WO2019098944A1 - Systèmes de développement d'îlots pancréatiques et leur transplantation - Google Patents

Systèmes de développement d'îlots pancréatiques et leur transplantation Download PDF

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WO2019098944A1
WO2019098944A1 PCT/SG2018/050567 SG2018050567W WO2019098944A1 WO 2019098944 A1 WO2019098944 A1 WO 2019098944A1 SG 2018050567 W SG2018050567 W SG 2018050567W WO 2019098944 A1 WO2019098944 A1 WO 2019098944A1
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laminin
islets
pancreatic islets
matrix
cell culture
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PCT/SG2018/050567
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English (en)
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Karl Tryggvason
Kristmundur SIGMUNDSSON
Juha Ojala
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National University Of Singapore
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    • 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
    • A61K35/37—Digestive system
    • A61K35/39—Pancreas; Islets of Langerhans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin

Definitions

  • the present disclosure relates to methods for supporting long-term adhesion and expansion / proliferation of whole pancreatic islets containing one or more cell types, (i.e. alpha, beta, delta, gamma, or epsilon cells) that, respectively, produce glucagon, insulin, pancreatic peptide (PP), somatostatin, or ghrelin.
  • cell types i.e. alpha, beta, delta, gamma, or epsilon cells
  • PP pancreatic peptide
  • somatostatin ghrelin
  • Compositions and matrices for use in such methods are also disclosed, as well as devices that include such pancreatic islets. Transplantation of the expanded hormone producing pancreatic islets is further disclosed and included herein.
  • Type 1 diabetes mellitus is a metabolic disease in which the pancreas fails to produce enough insulin, resulting in high blood glucose levels over a prolonged period. Symptoms of high blood glucose levels include frequent urination, increased thirst and hunger, blurry vision, and fatigue.
  • Type 2 diabetes is usually found in people aged 40 and above, but it can also develop in younger individuals who are overweight and physically inactive. In some people, the condition is mild and they are able to control their blood glucose with just diet and exercise. However, if the condition gets worse, oral medication or insulin injections may be required in addition to making lifestyle changes. Left untreated, diabetes can cause many complications, including heart disease, stroke, chronic kidney failure, foot ulcers, eye damage, and death. In some cases, Type 2 diabetes also benefits from treatment with exogenic insulin.
  • compositions for expanding pancreatic islets using laminin matrices. It is contemplated that devices made from a laminin matrix and pancreatic islets can also be transplanted into mammals, such as humans, as a treatment for Type 1 diabetes or Type 2 diabetes.
  • the expansion media are fully defined and xeno-free, enabling at least five weeks of in vitro expansion of whole functional islets on the laminin matrices. Longer culture and expansion periods are also possible.
  • Murine and non-human primate islets have been shown to attach and expand on laminin matrices. Generally speaking, pancreatic islets are isolated and then expanded on the laminin matrix.
  • the present disclosure is directed to compositions, and methods of utilizing the same, to expand pancreatic islets.
  • the islets are plated on a laminin matrix.
  • the laminin matrix may contain a mixture of several laminins, or be formed from a single laminin.
  • the laminin matrix may be affixed to a substrate if desired.
  • pancreatic islets comprising: plating pancreatic islets on a laminin matrix; and culturing the pancreatic islets in a cell culture medium to expand the pancreatic islets.
  • the cell culture medium contains 15 mM or less of glucose and is free of Phenol red.
  • the laminin matrix may contain one of more of the following laminin isoforms: laminin-521 , laminin-511 , laminin-421 , laminin-411 , laminin-332, or laminin-1 11.
  • the laminin matrix contains a single laminin isoform, and the single laminin is a complete laminin trimer (of alpha, beta and gamma chains) or a laminin chain, or a laminin fragment.
  • the single laminin can be laminin-521 , laminin- 511 , laminin-421 , laminin-411 , or laminin-332.
  • the culturing of the pancreatic islets may be performed in an atmosphere that is enriched in C0 2 .
  • the culturing may occur in an atmosphere containing from about 5% C0 2 to about 10% C0 .
  • the cell culture medium is made from at least one RPMI medium and also contains 10 vol% serum, but in certain cases the serum may not be needed.
  • the cell culture medium contains no unknown proteins, lipids, or growth factors; and contains glutathione, biotin, vitamin B12, PABA, inositol, and choline.
  • the cell culture medium is mTeSRI medium, or a similar cell culture medium.
  • the pancreatic islets may be cultured for at least one day.
  • the pancreatic islets are cultured for a period of about three (3) days to about 40 days, and can be cultured for longer than 40 days as well.
  • the pancreatic islets may have a size of about 50 pm to over 500 pm.
  • Such a device comprises a laminin layer containing a laminin matrix, preferably coated upon a surface of a solid substrate; and a layer of pancreatic islets upon the laminin layer.
  • the laminin matrix contains laminin-521 , laminin-511 , laminin-421 , laminin- 411 , laminin-332, or laminin-111.
  • the laminin matrix contains a single laminin, and the single laminin is a complete laminin trimer, or is a laminin chain, or is a laminin fragment.
  • the single laminin can be laminin-521 , laminin- 511 , laminin-421 , laminin-411 , or laminin-332.
  • the solid substrate is used to support the laminin layer, and can be made of polydimethylsiloxane (PDMS) or other similar biocompatible plastic materials, or made of biodegradable materials, as discussed further herein.
  • PDMS polydimethylsiloxane
  • the solid substrate can be in the shape of a circular film, but other sizes or forms of solid substrates such as spheres can be used.
  • the laminin layer does not have to coat the entire surface of the solid substrate. For instance, a perimeter that is laminin-free may be present.
  • the layer of pancreatic islets may contain a varying amount of islets, and in some particular embodiments has a total of from about 100 to about 200 islets. These islets can vary in size, and can range from about 50 pm to about 500 pm, or greater.
  • the present disclosure is related to methods for treating a mammal with a metabolic disorder such as diabetes.
  • the methods comprise transplanting the pancreatic islets described above into the mammal, sometimes along with the laminin matrix, or devices including the same.
  • the device can be implanted or grafted into a kidney of the mammal, or any other location that provides a vascular bed for the transplanted islets.
  • the mammal may be a human.
  • the pancreatic islets of the device can be human pancreatic islets, mouse pancreatic islets, monkey pancreatic islets, pig pancreatic islets, or rat pancreatic islets, or pancreatic islets from other mammals.
  • FIG. 1A is a set of images arranged in two rows and six columns showing the adhesion, migration, and expansion of isolated mouse pancreatic islets plated onto plastic culture plates coated with, in order from left to right, laminin-111 , laminin-332, laminin-411 , laminin-421 , laminin-511 or laminin-521.
  • the top row is day 0 (top left insert) and day 5.
  • the bottom row is day 14.
  • FIG. 1 B is a set of six images comparing islets spreading for five days on LN- 521 or EHS mouse-tumor-derived Matrigel in a 96-well microtiter plate.
  • the top row is LN-521
  • the bottom row is Matrigel.
  • the leftmost column is day 1.
  • the middle column is day 5, and the rightmost column is a magnified view of day 5.
  • FIG. 1 C indicates the spreading efficiency in relation to initial islet size, applying the islets from FIG. 1 B.
  • the y-axis is area increase per islet, and runs from 0 to 15 in intervals of 5.
  • the x-axis is the islet initial area, in pm 2 , and is logarithmic.
  • the x-axis indicates values from 5x10 3 to 4x10 4 .
  • Triangles are for LN-521 , and circles are for Matrigel.
  • FIG. 1 D shows islets from the same batch as in FIG. 1 B after cultivation on LN-521 for three weeks.
  • the top picture is day 1
  • the bottom picture is day 18.
  • FIG. 1 E illustrates that islets that were expanded for 18 days in culture show active synthesis of insulin and DNA.
  • the images are arranged in two rows and four columns.
  • the top row is anti-C-peptide antibody, and the bottom row is isotype control.
  • the far left column shows the culture.
  • the center left column shows C- peptide/DAPI/EdU staining.
  • the center right column shows DAPI/EdU staining.
  • the right column shows EdU staining.
  • FIG. 1 F is a quantification of the percentage of b-cells per islet and of proliferating b-cells from FIG. 1 E.
  • the left graph is % b-cells per islet, and the axis ranges from 0% to 100% in intervals of 20%.
  • the right graph is % of proliferating b- cells, and the axis ranges from 0% to 20% in intervals of 5%.
  • FIG. 1G is an illustration of non-attached spherical islets from mTomato mouse that developed central hypoxia, which induced necrosis within 18 hours.
  • the images are arranged in two rows and six columns.
  • the top row is stained with Hoechst (blue), MAR (green), and DRAQ7 (red).
  • the bottom row is stained with Hoechst (blue), mTomato (orange), and DRAQ7 (red).
  • the columns are labeled, from left to right, 1 hour, 10 hours, 18 hours, 36 hours, 58 hours, and 70 hours.
  • FIGS. 2A-2L are a set of images showing the staining of islet cells, at day five of islet culturing on Laminin 521 , addressing hormone production and quantification of islet composition thereof.
  • FIG. 2A shows C-peptide staining of insulin producing b-cells, in red.
  • FIG. 2B shows glucagon staining of a-cells, in blue.
  • FIG. 2C shows somatostatin staining of d-cells, in green.
  • FIG. 2D shows the merged view of C-peptide and somatostatin staining (FIG. 2A + FIG. 2C), indicating no overlapping in production of these hormones within islet cells.
  • FIG. 2G shows a merged view for the four hormones, indicating in addition to glucagon and pp-peptide, these a-cells also express insulin.
  • FIG. 2H shows the same as FIG. 2G, with a nuclear DAPI (cyan) staining added to the merged view, providing the total amount of endocrine cells for this islet counting 400 cells.
  • FIG. 21 shows the same as FIG. 2H, with the addition of membrane-Tomato to the merged view.
  • FIG 2M provides the DAPI based cell counting for 23 islets that were analyzed with the Columbus software (PerkinElmer) applying a segmentation algorithm based on the immunofluorescence approach described above, which results are provided in FIG 2N, with average values and SEM provided as black crosses. Image data was collected on an Operetta high content screening microscope, applying a 20x NA objective.
  • the axis is labeled“endocrine cells per islet”, and runs from 0 to 1200 in intervals of 200.
  • the axis is labeled“cell type per islet (%)”, and runs from 0 to 100 in intervals of 20. Five sets of results are seen in FIG. 2N. The leftmost is for alpha cells, the center left is for beta cells, the center is for gamma cells, the center right is for PP-cells, and the right is triple positive.
  • FIG. 3A is a bar graph illustrating the gene expression levels (FPKM) of integrin genes in freshly isolated mouse islets (day 0). Previously reported laminin binding integrins are highlighted in red (integrin alpha 6, Itga6; integrin alpha 3, Itga3 and integrin beta 4, Itgb4).
  • FIG. 3B is a bar graph indicating the number of differentially expressed genes after multiple testing correction with each laminin coating at day 12 compared to day 3 (Benjamini-Flochberg adjusted P ⁇ 0.05).
  • FIG. 3C is a graphic expressing the top five functionally enriched KEGG pathways and GO terms when running Gene Set Enrichment Analysis (GSEA) ranking the mouse genome by differential expression at day 12 versus day 3 with LN-521 , LN- 421 , and LN-111.
  • GSEA Gene Set Enrichment Analysis
  • FIG. 3D is a graphic illustrating known connections among the LN-521 gene signature retrieved from the STRING database.
  • the downregulated genes are Erol lb, Uggtl , Magtl , Hspa5, Pyy, and Gfptl
  • the genes identified as“focal adhesion” are Fine, Flna, Mylk, Myl9, Ltgal 1 , Thbsl , Spp1 , and Fn1. All other genes are upregulated.
  • FIG. 4A shows two schematic descriptions.
  • the top is a schematic description of how islets are cultured on LN-521 -coated PDMS membranes.
  • the bottom is a schematic description showing how the membranes are transplanted into the kidney capsule of a mouse.
  • FIG. 4B is a set of two pictures eight weeks after implantation.
  • the top picture shows the implanted device on the mouse kidney capsule.
  • the bottom picture shows the implanted device removed, and the original area where the device was implanted showing the red fluorescence of transplanted ActinDs-Red islets.
  • FIG. 4C is a set of images indicating that, 8 weeks after transplantation, the kidney is removed and ActinDs-Red positive transplanted islets produce C-peptide; i.e proving that the islets are functional and produce insulin.
  • the islets are well vascularized by the endothelial cells derived from the host.
  • FIG. 4D is a set of two graphs indicating the glucose curves of Streptozotocin induced T1 D mice before and after the transplantation of PDMS membranes with 1 1 Q- 130 islets growing on LN-521 into the kidneys thereof.
  • Streptozotocin was administered 2 weeks before transplantation, and kidneys were removed after 8 weeks.
  • the top graph shows the results for each individual mouse.
  • the bottom graph shows aggregate results for female mice receiving membranes cultured for 3 days, male mice receiving membranes cultured for 3 days, and male mice receiving membranes cultured for 7 days.
  • FIG. 4E is a graph comparing male mice to female mice
  • FIG. 4F is a graph aggregating all of the mice together.
  • FIG. 5A is a graph indicating the glucose curves of Streptozotocin induced T1 D mice before and after the transplantation of 75 medium size (LM-M75 80 %, 100- 150 mih) and 32 large (LM-L32 30%, 150-250 miti) islets growing on LN-521 into the kidneys thereof after scraping the islets off the cell culture plastic with a cell scraper and settling the islets into sterile PE 50 polyethylene tubing (Intramedic) suitable for injection under the kidney capsule.
  • the success rate of injection is estimated to be 80% for the medium-size islets and 30% for the large-size islets.
  • FIG. 5B is a graph indicating the glucose curves of Streptozotocin induced T1 D mice before and after the transplantation of 150 small (S150 100%, 70-100 miti), 150 medium size (M150 90 %, 100-150 miti), or 64 large (L64 100%, 150-250 miti) freshly isolated islets into the kidneys thereof after settling the islets into sterile PE 50 polyethylene tubing (Intramedic) suitable for injection under the kidney capsule.
  • the success rate of injection is estimated to 100%, 90% and 100% for the small, medium, and large islets respectively.
  • FIG. 5C is a graph indicating the glucose curves of Streptozotocin induced T1 D mice before and after a sham transplantation operation of the kidneys (i.e. no islets were transplanted).
  • Streptozotocin was administered 28 days before transplantation, and kidneys were removed after 49 days. No improvement of blood glucose was observed, indicating that all the islets in the mice receiving transplants in FIG. 5A and FIG. 5B were rendered non-functional by streptozotocin and any improvement of blood glucose seen was due to the transplanted islets.
  • FIG. 6 is a graph indicating the glucose curves of two Streptozotocin induced T1 D mice before and after the transplantation of PDMS membranes with 140 islets growing on LN-521 under the skin over left shoulder blade thereof.
  • Streptozotocin was administered 7 days before transplantation, and the blood glucose was followed for 56 days. Both mice showed blood glucose improvement close to normal values 8 weeks after transplantation.
  • FIGS. 7A-7G are a set of images following a glucose-stimulated insulin secretion (GSIS) assay.
  • FIG. 7A is a graphic of a GSIS assay, indicating the ratio of glucose input to insulin output.
  • FIG. 7B is an image of a Western blot analysis of GSIS assay samples for large size (150 pm to 250 pm) islets cultured on LN-521 or uncoated plastic, and exposed to either 2 mM or 25 mM glucose.
  • FIG. 7C is a bar graph of a GSIS assay indicating insulin released (ng/mL) per large size islet cell (150 pm to 250 pm) cultured on LN-521 or LN-1 11 at day 3 and day 12, with the capacity to secrete insulin, stimulated by glucose levels of 25 mM per islet.
  • the dark bar is LN-521
  • the white bar is LN-111.
  • FIG. 7D is a bar graph of a GSIS assay indicating insulin released (ng/mL) per medium size islet cell (100 pm to 150 pm) cultured on LN-521 or LN-1 11 at day 3 and day 12.
  • the dark bar is LN-521
  • the white bar is LN-11 1.
  • FIG. 7E is a bar graph of a GSIS assay indicating insulin released (ng/mL) per small size islet cell (70 pm to 100 pm) cultured on LN-521 or LN-111 at day 3 and day 12, with the capacity to secrete insulin, stimulated by glucose levels of 25 mM per islet.
  • the dark bar is LN-521
  • the white bar is LN-11 1.
  • FIG. 7F is a bar graph of the ratio of insulin released upon exposure to 25 mM glucose to insulin released upon exposure to 2 mM glucose of large, medium, and small islets cultured on LN-521 or LN-111.
  • the dark bar is LN-521
  • the white bar is LN- 111.
  • FIG. 7G is a bar graph of insulin released at 25 mM glucose, in picograms (pg), per beta cell of islet cells cultured on LN-521 or LN-111. The dark bar is LN-521 , and the white bar is LN-111.
  • Statistical significance for FIGS. 7C-7G is indicated by: ( * ) P ⁇ 0.05, ( ** ) P ⁇ 0.01 , and ( *** ) P ⁇ 0.001.
  • the term “comprising” may include the embodiments “consisting of” and “consisting essentially of.”
  • the terms “comprise(s),”“include(s),”“having,”“has,”“can,”“contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients/steps and permit the presence of other ingredients/steps.
  • compositions or processes as “consisting of” and “consisting essentially of” the enumerated ingredients/steps, which allows the presence of only the named ingredients/steps, along with any impurities that might result therefrom, and excludes other ingredients/steps.
  • the term“about” can be used to include any numerical value that can vary without changing the basic function of that value. When used with a range,“about” also discloses the range defined by the absolute values of the two endpoints, e.g.“about 2 to about 4” also discloses the range“from 2 to 4.” The term“about” may refer to plus or minus 10% of the indicated number.
  • pancreatic islet cells or“pancreatic islets.” These references should be understood as referring to the alpha, beta, gamma, delta, or epsilon cells in the pancreas that produce different hormones.
  • T1 D Type 1 diabetes
  • CGM continuous glucose monitoring
  • the current cadaveric islet transplant approach can both reduce severe hypoglycemic events (SHEs) and improve hypoglycemic awareness. It is also much less invasive compared to whole pancreas transplantation. However, it is limited by the lack of available donor organs, with less than on average 30-40 transplantations per year in the United Kingdom. The current cost of the procedure, which typically requires transplantation from minimum two separate donors, is also prohibitively expensive.
  • SHEs severe hypoglycemic events
  • T1 D and T2D are major focus for both multinational pharmaceutical corporations as well as emerging biotechnology and biomedical companies. These efforts are focused on the following areas: long-acting insulin derivatives and insulin biosimilars; therapeutics to increase insulin production; advanced insulin pumps combined with continuous glucose monitoring; autoimmune and immunomodulatory therapies; transplantation strategies including generation of insulin producing cells, such as xenogenic islets, human stem cell and iPSC approaches; encapsulation technologies for enhancement of transplantation strategies; generation of artificial pancreas, including repopulation of decellularized pancreas and 3D organ bioprinting; and therapeutics for stimulation of regeneration of beta cells in situ.
  • pancreatic islets can be grown, expanded and further transplanted into human subjects with the methods of the present disclosure.
  • the present disclosure provides a laminin matrix, upon which pancreatic islets are plated and cultured. This can take the form of a substrate support that is coated with specific basement membrane laminins.
  • a xeno-free and chemically defined cell culture medium system is used with the laminin matrix to expand the pancreatic islets, including all possible cell types.
  • a laminin protein comprises one a-chain subunit, one b-chain subunit, and one g-chain subunit, all joined together in a trimer through a coiled-coil domain.
  • the twelve known laminin subunit chains can form at least 16 trimeric laminin types in native mammalian tissues.
  • Within the trimeric laminin structures are identifiable domains that possess binding activity towards other laminin and basal lamina molecules, as well as cell plasma membrane-bound receptors.
  • domains VI, IVb, and IVa form globular structures
  • domains V, lllb, and Ilia which contain cysteine-rich EGF-like elements
  • Domains I and II of the three chains participate in the formation of a triple-stranded coiled-coil structure (the long arm).
  • One nomenclature for these laminin proteins describes the isoforms based on their chain compositions, e.g., laminin-111 (laminin-1 ) that contains alpha-1 , beta-1 , and gamma-1 chains.
  • the term“laminin-521” refers to the protein isoform formed by joining a5, b2, and y1 chains together.
  • the term“laminin-511” refers to the protein formed by joining a5, b1 , and y1 chains together.
  • the term“laminin-421” refers to the protein formed by joining a4, b2, and g1 chains together.
  • the term“laminin-411” refers to the protein formed by joining a4, b1 , and y1 chains together.
  • the term“laminin-332” refers to the protein formed by joining a3, p3, and y2 chains together.
  • the laminin can be a complete protein trimer, or a protein chain, or a protein fragment.
  • the term“complete” refers to the protein being composed of all the domains of the a-chain, one b-chain, and g-chain, with the three chains being joined together to form the heterotrimeric structure.
  • the protein is not broken down into separate chains, fragments, or functional domains.
  • the term“chain” refers to the entirety of the individual alpha, beta, or gamma chain of the laminin protein.
  • fragment refers to any protein fragment that contains one, two, or three functional domains that possesses binding activity to another molecule or receptor. However, a chain should not be considered a fragment because each chain possesses more than three such domains. Similarly, a complete laminin protein trimer should not be considered a fragment. Examples of functional domains include Domains I, II, III, IV, V, VI, and the G domain.
  • the present disclosure relates to methods for culturing and expanding pancreatic islet cells, i.e. to obtain a greater number of islet cells from a smaller number of cells, also known as proliferation.
  • the pancreatic islets are cultured on a laminin matrix which can be affixed to a solid substrate, which acts as a support.
  • the term“solid” refers to the state of matter, i.e. the substrate is not liquid or gas or plasma.
  • the substrate can be rigid / hard or very flexible (such as a film), and the term “solid” should not be construed as requiring a particular degree of rigidity.
  • the laminin matrix may contain a mixture of multiple laminins, or may contain a single laminin trimer, or a single laminin chain, or a single laminin fragment.
  • the term “single” is used herein to mean that the laminin matrix contains only one laminin trimer or chain or fragment, but permits other ingredients to still be present in the laminin matrix.
  • the pancreatic islets are then nourished using a cell culture medium that contains a small amount of glucose, fifteen (15) millimolar (mM) or less.
  • the cell culture medium also does not contain Phenol red, which is also known as phenolsulfonphthalein.
  • the structural support for the cells is a laminin matrix.
  • the pancreatic islets are then deposited upon the laminin matrix.
  • the laminin matrix can be formed upon a substrate, which can be a rigid or flexible material that provides a surface upon which the laminin matrix is formed.
  • the substrate can be, for example, a petri dish or the well of a multi-well plate.
  • the substrate can also be a thin film or membrane made of a biocompatible polymer like polydimethylsiloxane (PDMS), polyvinyl chloride (PVC), polytetrafluoroethylene (PTFE), polyethersulfone (PES), polyethylene (PE), polyetheretherketone (PEEK), polysulfone (PS), polypropylene (PP), polyethylene glycol (PEG), polyvinyl alcohol (PVOFI), polymethyl methacrylate (PMMA), polyethylene vinyl acetate (EVA), poly(ether urethane), polyethylene terephthalate, polyethylene oxide, polyethylene oxide-co- polypropylene oxide, or polyacrylamide.
  • PDMS polydimethylsiloxane
  • PVC polyvinyl chloride
  • PTFE polytetrafluoroethylene
  • PES polyethersulfone
  • PE polyethylene
  • PEEK polyetheretherketone
  • PS polypropylene
  • PP polyethylene glycol
  • PVOFI polyvin
  • the substrate could alternatively be made from a biodegradable material like polylactic acid, polyglycolic acid, poly( -caprolactone), poly(dioxanone), poly(lactide-co-glycolide), polyglyconate, or polyorthoester, or a hydrogel.
  • a biodegradable material like polylactic acid, polyglycolic acid, poly( -caprolactone), poly(dioxanone), poly(lactide-co-glycolide), polyglyconate, or polyorthoester, or a hydrogel.
  • the laminin matrix on the substrate can contain any effective laminin. It is specifically contemplated that the laminin matrix contains only one particular laminin (i.e. one single laminin), though other ingredients may also be present in the laminin matrix.
  • the laminin is Laminin-511 (LN-511 ), Laminin-521 (LN-521 ), Laminin-411 (LN-411 ), Laminin-421 (LN-421 ), or Laminin-332 (LN-332). It is particularly contemplated that the laminin matrix does not contain a cadherin. Cadherins are also known as desmogleins and desmocollins, and are absent from the laminin matrix.
  • the laminin matrix is used in combination with a cell culture medium.
  • cell culture media include a large number and a large amount of various growth factors and cytokines to inhibit differentiation and improve proliferation.
  • glucose concentration can range from 3.9 mM to 7.7 mM in plasma, depending on fasting conditions and time measured after eating.
  • Glucose concentration in serum can range from 3.9 mM to 11.0 mM.
  • the beta cells of the pancreas are exceptionally sensitive to glucose, and can become stressed after long-term exposure to glucose levels that are above physiological concentrations, which in serum is about 3.9 to 11 mM.
  • the cell culture media used in the present methods have a glucose level that is within physiological glucose concentrations and are free of Phenol Red.
  • the cell culture media have a glucose level of 15 mM or lower, including 11 mM or lower, and including 5 mM or lower.
  • media containing higher concentrations of glucose may also be used.
  • the cell culture medium of the present disclosure is obtained by mixing at least one RPMI 1640 medium together with serum. More specifically, the following reagents, available from GIBCO and listing the GIBCO catalog number, are used:
  • the two RPMI 1640 media are mixed together in a 1 :1 volume ratio to make 90 mL to decrease the glucose concentration.
  • 10 mL of FBS is then added to obtain a serum amount of 10% of final volume, resulting in 10% FBS/RPMI 1640 medium.
  • This cell culture medium is designed to meet the needs of pancreatic islets.
  • the final glucose concentration of the cell culture medium should be in desired embodiments 15 mM or less, and in particular embodiments be 1 1 mM or less, or even 5 mM or less.
  • the glucose concentration is at least 3.5 mM, which is just under the low end of physiological concentrations listed above.
  • RPMI 1640 medium requires supplementation, commonly with 10% FBS, because no proteins, lipids, or growth factors are present.
  • the serum may be from about 8 vol% to about 12 vol% of the cell culture medium.
  • RPMI 1640 medium uses a sodium bicarbonate buffer system (2.0 g/L), and therefore requires a 5-10% C0 2 environment to maintain physiological pH. In other embodiments, however, it is contemplated that serum is not needed.
  • another cell culture medium that can be used contains RPMI 1640 medium and a Connaught Medical Research Laboratories (CMRL) medium mixed together in a 1 :1 ratio and supplemented with fetal calf serum to obtain a serum amount of 10% of the final volume, and containing 10 mM HEPES, 1% GlutaMax, 0.5% penicillin, and 0.5% streptomycin.
  • This cell culture medium has a glucose concentration of -7.43 mM.
  • the cell culture medium will be completely defined and xeno-free. Therefore, desirably it should not contain sera that are usually from animals (e.g. bovine serum) that can vary from batch to batch.
  • the medium should also be devoid of any differentiation inhibitors, feeder cells, or differentiation inductors, or apoptosis inhibitors.
  • feeder cells include mouse fibroblasts or human dermal fibroblasts.
  • differentiation inductors include Noggin or keratinocyte growth factor.
  • the cell culture medium may be mTeSRI cell culture medium, which can contain up to 4-6 mM glucose.
  • the ingredients of this cell culture medium are listed in the following Table 1 , though it is contemplated that the amount of each individual ingredient can vary up to 5% in each direction:
  • the source of the pancreatic islets can be any animal. However, in particular embodiments, the source is a mammal, such as a human, mouse, monkey, pig, or rat. After being isolated from their source, the pancreatic islets are plated on the laminin matrix, which is itself usually coated upon the substrate. The pancreatic islets are then cultured in the cell culture medium and expanded, typically for a period of at least 3 days and lasting as long as 35 days in culture, or 40 days in culture, or even longer. As mentioned above, this may be done in an atmosphere containing about 5% to about 10% CO 2 when the RPMI-based cell culture medium is used.
  • the culturing also occurs at elevated temperatures within ⁇ 5°C of normal body temperature (37°C).
  • Cell culture medium can be exchanged as desired.
  • the expansion of islets occurs without removing them from the substrate and without dispersing the cells. Under those conditions all islet cell types proliferate at a similar rate. It is also possible to disperse the islets into single cells, then replate them and grow, but it is not necessary for expanding the islet cells (and results in unnecessary cell losses).
  • pancreatic islets have normoglycemic function, i.e. they produce insulin in lesser or greater quantities depending on the amount of glucose in their environment.
  • the pancreatic islets may have a size ranging from about 50 pm to about 500 pm, as measured by their diameter. Some human islets are even larger.
  • a laminin layer containing a laminin matrix can comprise a laminin layer containing a laminin matrix; and a layer of pancreatic islets upon the laminin layer.
  • the laminin layer is formed upon a solid substrate. If desired, and depending on the shape of the substrate, there can be two laminin layers on the substrate, one layer on each of the opposite surfaces of the substrate, and two layers of pancreatic islets (one layer of pancreatic islets on each of the laminin layers). It is contemplated here that the substrate is in the shape of a thin film or membrane, or is in the shape of a sphere or a fiber.
  • a sphere or fiber would be considered to have only one surface, whereas a thin film or membrane would be considered to have two opposite surfaces.
  • the substrate can be coated with the laminin matrix and used as surface(s) for adhesion and expansion of islets in vitro. Suitable materials for the membrane include polydimethylsiloxane (PDMS) and other biologically compatible substrates or biodegradable substrates as described above.
  • PDMS polydimethylsiloxane
  • the laminin layer does not have to cover the entire surface of the flexible substrate, as will be further explained in the Examples.
  • the layer of pancreatic islets can contain a total of about 50 to several hundreds of whole islets, and even up to 1000 islets.
  • the layer of pancreatic islets can contain from about 50 to about 1000 islets, or from about 50 to about 200 islets.
  • two layers of pancreatic islets could be placed on opposite surfaces of the laminin layer.
  • these devices can be transplanted into a mammal, such as a human, who is suffering from diabetes (Type 1 or Type 2).
  • the device can be implanted into any suitable location or organ or tissue with sufficient vascular bed, for example the pancreas, the kidney, muscle or subcutaneous tissue.
  • Pancreatic islets were isolated from mice and monkeys, and plated onto wells containing 5 pg/cm 2 of one of six laminins (LN-111 , LN-332, LN-411 , LN-421 , LN-511 , LN-521 ) or Matrigel (an EHS mouse sarcoma tissue extract that contains several basement membrane components). mTeSRI cell culture medium was used.
  • FIG. 1A is a set of images showing the adhesion, migration, and expansion of isolated mouse pancreatic islets. Morphology of islets plated right after plating is provided in the top-left insert, except for islets plated on LN-332, where zero-hour islets are provided as overlay in green.
  • the islets did not attach to LN-11 1. Islets plated on LN-511 and LN-521 attached and effectively spread in a chemically defined serum-free medium, LN-521 providing more rapid attachment and expansion than LN-511. The islets unwrapped from spheres into 2-3 cell layer sheets in just a few days while maintaining cell-cell contacts. Islets attaching to LN-332, LN-411 and LN-421 supported adhesion at day 5, but the spreading was not extensive and the islets shrank and developed gaps by day 14. At day 14, islets on LN-332, LN-411 , and LN-421 undertook re-clustering (and central necrosis). Red coloring indicates DRAQ7 that binds to DNA in leaky and dying cells.
  • FIG. 1 B illustrates the comparison of islets spreading for five days on LN-521 to Matrigel in a 96-well microtiter plate. For each condition, a typical islet is indicated with an arrow and provided at high magnification. Nuclei are stained with DAPI (blue) and p-cells spotted with anti-C peptide antibody (red). The islets on Matrigel did not expand, whereas those on LN-521 did.
  • FIG. 1C indicates the spreading efficiency in relation to initial islet size for the islets from FIG. 1 B.
  • FIG. 1 D shows islets from the same batch as in FIG. 1 B after cultivation on LN-521 for three weeks.
  • FIG. 1 E illustrates the islets expanded for 18 days in culture, and shows active synthesis of insulin (green coloring indicates C-peptide staining) and DNA (red coloring indicates EdU incorporation to nuclei).
  • 1G is an illustration of non-attached spherical islets from mTomato mouse (orange cell membranes) that developed central hypoxia (top row, green: MAR), which induced necrosis within 18 hours (red: DRAQ7). Leaky and dead cells are indicated by blue Hoescht staining of nuclei. Non-attached whole islets rapidly expel the necrotic cells as clumps, indicating that a large proportion of b-cells are disabled early. In contrast, b-cells, mainly located on or close to the islet surface, survive better.
  • FIGS. 2A-2N are images showing the staining of islet cells, at day five of islet culturing on Laminin 521 , addressing hormone production and quantification of islet composition thereof.
  • FIG. 2A shows C-peptide staining of insulin producing b-cells, in red.
  • FIG. 2B shows glucagon staining of a-cells, in blue.
  • FIG. 2C shows somatostatin staining of d-cells, in green.
  • FIG. 2D shows the merged view of C-peptide and somatostatin staining (A+C), indicating no overlapping in production of these hormones within islet cells.
  • FIG. 2G shows a merged view for the four hormones, indicating in addition to glucagon and pp- peptide, these a-cells also express insulin.
  • FIG. 2H shows the same as G, with a nuclear DAPI (cyan) staining added to the merged view, providing the total amount of endocrine cells for this islet counting 400 cells.
  • FIG. 2I shows the same as H, with the addition of membrane-Tomato to the merged view.
  • FIG. 2J, FIG. 2K, and FIG. 2L provide the merged views (as for FIG. 2G, FIG. 2H, and FIG. 21, respectively) for the non-specific background control, where the four secondary antibodies were added to the islets in absence of the primary antibodies, keeping the same microscopy settings.
  • FIG 2M provides the DAPI based cell counting for 23 islets that got analized with the Columbus software (PerkinElmer) applying a segmentation algorithm based on the immunofluorescence approach described above, which results are provided in FIG 2N, with average values and SEM provided as black crosses. Image data was collected on a Operetta high content screening microscope, applying a 20x NA objective.
  • FIG. 3A reveals that integrin beta-1 (Itgbl ) has the greatest basal gene expression levels (FPKM) in freshly isolated mouse islets (day 0) followed by integrin alphaV (Itgav) and integrin alpha-6 (Itga6). Previously reported laminin-binding integrins are highlighted in red (integrin alpha 6, Itga6; integrin alpha 3, Itga3 and integrin beta 4, Itgb4).
  • RNA sequencing was performed in order to look at differential gene expression on day 3 and day 12 in islets cultured on LN-521.
  • FIG. 3B indicates the number of differentially expressed genes after multiple testing correction with each laminin coating at day 12 compared to day 3 (Benjamini-Hochberg adjusted P ⁇ 0.05).
  • LN-521 demonstrated large numbers of upregulated and downregulated differentially expressed genes, while LN-421 showed extremely limited upregulated but fairly moderate downregulated numbers of differentially expressed genes.
  • LN-111 also showed an extremely limited upregulated number of differentially expressed genes but demonstrated an extremely high number of downregulated differentially expressed genes.
  • LN-521 resulted in the up-regulation of several genes enriched for KEGG pathways and molecular processes related to adhesion and cell-to-cell interaction, including “focal adhesion”, “vascular smooth muscle contraction”, “regulation of cytoskeleton” and“ECM-receptor interactions” pathways.
  • coating with LN- 111 or LN-421 resulted in down-regulation (or no significant change) of these processes and pathways.
  • Integrin a1 1 as well as other integrins are reported to bind collagens I, IV and IX, and other proteins such as fibronectin or VCAM.
  • FIG. 3C The top five functionally enriched KEGG pathways and GO terms are graphed in FIG. 3C. These were determined by running Gene Set Enrichment Analysis (GSEA) ranking the mouse genome by differential expression at day 12 versus day 3 with LN-521 , LN-421 , and LN-11 1. In the level graph, color is mapped to GSEA normalized enrichment score (NES) of -5 to 5. The higher the NES value, the stronger the enrichment for the pathway that contains the genes that are downregulated. Results are displayed as sorted by LN-521 NES values. Significant results after multiple testing correction are denoted by ** (GSEA FDR ⁇ 0.05). Nominally significant results are denoted by * (GSEA P ⁇ 0.05).
  • each node represents a gene from the LN-521 gene signature
  • each edge (connection) represents an interaction between a pair of genes as reported in the STRING database.
  • STRING protein-protein interactions are highlighted in red.
  • Node size is mapped to the absolute Log2 gene expression fold change in day 12 compared to day 3. Up regulated genes (positive Log2 fold change) at day 12 compared to day 3 with LN-521 coating are colored in red, whereas down regulated genes (negative Log2 fold chance) at day 12 compared to day 3 with LN-521 coating are colored in blue.
  • pancreatic islets were grown on laminin coated films and transplanted under the kidney capsule of streptozotocin induced T1 D mice.
  • a schematic description of islets cultured on LN-521 coated PDMS membranes is shown at the top of FIG. 4A.
  • a PDMS membrane blue
  • the inner 3 mm cast is used for laminin coating (yellow), and it is removed after overnight adhesion of the plated islets (black dots).
  • the outer cast is used to facilitate the punching of the membrane into 5 mm diameter membranes used for the transplantation.
  • 50-70 islets isolated from Actin- DsRed transgenic mice or normal C57BI/6N mice were plated onto PDMS films coated overnight with LN-521 , and then cultured for 3 days or 7 days prior to transplantation. The islets attached, flattened out, and expanded on the PDMS film.
  • FIG. 4A The bottom of FIG. 4A is a schematic description of the transplantation procedure under the kidney capsule.
  • a small incision is made to the dorsal left flank of the mouse under anesthesia. Through the opening, the left kidney is exposed and a needle is used to make an opening through the kidney capsule.
  • Two PDMS membranes with cultured islets exposed towards kidney parenchyma are placed under the capsule on both sides of the kidney. The kidney is carefully pushed back into the peritoneum and the opening through the peritoneal wall, and the skin is sealed with sutures.
  • FIG. 4B The top picture indicates where the PDMS membrane was placed.
  • the transplanted islets originated from Actin-DsRed mice showing red fluorescence that can be seen attached to the kidney parenchyme in the lower panel after the PDMS membrane is removed. This indicates that the membrane can be removed if desired without removing the transplanted islets.
  • FIG. 4C is a set of stains indicating the presence of transplanted islets originating from Actin-DsRed mice, C-peptide, and CD31 separately, then merged into one, and a flattened CD31 stain (showing a Z-stack of many layers combined).
  • the yellow dotted line indicates the boundary between the transplanted islets isolated from Actin-DsRed mice (red cells, top right) and the kidney cortex tissue of a mouse to which the expanded islets were transplanted.
  • FIG. 4D indicates the glucose curves of streptozotocin induced T1 D mice before and after transplantation of PDMS membranes with 110-130 mouse islets grown on LN-521 for 3 or 7 days. Streptozotocin was administered 2 weeks before transplantation. A spike in blood glucose levels is seen, indicating death of the natural beta cells in the pancreas. Regardless of the sex of the animals, within 2-3 weeks, the blood glucose decreased from >28 mM down to normal values ( ⁇ 11.1 mM) where it stayed until the transplant was removed. This indicated that the transplanted islets were producing insulin and responding physiologically to feeding. The islet transplant receiving kidneys were removed after 8 weeks, and the glucose levels spiked back up.
  • FIG. 4E is a graph comparing male mice to female mice
  • FIG. 4F is a graph aggregating all of the mice together.
  • FIG. 5A is a graph showing the glucose levels (mM) over time (days). The success of transplantation was estimated to be 80% and 30%, respectively. Prior to the transplantation the blood glucose was stably over 33.3 mM for one week and therefore any improvement in the blood glucose values is expected to come from the insulin produced by the small number of transplanted islets.
  • FIG. 5B is a graph showing the glucose levels (mM) over time (days) for round islets freshly isolated prior to transplantation in a separate experiment.
  • One mouse received 64 large islets (150-250 miti, L64), one mouse received 150 medium islets (100-150 miti, M150), and one mouse received 150 small islets( 70-100 miti, S150).
  • the success of transplantation was estimated to be 100%, 90%, and 100% respectively.
  • Medium size islets seemed to work better than small islets in improving the blood glucose level. Due to lower number of large islets, the comparison is not as clear.
  • the normalization of blood glucose requires at least 7 weeks or higher number of transplanted islets when using freshly isolated islets.
  • FIG. 5C is a graph showing the glucose levels over time for three“dummy” mice that were operated upon, but received no islets (i.e. a sham operation). Prior to the transplantation the blood glucose was high, but still fluctuating below 33.3 mM, therefore any improvement in the blood glucose values after the sham operation is expected to follow the same pattern confirming that no real improvement of blood glucose occurred during the 7 week follow up time.
  • FIG. 6 is a graph showing the glucose levels (mM) over time (days).
  • Normoglycemia limit 1 1.1 mM is marked “LIMIT”. Both mice show improvement in the blood glucose values close to normal levels at week 8 post-transplantation.
  • the subcutaneous model of laminin-cultured islets has therefore similar efficacy as transplantation of round freshly isolated islets depicted in FIG. 5B, indicating that the flat islets withstand well the relatively hypoxic environment provided by the subcutaneous vascular bed in comparison to the kidney parenchyma.
  • Islets were cultured at 8 mM glucose levels then exposed to 2 mM for 1 hour. After collecting a sample, islets were then exposed to 25 mM glucose for 1 hour, after which another sample was collected and the culture continued. As shown in FIG. 7A, insulin output increased proportionally to glucose input, with increased beta cell efficacy at 25 mM glucose levels compared to 2 mM glucose levels.
  • a Western blot analysis of the GSIS assay was conducted for samples of large size islets (150-250 pm) cultured on either LN-521 or uncoated plastic. As shown in FIG. 7B, insulin output was observed in large size islets after exposure to 25 mM glucose and cultured on LN-521. However, no significant output (i.e. no visible insulin blot) was observed in islets cultured on LN-521 with 2 mM glucose or in islets cultured on uncoated plastic (not shown).
  • a GSIS assay was performed on large size islets (150-250 pm) cultured on either LN-521 or LN-111. Islets were observed for their capacities to secrete insulin potentiallymulated by glucose levels of 25 mM per islet. A graph comparing these capacities is shown in FIG. 7C, which indicates that islets cultured on LN-521 released more insulin than islets cultured on LN-111. From day 3 of culture to day 12 of culture, the amount of insulin released from islets cultured on LN-521 increased by a third, while the amount of insulin released from islets cultured on LN-111 remained the same.
  • a GSIS assay was performed on medium size islets (100-150 pm) cultured on either LN-521 or LN-111. Islets were observed for their capacities to secrete insulin potentiallymulated by glucose levels of 25 mM per islet. A graph comparing these capacities is shown in FIG. 7D, which indicates that islets cultured on LN-521 released more insulin than islets cultured on LN-111. From day 3 of culture to day 12 of culture, the amount of insulin released from islets cultured on LN-521 doubled, while the amount of insulin released from islets cultured on LN-1 11 minimally increased.
  • a GSIS assay was performed on small size islets (70-100 pm) cultured on either LN-521 or LN-111. Islets were observed for their capacities to secrete insulin stimulated by glucose levels of 25 mM per islet. A graph comparing these capacities is shown in FIG. 7E, which indicates that islets cultured on LN-521 released more insulin than islets cultured on LN-111. From day 3 of culture to day 12 of culture, the amount of insulin released from islets cultured on LN-521 and LN-111 remained approximately the same.
  • FIG. 7F is a graph comparing the results from the GSIS assays. Overall, islets cultured on LN-521 secreted more insulin than islets cultured on LN-111. Further, the larger the islet, the greater the amount of secreted insulin. Therefore, the greatest insulin secretion was observed in large islets cultured on LN-521. A broad comparison may be found in FIG. 7G.
  • FIGS. 7C-7G Statistical significance for FIGS. 7C-7G is indicated by: ( * ) P ⁇ 0.05, ( ** ) P ⁇ 0.01 , and ( *** ) P ⁇ 0.001.

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Abstract

L'invention concerne des procédés de culture d'îlots pancréatiques fonctionnels in vitro et de transplantation de ces derniers dans une espèce mammifère pour traiter des troubles métaboliques tels que la déficience en insuline. Généralement, les îlots sont isolés, plaqués sur une matrice de laminine et cultivés dans un milieu de culture cellulaire en vue de développer les îlots pancréatiques, la matrice de laminine contenant de la laminine-521, de la laminine-51 1, de la laminine-421, de la laminine-41 1 ou de la laminine-332, ou un mélange de ces dernières. Des dispositifs comprenant une matrice de laminine et des îlots pancréatiques peuvent être transplantés chez un mammifère diabétique, et il a été montré qu'ils régulent les taux de glucose.
PCT/SG2018/050567 2017-11-15 2018-11-14 Systèmes de développement d'îlots pancréatiques et leur transplantation WO2019098944A1 (fr)

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CN113308433A (zh) * 2021-04-27 2021-08-27 立沃生物科技（深圳）有限公司 一种胰岛体外培养表面杂细胞处理方法

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