solution and finally Hank's Balanced Salt Solution (HBSS, 300 cc).
  • Betadine 300 cc, 5%
  • Kefzol/Fungizone 150 cc HBSS, lgr Kefzol, lOOmg Fungizone
  • HBSS Hank's Balanced Salt Solution
  • pancreata were digested using a modified automated method as described in Ricordi et al. (1998, Diabetes 37: 413-420). Briefly, pancreatic ducts were perfused in controlled fashion with 300 mL of cold enzyme solution (0.5 g Liberase-HI, Roche Molecular, Indianapolis, IN, or Collagenase NBl, Serva, EU) in indicator- free HBSS, supplemented with HEPES and calcium chloride. A 6OmL syringe and two 16 gauge angiocatheters were used to infuse the enzyme solution. After delivery of enzyme solution through the pancreatic ducts, the pancreas was divided into 6-10 sections and transferred to a sterile container designed to facilitate tissue degradation.
  • cold enzyme solution 0.5 g Liberase-HI, Roche Molecular, Indianapolis, IN, or Collagenase NBl, Serva, EU
  • indicator- free HBSS supplemented with HEPES and calcium chloride.
  • the islets were then separated by gentle mechanical dissociation provided by manually shaking the stainless-steel chamber (Ricordi chamber), with a closed circulation flow-through system to bathe the tissue in the digestive enzymes at 37°C. At intervals during the digestion, samples were taken to assess the progress of the dissociation of islets from the acinar tissue. Free floating islets were detected at 10-20 min of digestion. When an adequate number of acinar- free islets were detected (>50% free islets), the enzymatic process was stopped by cooling and dilution with 6-10 L of M199 medium (Media Tech) supplemented with 250 mL of 20% human albumin.
  • M199 medium Media Tech
  • the crude cellular fraction was then collected in 500 mL conical tubes and concentrated by centrifugation for 1 minute at 1,000 rpm. The number and size of the islets were determined by dithizone staining, the volume of the pellet was measured and the total number of islet equivalents (IE) units calculated.
  • IE islet equivalents
  • the Cobe device was pre-loaded with 150 mL Biocoll having a density of 1.10 g/mL and centrifuged at 1500 rpm. Thereafter, 130 mL Biocoll (density 1.10 g/mL) was added to the front beaker as the "heavy" gradient and 140 mL 1.077 Biocoll (density 1.077 g/mL) was added to the rear beaker as the "light" gradient.
  • the tissue was loaded after loading the gradient mixture and islets separated at 2000 rpm. In this method, up to 20 mL tissue was used to isolate islets at each iteration of the gradient separation. After 5 minutes, the tissue was collected in 12 fractions (250 mL tubes pre-f ⁇ lled with 200 wash media).
  • the Cobe device was loaded with 110 mL Biocoll having a density of 1.10 g/mL and centrifuged at 1500 rpm. Then, 130 mL "heavy gradient” (49% Biocoll, 51% UW solution, density 1.079 g/mL) was added to the front beaker and 140 mL "light” gradient (30% Biocoll, 70% UW, density 1.068 g/mL) to the rear beaker (UW solution has density of 1.046 g/mL).
  • pancreatic islet cells After first incubating in UW solution, the unpurif ⁇ ed pancreatic islet cells in UW solution were loaded onto the gradient once the gradient is almost completed. The pancreatic islet cells were centrifuged and separated at 3000 rpm. Using this method, up to 50 mL of tissue (packed volume) can be loaded to each run of the gradient separation. After 5 minutes, tissue was collected in 12 fractions (30 mL into tubes pre-f ⁇ lled with 200 wash media). Wash media used was HBSS-based solution (Hank's Buffered Salt Solution). In both methods, fractions with purity > 60% were collected as "top” and ⁇ 60% as "bottom” layers (up to 7 mL tissue).
  • Isolation outcome was assessed through quantification of islet mass by dithizone staining, expressed in equivalent islet numbers (EIN) according to the method of Ricordi et al. (1990, Acta Diabetol Lat.. 27: 185-95). Islet recovery rate was calculated as ratio of the islets number after purification to the islets number after digestion x 100. Islet viability was determined by fluorescent staining with Syto-Green/ Ethidium Bromide as described in Yang et al. (1998, Cell Transplant 7: 443-51); percentage of dead and live cells was estimated according to the methods of Avila et al. (2003, Cell Transplant 12: 877-81).
  • the islets purified with SM were collected in only two fractions (average purity of 68.9% and 36.3%); in contrast, using the UIC-UB method, highly purified islets were consistently collected in 6 separate fractions (with purity of 84.8%, 82.5%, 72.0%, 59.3%, 46.8% and 36.2%).
  • Overall purity of the "top" layer was 77.5 ⁇ 13.4% in SM and 82.9 ⁇ 11.4% in UIC-UB method.
  • the stimulation index showed equivalence between these groups (5.3 ⁇ 0.7 versus 4.5 ⁇ 0.6).

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Abstract

La présente invention concerne des procédés d'isolement de cellules ou de groupes de cellules à partir de composants du stroma suite à un traitement enzymatique et/ou une interruption physique d'un organe ou d'un tissu au moyen de gradients d'intensité et des trousses pour isoler de telles cellules. L'invention concerne également des cellules ou des groupes de cellules, notamment, des cellules d'îlot pancréatique, produites par ces procédés, et des procédés d'utilisation desdites cellules pour la transplantation.
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EP2370565A2 (fr) * 2008-12-26 2011-10-05 Baylor Research Institute Purification à grande échelle d'îlots pancréatiques
CN109468266A (zh) * 2018-11-26 2019-03-15 温州医科大学 动物和人胰腺器官消化后组织、细胞洗涤溶液及制备方法
CN113046305A (zh) * 2021-04-29 2021-06-29 佛山市第一人民医院（中山大学附属佛山医院） 一种胰岛细胞的分离纯化方法
CN114196614A (zh) * 2021-12-14 2022-03-18 福建省医学科学研究院 pAdM3C感染大鼠胰腺导管细胞促进转分化方法

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2370565A2 (fr) * 2008-12-26 2011-10-05 Baylor Research Institute Purification à grande échelle d'îlots pancréatiques
EP2370565A4 (fr) * 2008-12-26 2012-08-22 Baylor Res Inst Purification à grande échelle d'îlots pancréatiques
CN109468266A (zh) * 2018-11-26 2019-03-15 温州医科大学 动物和人胰腺器官消化后组织、细胞洗涤溶液及制备方法
CN113046305A (zh) * 2021-04-29 2021-06-29 佛山市第一人民医院（中山大学附属佛山医院） 一种胰岛细胞的分离纯化方法
CN114196614A (zh) * 2021-12-14 2022-03-18 福建省医学科学研究院 pAdM3C感染大鼠胰腺导管细胞促进转分化方法

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