WO2023234421A1 - Feuillet cellulaire réparateur de cartilage et son procédé de fabrication - Google Patents

Feuillet cellulaire réparateur de cartilage et son procédé de fabrication Download PDF

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WO2023234421A1
WO2023234421A1 PCT/JP2023/020854 JP2023020854W WO2023234421A1 WO 2023234421 A1 WO2023234421 A1 WO 2023234421A1 JP 2023020854 W JP2023020854 W JP 2023020854W WO 2023234421 A1 WO2023234421 A1 WO 2023234421A1
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cells
sheet
cell
cartilage
cell sheet
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PCT/JP2023/020854
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Japanese (ja)
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正人 佐藤
匠 高橋
範行 妻木
恵利子 豊田
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学校法人東海大学
国立大学法人京都大学
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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
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material

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  • the present invention relates to a cell sheet for cartilage repair produced from pluripotent stem cell-derived chondrocytes.
  • Non-Patent Document 1 and Patent Document 1 describe a method in which cartilage particles are produced from pluripotent stem cells and transplanted to a defective site of articular cartilage, so that the cartilage particles combine with surrounding tissues and compensate for the defective articular cartilage.
  • a defective site of articular cartilage it is necessary to fix the surrounding area with fibrin glue, but because the fixing force is weak, there is a possibility that the cartilage particles will fall off after transplantation.
  • fixation is difficult, it is difficult to transplant for extensive and worn-out cartilage defects or degeneration, such as cartilage defects caused by osteoarthritis.
  • cartilage particles are expected to compensate for defective articular cartilage, and the cartilage particles themselves are expected to function as articular cartilage.
  • the mechanism of action of cartilage cell sheets is that they have a paracrine effect as well as an effect to cover the defect. It is considered to be the main.
  • the chondrocyte sheet does not have the characteristics of articular cartilage when transplanted, it is thought to activate cells on the host side and promote repair and regeneration of hyaline cartilage-like tissue similar to conventional articular cartilage.
  • Non-Patent Document 2 Non-Patent Document 3, Patent Document 2, and Patent Document 3 disclose a cartilage cell sheet using cartilage tissue derived from a living body such as a patient with polydactyly as a cell source, and a method for manufacturing the same. . It is also stated that such a chondrocyte sheet can be applied to knee osteoarthritis, and that its safety and effectiveness have been confirmed in clinical studies. However, when using chondrocyte sheets that use cartilage tissue derived from living bodies such as polydactyly patients as a cell source for allogeneic transplantation, there are issues such as a shortage of donors and the quality of the chondrocyte sheets varying depending on the characteristics of the donor cells.
  • pluripotent stem cells as a cell source, it is possible to stabilize the quality of the chondrocyte sheet, and since pluripotent stem cells can be expanded without limit, it is also possible to secure the required number of cells. It's easy.
  • An object of the present invention is to provide a chondrocyte sheet using pluripotent stem cells as a cell source.
  • the present inventors have proposed that in the conventional method for producing a chondrocyte sheet using cartilage tissue derived from a living body such as a patient with polydactyly as the cell source, polydactyly can be used instead of the living body-derived cartilage tissue as the cell source.
  • chondrocytes derived from pluripotent stem cells The present inventors have discovered that by culturing chondrocytes (hereinafter also referred to as induced chondrocytes) in a low serum medium, it is possible to produce a chondrocyte sheet suitable for the repair and regeneration of articular cartilage, and have completed the present invention.
  • the present invention includes the following aspects.
  • a cell sheet for cartilage repair is formed from a culture of induced chondrocytes derived from pluripotent stem cells, The cultured product is a cell sheet for cartilage repair, which is obtained by culturing the induced chondrocytes in a low serum medium.
  • the cell sheet for cartilage repair according to [1] which satisfies all of the following (1) to (3): (1) The amount of transforming growth factor- ⁇ 1 (TGF- ⁇ 1) secreted per unit area during 72 hours of culture is 3.0 ng/cm 2 or more. (2) The amount of melanoma inhibitory activity (MIA) secreted per unit area during 72 hours of culture is 10.0 ng/cm 2 or more.
  • TGF- ⁇ 1 transforming growth factor- ⁇ 1
  • MIA melanoma inhibitory activity
  • the ratio of the number of CD56 positive cells to the total number of cells contained in the cell sheet is 85% or more.
  • [4] The cell sheet for cartilage repair according to any one of [1] to [3], which satisfies all of the following (A) to (D): (A) Positive in immunostaining using an antibody against type I collagen. (B) Immunostaining using an antibody against type II collagen is negative. (C) Negative in Safranin O staining. (D) Positive in immunostaining using an antibody against aggrecan. [5] The cell sheet for cartilage repair according to any one of [1] to [4], wherein the serum concentration of the low serum medium is 5 v/v % or less. [6] The cell sheet for cartilage repair according to any one of [1] to [5], wherein the pluripotent stem cells are human-derived iPS cells.
  • the hypoxic conditions are conditions in which the oxygen concentration in the culture atmosphere is 5 v/v% or less.
  • a method for producing a cell sheet for cartilage repair comprising: A method comprising culturing induced chondrocytes derived from pluripotent stem cells in a low serum medium. [11] The method according to [10], wherein the serum concentration of the low serum medium is 5 v/v% or less. [12] [10] or [11], wherein the step of culturing the induced chondrocytes in a low serum medium includes the step of culturing the induced chondrocytes under conditions where the oxygen concentration in the culture atmosphere is 5 v/v% or less. Method described.
  • a method for culturing induced chondrocytes derived from pluripotent stem cells comprising: A method comprising culturing the induced chondrocytes in a low serum medium.
  • the step of culturing the induced chondrocytes in a low serum medium includes the step of culturing the induced chondrocytes under conditions where the oxygen concentration in the culture atmosphere is 5 v/v% or less.
  • a chondrocyte sheet suitable for repairing and regenerating articular cartilage can be produced using pluripotent stem cells as a cell source.
  • pluripotent stem cells as a cell source
  • using pluripotent stem cells as a cell source may reduce costs.
  • the chondrocyte sheet of the present invention has excellent adhesive properties and can be easily transplanted into extensive and worn cartilage defects or degeneration sites.
  • HE Hematoxylin and eosin staining
  • iPSC sheet D a chondrocyte sheet prepared using a DMEM/F12 medium containing 20 v/v% FBS and a chondrocyte sheet (iPSC sheet M) prepared by the method of the present invention
  • Photographs of safranin O staining (SafO), toluidine blue staining (TB), type I collagen immunostaining (COL1), type II collagen immunostaining (COLII), aggrecan staining (ACAN), and fibronectin staining (FN) Photographs of safranin O staining (SafO), toluidine blue staining (TB), type I collagen immunostaining (COL1), type II collagen immunostaining (COLII), aggrecan staining (ACAN), and fibronectin staining (FN) (photos in place of drawings) ).
  • the scale bar indicates 100 ⁇ m.
  • HE hematoxylin and eosin staining
  • TB toluidine blue staining
  • SafO safranin O staining
  • COL1 type I collagen immunostaining
  • COL2 type II collagen immunostaining
  • hVim human-specific vimentin staining.
  • the bottom row of iPSC sheet D and iPSC sheet M is an enlarged frame of the hVim photo.
  • Photographs after staining of knee joint tissue 12 weeks after transplantation in a heterogeneous orthotopic transplantation experiment using iPSC sheet D and iPSC sheet M.
  • HE hematoxylin and eosin staining
  • TB toluidine blue staining
  • SafO safranin O staining
  • COL1 type I collagen immunostaining
  • COL2 type II collagen immunostaining
  • hVim human-specific vimentin staining.
  • the bottom row of iPSC sheet D and iPSC sheet M is an enlarged frame of the hVim photo.
  • the numbers on each column indicate the average secreted amount (ng) per sheet (4.2 cm 2 ).
  • HE hematoxylin and eosin staining
  • TB toluidine blue staining
  • SafO safranin O staining
  • COL1 type I collagen immunostaining
  • COL2 type II collagen immunostaining
  • hVim human-specific vimentin staining.
  • the bottom row of PDC sheet and iPSC sheet D is an enlarged frame of the hVim photo.
  • FIG. 2 is a diagram showing the changes in temperature as a heat map.
  • GO Gene Ontology
  • v/v% means "volume percent”.
  • range of each numerical value in the present invention does not exclude that it is substantially within the numerical range, and may mean, for example, up to ⁇ 10% of the numerical value.
  • One embodiment of the method of the present invention is a method for producing a cell sheet for cartilage repair, which includes a step of culturing induced chondrocytes derived from pluripotent stem cells in a low serum medium.
  • the chondrocyte sheet produced by the method of the present invention may be a chondrocyte sheet suitable for cartilage repair and regeneration. Such a chondrocyte sheet is also referred to as a cartilage repair cell sheet. Further, the chondrocyte sheet produced by the method of the present invention may specifically be the chondrocyte sheet of the present invention described below.
  • Another embodiment of the method of the present invention is a method of culturing induced chondrocytes derived from pluripotent stem cells, which comprises a step of culturing the induced chondrocytes in a low serum medium.
  • Pluripotent stem cells refer to stem cells that have pluripotency that can differentiate into all cells existing in a living body and also have the ability to proliferate.
  • Pluripotent stem cells include, but are not particularly limited to, embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transfer, spermatogonial stem cells (GS cells), and embryonic germ cells (EG cells).
  • pluripotent cells pluripotent cells
  • iPS induced pluripotent stem
  • the pluripotent stem cells are preferably ES cells, ntES cells, and/or iPS cells, particularly preferably iPS cells.
  • pluripotent stem cells are not particularly limited as long as the effects of the present invention can be obtained, but mammals are preferred, including primates such as humans, rodents such as mice and rats, dogs, cats, rabbits, and cows. , a horse, a goat, a sheep, a pig, or a chicken, and even more preferably a human.
  • the pluripotent stem cells may be any combination of the above pluripotent stem cell examples and origins.
  • Pluripotent stem cells are preferably ES cells, ntES cells, and/or iPS cells derived from mammals, and more preferably from primates such as humans, rodents such as mice or rats, dogs, cats, and rabbits. , bovine, horse, goat, sheep, pig, or chicken-derived ES cells, ntES cells, and/or iPS cells, and even more preferably human-derived ES cells, ntES cells, and/or iPS cells. .
  • the pluripotent stem cells are iPS cells, they are preferably iPS cells derived from mammals, more preferably from primates such as humans, rodents such as mice or rats, dogs, cats, rabbits, etc. iPS cells derived from cows, horses, goats, sheep, pigs, or chickens, and even more preferably human iPS cells.
  • the initialization factors are, for example, Oct3/4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15.
  • Examples include genes or gene products such as -2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, or Glis1, and these reprogramming factors may be used alone or in combination. Good too.
  • Combinations of initialization factors include WO2007/069666, WO2008/118820, WO2009/007852, WO2009/032194, WO2009/058413, WO2009/057831, WO2009/075119, WO2009/079007, W O2009/091659, WO2009/101084, WO2009/ 101407, WO2009/102983, WO2009/114949, WO2009/117439, WO2009/126250, WO2009/126251, WO2009/126655, WO2009/157593, WO2010/009015, WO 2010/033906, WO2010/033920, WO2010/042800, WO2010/050626, WO2010/056831, WO2010/068955, WO2010/098419, WO2010/102267, WO2010/111409, WO2010/111422, WO2010/115050, WO2010/124290, WO2010/14 7395, WO2010/147612,
  • somatic cells used to obtain iPS cells are not particularly limited, and include, for example, fetal (child) somatic cells, newborn (child) somatic cells, and mature, healthy or diseased somatic cells. It may also include any of primary cultured cells, subcultured cells, and established cell lines.
  • somatic cells include (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells, (2) tissue progenitor cells, and (3) blood cells (peripheral stem cells).
  • lymphocytes epithelial cells, endothelial cells, muscle cells, fibroblasts (skin cells, etc.), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (pancreatic exocrine cells) etc.), differentiated cells such as brain cells, lung cells, kidney cells, and adipocytes, but are not limited to these.
  • Chondrocyte means a cell that produces extracellular matrix such as collagen that constitutes cartilage, or a precursor cell that becomes such a cell. Further, such chondrocytes may be cells expressing chondrocyte markers, and examples of chondrocyte markers include type II collagen (COL2A1) and SOX9.
  • “Induced chondrocytes” means chondrocytes obtained by being induced from undifferentiated cells. In this case, “undifferentiated cells” refer to cells that can differentiate into chondrocytes, and it is preferable to use pluripotent stem cells as the cell source. Moreover, "induced” may mean that differentiation is induced by any method.
  • induced chondrocytes derived from pluripotent stem cells may be chondrocytes obtained by inducing differentiation by any method using pluripotent stem cells as a cell source, and specifically, For example, they may be chondrocytes obtained by inducing differentiation from pluripotent stem cells in vitro. Any known method can be used to induce induced chondrocytes from pluripotent stem cells, for example, Yamashita, A. et al. Generation of Scaffoldless Hyaline Cartilaginous Tissue from Human iPSCs. Stem Cell Reports 4, 404 -418 (2015), WO2015/064754, or WO2016/133208.
  • the method of the present invention includes the step of culturing induced chondrocytes derived from pluripotent stem cells in a low serum medium.
  • the "low serum medium” refers to a conventional chondrocyte sheet manufacturing method, for example, Maehara, M. et al. Characterization of polydactyly-derived chondrocyte sheets versus adult chondrocyte sheets for articular cartilage repair. 22 (2017), Sato, M. et al. Combined surgery and chondrocyte cell-sheet transplantation improves clinical and structural outcomes in knee osteoarthritis.
  • npj Regenerative Medicine 4, 1-11 (2019), WO2006/093151, or JP 2020- Refers to a medium with a lower serum concentration compared to the medium for culturing chondrocytes described in No. 006207.
  • the serum concentration of the medium in the conventional chondrocyte sheet manufacturing method may typically be 20% v/v.
  • the serum concentration of the low serum medium is, for example, 10v/v% or less, 5v/v% or less, 4v/v% or less, 3v/v% or less, 2.5v/v% or less, 2.2v/v% or less, It may be 2v/v% or less, 0v/v% or more, 0.5v/v% or more, 1v/v% or more, 1.5v/v% or more, 1.8v/v% or more, 2v/v % or more, or a consistent combination thereof.
  • the serum concentration of the low serum medium in the present invention is, for example, 0 v/v% or more and 10 v/v% or less, 0.5 v/v% or more and 5 v/v% or less, and 0.5 v/v% or more and 2 .5v/v% or less, 0.5v/v% or more and 2v/v% or less, 1.5v/v% or more and 2.5v/v% or less, 1.8v/v% or more and 2.2v/v% or less, Or it may be 2v/v%.
  • the fact that the serum concentration of the low serum medium in the present invention is 2v/v% does not exclude an equivalent range in which the serum concentration is substantially 2v/v%. Such a range may be, for example, 1.8 v/v% or more and 2.2 v/v% or less, but is not limited to this.
  • serum examples include fetal bovine serum (FBS), human serum such as patient's own serum, horse serum, sheep serum, goat serum, pig serum, chicken serum, rat serum, mouse serum, etc. Although not limited to, FBS or patient autologous serum is preferred.
  • FBS fetal bovine serum
  • human serum such as patient's own serum, horse serum, sheep serum, goat serum, pig serum, chicken serum, rat serum, mouse serum, etc.
  • the low serum medium of the present invention is not particularly limited except for the serum concentration, and may be any known medium, but a medium suitable for mesenchymal stem cells is preferred.
  • media suitable for mesenchymal stem cells include, but are not limited to, MesenPRO and StemPRO.
  • the step of culturing the induced chondrocytes derived from the pluripotent stem cells of the present invention in a low serum medium includes the step of culturing the induced chondrocytes using a culture vessel whose surface is coated with a temperature-responsive polymer. may be included.
  • Temperature-responsive polymers include, but are not particularly limited to, poly-N-isopropylacrylamide.
  • the step of culturing the induced chondrocytes derived from the pluripotent stem cells of the present invention in a low serum medium involves culturing the induced chondrocytes under conditions where the culture atmosphere has a low oxygen concentration (i.e., under hypoxic conditions). It may include a step of. "Culture under hypoxic conditions” means that the oxygen concentration in the atmosphere when cells, particularly chondrocytes, are cultured is lower than the oxygen concentration at which the cells are normally cultured. "Oxygen concentration at which cells are normally cultured” is not uniquely determined as it varies depending on the cell and the species from which it is derived, but as an example, for general mammalian chondrocytes, it is 20v/v%.
  • hypoxic conditions may be conditions in which the oxygen concentration in the culture atmosphere is less than 20 v/v%.
  • the hypoxic conditions are preferably conditions in which the oxygen concentration in the culture atmosphere is 10 v/v% or less, more preferably 7 v/v% or less, and even more preferably 5 v/v%.
  • the conditions are as follows, particularly preferably 3v/v% or less, and even more preferably 2v/v% or less.
  • conditions where the oxygen concentration in the culture atmosphere is less than XXv/v% or XXv/v% or less means that the culture atmosphere does not contain oxygen at the lower limit, unless otherwise specified, that is, the oxygen concentration in the culture atmosphere is It refers to conditions where the oxygen concentration in the culture atmosphere is 0 v/v% or more, including cases where the concentration is 0%.
  • the lower limit of the oxygen concentration in the culture atmosphere under hypoxic conditions is not particularly limited as long as it does not contradict the above range, but for example, the oxygen concentration in the culture atmosphere is 0 v/v% or more, 0.5 v/v% or more. , or 1v/v% or more.
  • hypoxic condition may be a combination within the above range, and specifically, for example, 0.5 v/v% or more and 10 v/v% or less, 0.5 v/v% or more and 5 v/v% Below, it may be 0.5 v/v% or more and 3 v/v% or less, 0.5 v/v% or more and 2 v/v% or less, or 1 v/v% or more and 5 v/v% or less.
  • the culturing period is not particularly limited as long as the chondrocyte sheet of the present invention can be obtained, but for example, 7 days. It may be more than 21 days.
  • the number of cells to be seeded at the start of culture is not particularly limited as long as the chondrocyte sheet of the present invention can be obtained.
  • it may be 1000 cells/ cm2 or more, 10000 cells/cm2 or more, 30000 cells/cm2 or more , or 50000 cells/cm2 or more, and 1000000 cells/cm2 or less, 100000 cells/ cm2 or less, It may be 70,000 cells/cm 2 or less, or 50,000 cells/cm 2 or less.
  • the chondrocyte sheet of the present invention is formed from a culture of induced chondrocytes derived from pluripotent stem cells, and the culture is formed by culturing the induced chondrocytes in a low serum medium. This is a chondrocyte sheet obtained by culturing.
  • the chondrocyte sheet of the present invention is suitable for cartilage repair and regeneration. Therefore, the chondrocyte sheet of the present invention may be a cell sheet for cartilage repair.
  • the pluripotent stem cells and the induced chondrocytes derived from the pluripotent stem cells are as described above.
  • the chondrocyte sheet of the present invention has the following characteristics: "transforming growth factor- ⁇ 1 (TGF- ⁇ 1) secretion amount”, “melanoma inhibitory activity (MIA) secretion amount", and "CD56 per total number of cells included in the cell sheet”. It may be characterized by one or more selected from the group consisting of "percentage of positive cells”.
  • TGF- ⁇ 1 is a growth factor involved in the proliferation and differentiation of articular cartilage, and may affect the therapeutic effects brought about by chondrocyte sheets.
  • MIA is also known as a cartilage-derived retinoic acid-sensitive protein, and it has been reported that chondrocytes specifically express MIA and that MIA has an anabolic effect on chondrocytes.
  • CD56 is a glycoprotein on the cell surface known as neural cell adhesion molecule (N-CAM) or neural cell adhesion molecule. It is known that natural killer cells, nerve cells, skeletal muscle cells, etc. express it, but it has also been reported that it is expressed in some bone marrow-derived mesenchymal stem cells.
  • N-CAM neural cell adhesion molecule
  • the secretion amount of TGF- ⁇ 1 and the secretion amount of MIA are, for example, the amount of TGF- ⁇ 1 protein per unit area secreted by the chondrocyte sheet into the medium during a certain culture period when a chondrocyte sheet is cultured, and the amount of MIA protein.
  • the culture conditions are not particularly limited as long as they are the culture conditions normally used when measuring the secretion amount.
  • a medium may be used, it is preferable to use a DMEM/F12 medium containing 1% FBS.
  • Other culture conditions may also be those commonly used when measuring the amount of secretion.
  • the temperature is 34°C or more and 40°C or less, preferably 35°C or more and 39°C or less, more preferably 36°C or more and 38°C or less, and the carbon dioxide (CO 2 ) concentration is 0v/v% or more and 10v/v.
  • the oxygen concentration is preferably 3 v/v% or more and 7 v/v% or less, and the oxygen concentration is 0 v/v% or more and 25 v/v% or less, preferably 15 v/v% or more and 25 v/v% or less.
  • the culture time may be, for example, 1 hour, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, or 96 hours, and the upper and lower limits of these may be consistent. You may choose a combination.
  • the culture time may be the culture time when the time when the medium for measuring the secretion amount is applied to the chondrocyte sheet is set to 0. For example, "72 hours of culture” in this specification is , is "72 hours of culture" when the time is set to 0.
  • the chondrocyte sheet of the present invention may have a secretion amount of TGF- ⁇ 1 per unit area after 72 hours of culture, for example, of 2.0 ng/cm 2 or more, and 3.0 ng/cm 2 It is preferably at least 3.5 ng/cm 2 , more preferably at least 3.5 ng/cm 2 .
  • the chondrocyte sheet of the present invention may have an MIA secretion amount per unit area of 6.0 ng/cm 2 or more, for example, 10.0 ng/cm 2 after 72 hours of culture. It is preferably at least 12.5 ng/cm 2 , more preferably 12.5 ng/cm 2 .
  • a “CD56 positive cell” is a cell that expresses CD56, such as a cell that expresses CD56 on the cell surface, a cell that expresses CD56 inside the cell, or a cell that expresses CD56 on both the cell surface and inside the cell.
  • the cell expresses CD56 at least on the cell surface.
  • “Expressing CD56” may mean the presence of CD56 protein on the cell surface, or the presence of CD56 protein or mRNA inside the cell. Therefore, a method for detecting CD56-positive cells may be a method of detecting cells in which CD56-positive cells are present by detecting CD56 protein on the cell surface or CD56 protein or mRNA inside the cell.
  • the method may be to detect cells in which CD56 protein is present on the cell surface by detecting CD56 protein.
  • a method for detecting CD56 protein on the cell surface for example, an antibody that specifically binds to CD56 protein can be used.
  • a method for measuring CD56-positive cells is to use a fluorescently labeled antibody that specifically binds to the CD56 protein to calculate the number of cells that have the CD56 protein on the cell surface by flow cytometry. Examples include measurement methods.
  • the ratio of the number of CD56-positive cells to the total number of cells contained in the cell sheet may be 80% or more, preferably 85% or more, More preferably, it is 90% or more.
  • the chondrocyte sheet of the present invention has "TGF- ⁇ 1 secretion amount”, “MIA secretion amount”, and "ratio of the number of CD56-positive cells to the total number of cells contained in the cell sheet" within the above ranges. Any combination may be used. Specifically, the chondrocyte sheet of the present invention may satisfy all of the following (1) to (3), for example: (1) The amount of Transforming growth factor- ⁇ 1 (TGF- ⁇ 1) secreted per unit area during 72 hours of culture is 2.0 ng/cm 2 or more, preferably 3.0 ng/cm 2 or more, and more preferably It is 3.5 ng/cm 2 or more.
  • the amount of melanoma inhibitory activity (MIA) secreted per unit area during 72 hours of culture is 6.0 ng/cm 2 or more, preferably 10.0 ng/cm 2 or more, and more preferably 12.5 ng/cm 2 or more. cm2 .
  • the ratio of CD56-positive cells to all cells contained in the cell sheet is 80% or more, preferably 85% or more, and more preferably 90% or more.
  • the chondrocyte sheet of the present invention may further exhibit significantly higher COL2A1 gene expression compared to conventional chondrocyte sheets.
  • the chondrocyte sheet of the present invention has a COL2A1 gene expression level of 100 or more, more preferably a COL2A1 gene expression level of 1000 or more, when the expression level of the COL2A1 gene in conventional chondrocyte sheets is 1. It is.
  • Conventional chondrocyte sheets include, for example, a chondrocyte sheet derived from cartilage tissue of a human polydactyly patient; one example is the chondrocyte sheet derived from cartilage tissue of a human polydactyly patient described in JP-A No. 2020-006207. can be mentioned.
  • the chondrocyte sheet of the present invention may exhibit significantly higher COL2A1 gene expression compared to a chondrocyte sheet derived from cartilage tissue of a human polydactyly patient, and preferably, the chondrocyte sheet of the present invention
  • the expression level of the COL2A1 gene in the cartilage tissue-derived chondrocyte sheet is defined as 1, the expression level of the COL2A1 gene is 100 or more, more preferably 1000 or more.
  • the chondrocyte sheet of the present invention may further satisfy one or more of the following (A) to (D), and preferably satisfies all of the following (A) to (D): (A) Positive in immunostaining using an antibody against type I collagen. (B) Immunostaining using an antibody against type II collagen is negative. (C) Negative in Safranin O staining. (D) Positive in immunostaining using an antibody against aggrecan.
  • Immunostaining using antibodies refers to an antibody against a target protein, such as an antibody against type I collagen, an antibody against type II collagen, or an antibody against aggrecan, and if necessary, a secondary antibody that recognizes the antibody, an antibody ( Refers to the detection of cells containing target proteins that may be contained in cell sheets, tissues, etc. using molecules that label secondary antibodies (including secondary antibodies, etc.).
  • "positive in immunostaining” means that cells containing the target protein are detected in a cell sheet or tissue, and specifically, for example, all cells contained in a cell sheet or tissue, etc. 90% or more of the cells may be detected as cells containing the target protein.
  • negative in immunostaining refers to cells containing a target protein detected in a cell sheet or tissue below a certain level, or not detected. 10% or less of all cells contained in a tissue or the like may be detected as cells containing the target protein, but this also includes a case of 0%, that is, a case where no cells containing the target protein are detected.
  • Type I collagen is a collagen found in scar tissue, tendons, ligaments, endomysium of myofibrils, bone, dermis, dentin, and degenerated cartilage, and is expressed from the COL1A gene.
  • the chondrocyte sheet of the present invention is preferably positive in immunostaining using an antibody against type I collagen, especially during culture, but after transplantation into a living body, part or all of the transplanted sheet and its surrounding tissues. It may disappear.
  • Type II collagen is found in the cornea, vitreous body, and cartilage, is the main collagen that makes up articular cartilage, and is expressed from the COL2A gene.
  • the chondrocyte sheet of the present invention is preferably negative in immunostaining using an antibody against type II collagen, especially during culture, but after transplantation into a living body, part or all of the transplanted sheet and its surrounding tissues. May appear.
  • Safranin O is a dye that stains cell nuclei, and is known to also stain cartilage.
  • Safranin O staining can be performed by any known method, but specifically, for example, first treated with hematoxin, immersed in a fast green aqueous solution, treated with an acetic acid aqueous solution, and then immersed in a safranin O aqueous solution. It may be implemented by doing so.
  • the chondrocyte sheet of the present invention is preferably negative in Safranin O staining.
  • Aggrecan has a core protein consisting of three globular domains (G1, G2, and G3) and a glycosaminoglycan (keratan sulfate or chondroitin sulfate) binding region between G2 and G3, and has a molecular weight of approximately 2500 kDa. / chondroitin sulfate proteoglycan.
  • Aggrecan is present in cartilage tissue and is known to contribute to the physical strength and elasticity of cartilage tissue.
  • the chondrocyte sheet of the present invention is preferably positive in immunostaining using an antibody against aggrecan.
  • the chondrocyte sheet of the present invention includes CHI3L1, STEAP4, CEMIP, METTL7A, DPP4, CLGN, NDUFV3, ISM1, GPNMB, SCARA3, NUBPL, TNFAIP8, ABCC3, RGCC, DAPK2, LRRN4CL, EEPD1, C1QTNF1, PPM1H, SLC12A7, GMPR, High levels of one or more proteins selected from FHIT, PLXDC1, GLB1L, MMP13, TMEM119, SLC9A9, MATN2, CHST2, TLR2, CLU, VSNL1, TWIST1, FMNL2, CHCHD5, FABP4, CTSK, MCC, ABCA1, MID1, and STRIP2.
  • the protein is characterized by low expression of the above proteins.
  • the chondrocyte sheet of the present invention supports epithelial-mesenchymal transition in colorectal cancer, lipid transport, regulation of hormone levels, positive regulation of cell death, cellular response to tumor necrosis factor, positive regulation of secretion by cells, and angiogenesis.
  • IGF insulin-like growth factors
  • IGFBPs insulin-like growth factor binding proteins
  • the chondrocyte sheet of the present invention is characterized by low expression of proteins related to IGF transport regulation and uptake by IGFBPs, and/or hormonal responses, and/or extracellular matrix tissue, NABA matrisome-associated proteins, bone formation, and It is preferable that a protein associated with the positive regulation of protein phosphorylation/or have characteristics of both high expression and low expression.
  • the chondrocyte sheet of the present invention may be used to treat damaged or degenerated cartilage.
  • a method of treating cartilage for example, transplantation into damaged or degenerated cartilage can be considered.
  • the chondrocyte sheet of the present invention has a sheet-like shape and exhibits excellent adhesion and cartilage tissue repair/regeneration, so it can be easily transplanted into cartilage that has been extensively worn out, such as in cases of osteoarthritis. Yes, and we can also expect treatment for such cartilage.
  • Cartilage treatment using the chondrocyte sheet of the present invention may be performed not only by the chondrocyte sheet itself filling the damaged area, but also by promoting repair and regeneration of surrounding cartilage tissue and bone tissue.
  • the chondrocyte sheet of the present invention can be used to treat one or more diseases selected from the group consisting of traumatic cartilage damage, osteochondral damage, osteochondritis dissecans, meniscal damage, intervertebral disc degeneration, and osteoarthritis. May be used therapeutically.
  • the present invention has great significance as a treatment method for knee osteoarthritis for which no radical treatment has been established.
  • Example 1 Induction of differentiation of chondrocytes from pluripotent stem cells was carried out using the QHJI 01s04 strain, which is an iPS cell derived from human peripheral blood, established in the iPS cell stock project of the Kyoto University iPS Cell Research Institute. It was carried out according to the method described in WO2015/064754. Specifically, the iPS cells were cultured in a feeder-free state in a culture dish coated with Matrigel and maintained in Essential 8 (Invitrogen) medium.
  • DMEM/F12 medium containing 10 ng/ml Wnt3a (R&D), 10 ng/ml Activin A (R&D), 1% ITS (Invitrogen), and 1% FBS.
  • cartilage masses in suspension culture are also called cartilage particles.
  • induced chondrocytes derived from pluripotent stem cells contained in the cartilage particles are also simply referred to as induced chondrocytes.
  • Example 2 Preparation of chondrocyte sheet
  • the cartilage particles (wet weight 1-4 g) cultured for 19 weeks were dispersed in DMEM/F12 medium containing 30 ml of 20% FBS containing 5 mg of Liberase (Roche) and 1% antibiotic.
  • the mixture was dispersed using a stirrer bar at a rotation speed of 60 rpm for 2 and a half hours in an incubator at 37° C. and 5% CO 2 .
  • the medium containing the dispersed chondrocytes was passed through a 40 ⁇ m cell strainer, washed with DPBS, and centrifuged at 1500 rpm for 5 minutes at room temperature.
  • the supernatant was removed with an aspirator, washed with 35 ml of basal medium, and centrifuged at 1500 rpm for 5 minutes at room temperature.
  • the supernatant was removed with an aspirator, washed with 10 ml of basal medium, and centrifuged at 1500 rpm for 5 minutes at room temperature.
  • the supernatant was removed with an aspirator and suspended in 5 ml of MesenPRO medium (containing 2 v/v% fetal bovine serum (FBS); supplemented with 1:100 GlutaMAX and 1% antibiotic). A portion of the cell suspension was mixed 1:1 with trypan blue, and the cell number was determined using a Countess.
  • a 6-well plate for inserts (Falcon) and a temperature-responsive culture insert (UpCell insert, Cell Seed) as culture equipment for producing cell sheets, and add 3 ml of MesenPRO medium to each well of the 6-well plate. and the insert was installed. Based on the measured cell number, the cells were suspended in 2 ml of MesenPRO medium and seeded at approximately 50,000 cells/cm 2 in an insert. That is, approximately 210,000 cells were suspended in 2 ml of MesenPRO medium and seeded onto the insert. Thereafter, the cells were cultured for 14 days in an incubator at 37° C., 5% CO 2 and 2% O 2 , and the medium was replaced every other day. The chondrocyte sheet thus obtained is called iPSC sheet M. Furthermore, a chondrocyte sheet was prepared by the same method and named iPSC sheet D, except that DMEM/F12 medium containing 20 v/v% FBS was used instead of MesenPRO medium.
  • Eosin solution Prepare an eosin solution by mixing 30 mL of pure eosin solution (3204-2, Muto Chemical) and 120 mL of 95% ethanol. When used in staining, the eosin solution is diluted five times with 95% ethanol, and the diluted solution is used as the eosin solution.
  • Fast Green aqueous solution 80 mg of Fast Green (1A304, CHROMA) is dissolved in 100 mL of ion-exchanged water, and the solution is filtered to obtain a 0.08% by mass Fast Green aqueous solution.
  • Safranin O aqueous solution 100 mg of Safranin O (1B463, CHROMA) is dissolved in 100 mL of ion exchange water to obtain a 0.1% by mass Safranin O aqueous solution.
  • Citric acid buffer Dissolve citric acid monohydrate in ion exchange water to obtain a 0.01M citric acid aqueous solution (hereinafter referred to as solution A).
  • Trisodium citrate dihydrate is dissolved in ion-exchanged water to obtain a 0.1M aqueous sodium citrate solution (hereinafter referred to as liquid B).
  • liquid B a 0.1M aqueous sodium citrate solution
  • 95 mL of solution A and 415 mL of solution B are combined and stirred, and the pH is adjusted to 6.0 with 1N NaOH to obtain a 0.01M citrate buffer.
  • Example 4 Method for preparing frozen sections for histological analysis
  • Cell sheets were fixed with 4% paraformaldehyde solution and prepared using Tissue Tech O. C. T. It was embedded in compound (4583, Sakura Finetech Japan Co., Ltd.) and frozen sections were prepared.
  • the frozen section of the cell sheet was sliced perpendicularly to the surface that was in contact with the temperature-responsive culture dish.
  • the thickness of the sliced section was 20 ⁇ m when subjected to immunostaining, and 10 ⁇ m when subjected to staining other than immunostaining.
  • the frozen sections were subjected to the staining described below.
  • Example 5 Hematoxylin and eosin staining of cell sheets1. Pretreatment The sections were immersed once in 100% ethanol for 10 minutes. 2. Water washing treatment 1. The treated sections were washed with water. 3. Immersion in ion-exchanged water 2. above. After washing with water, the sections were immersed in ion-exchanged water for 5 minutes. 4. Hematoxylin treatment 3 above. After immersion, the section was immersed in the hematoxylin aqueous solution for 3 to 4 minutes. 5. Water washing treatment 4 above. After the treatment, the sections were washed in hot water (50°C) for 3-4 minutes. 6. Eosin treatment 5. After washing, the sections were immersed in the eosin solution for 10 minutes. 7.
  • Example 6 Safranin O staining of cell sheets 1. Pretreatment The sections were immersed once in 100% ethanol for 10 minutes. 2. Water washing treatment 1. The treated sections were washed with water. 3. Immersion in ion-exchanged water 2. above. After washing with water, the sections were immersed in ion-exchanged water for 5 minutes. 4. Hematoxylin treatment 3 above. After immersion, the section was immersed in the hematoxylin aqueous solution for 4 to 5 seconds. 5. Water washing treatment 4 above. After the treatment, the sections were washed in hot water (50°C) for 3-4 minutes. 6. Fast green processing 5. above. After washing, the section was immersed in the 0.08% by mass fast green aqueous solution for 10 minutes. 7.
  • Acetic acid treatment 6 above. After treatment, the sections were treated once or twice with a 1% by volume acetic acid aqueous solution. 8. Safranin treatment 7 above. After the treatment, the section was immersed in the 0.1% by mass safranin O aqueous solution for 10 minutes. 9. Dehydration, Clearing, and Encapsulation These treatments were performed as described in "Hematoxylin and eosin staining of cell sheets" above.
  • Example 7 Toluidine blue staining of cell sheets 1. Pretreatment The sections were immersed once in 100% ethanol for 10 minutes. 2. Water washing treatment 1. The treated sections were washed with water. 3. Immersion in ion-exchanged water 2. above. After washing with water, the sections were immersed in ion-exchanged water for 5 minutes. 4. Hematoxylin treatment 3 above. After immersion, the section was immersed in the hematoxylin aqueous solution for 3 to 4 minutes. 5. Water washing treatment 4 above. After the treatment, the sections were washed in hot water (50°C) for 3-4 minutes. 6. Toluidine blue treatment 5. above.
  • Example 8 Aggrecan staining of cell sheets 1.
  • Pretreatment Sections were immersed in 100% ethanol for 3 times for 5 minutes. Next, the sections were immersed once in 70% ethanol for 5 minutes. 2.
  • Water washing treatment 1. The treated sections were washed with water. 3. Immersion in ion-exchanged water 2. above. After washing with water, the sections were immersed in ion-exchanged water for 5 minutes. 4.
  • Antigen retrieval treatment 3. above. After immersion, the section was immersed in the citrate buffer at 98° C. for 10 minutes. 5.
  • Fibronectin Staining of Cell Sheets Aggrecan staining was performed in the same manner except for changing the reagents used in "10. Primary antibody reaction” and "12. Secondary antibody reaction” in "Aggrecan staining of cell sheets” above.
  • the antibody used in the primary antibody reaction was Anti-Fibronectin mIgG (MAB1940, Chemicon, diluted to 0.2 ⁇ g/mL with 1% Goat Normal Serum).
  • the reagent used in the secondary antibody reaction was F(ab')2-Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (Thermo Fisher Scientific Inc. ., A-11017).
  • Type I collagen staining of cell sheets Type II collagen staining was carried out using the same method except for changing the reagents used in “10. Primary antibody reaction” and “12. Secondary antibody reaction” in “Aggrecan staining of cell sheets” above. It was conducted.
  • the antibody used in the primary antibody reaction was an antibody against type I collagen (1310-01, SouthernBiotech, 1:100 dilution in 0.01M PBS).
  • the reagent used in the secondary antibody reaction was ImmPRESS Polymer Anti-Goat IgG Reagent (MP-7405, Vector Laboratories).
  • Type II collagen staining of cell sheets Type II collagen staining was performed using the same method except for changing the reagents used in “10. Primary antibody reaction” and “12. Secondary antibody reaction” in “Aggrecan staining of cell sheets” above. It was conducted.
  • the antibody used in the primary antibody reaction was type II collagen primary antibody (Kyowa Pharma Chemical Co., Ltd., diluted 1:100 with 0.01M PBS).
  • the reagent used in the secondary antibody reaction was ImmPRESS Polymer Anti-Mouse IgG Reagent (MP-7402, Vector Laboratories).
  • Example 12 Heterogeneous orthotopic transplantation experiment
  • the cell sheet was allowed to stand at room temperature for 30 minutes, then peeled off using a polyvinylidene fluoride (PVDF) membrane, and used in a xenogeneic orthotopic transplantation experiment.
  • PVDF polyvinylidene fluoride
  • Twelve-week-old nude rats F334/NJcl-rnu/rnu, Claire
  • a full-thickness cartilage defect (diameter 2 mm, depth 1 mm) was created in the articular cartilage of one knee, and half of the cell sheet was transplanted to cover the defect.
  • Knee joint tissues were collected 4 and 12 weeks after transplantation, and histological evaluation of the articular cartilage tissue was performed. Histological evaluation was performed by hematoxylin and eosin staining, toluidine blue staining, safranin O staining, type I collagen immunostaining, type II collagen immunostaining, and human-specific vimentin staining.
  • Example 13 Method for preparing sections for histological evaluation Cartilage sections were fixed by immersion in 20% formalin and embedded in paraffin. Embedding was performed using Histoprep 586 (415-25791, Wako Pure Chemical Industries, Ltd.) as an embedding agent and an embedding console system (Tissue-Tek, Sakura Fine Tech Japan Ltd.). The embedded samples were sectioned vertically while in the upright position. The thickness of the sliced section was 3 ⁇ m. The thinly sliced sections were placed on glass slides and subjected to the staining described below.
  • Example 14 Hematoxylin and eosin staining of cartilage1. Deparaffinization The sections were immersed in xylene for 3 times for 5 minutes. Next, the sections were immersed in 100% ethanol for 5 minutes three times. Next, the sections were immersed once in 70% ethanol for 5 minutes. 2. Water washing treatment, immersion in ion-exchanged water, hematoxylin treatment, water washing treatment, eosin treatment, dehydration, clearing, and encapsulation These treatments were performed as described in "Hematoxylin and eosin staining of cell sheets" above.
  • Toluidine blue staining of cartilage Except for the fact that "1. Deparaffin treatment” described in “Hematoxylin and eosin staining of cartilage” above was performed instead of "1. Pretreatment” in “Toluidine blue staining of cell sheets” above. The staining was performed in the same manner as in the above “Toluidine blue staining of cell sheets”.
  • Type I collagen staining of cartilage In place of "1. Pretreatment” in the above “Type I collagen staining of cell sheets", "1. Deparaffin treatment” described in the above “Hematoxylin and eosin staining of cartilage” was performed. Staining was performed in the same manner as in the above-mentioned "type I collagen staining of cell sheet” except for this.
  • Type II collagen staining of cartilage Instead of “1. Pretreatment” in “Type II collagen staining of cell sheets” above, “1. Deparaffin treatment” described in “Hematoxylin and eosin staining of cartilage” above was performed. Staining was performed in the same manner as in the above-mentioned "Type II collagen staining of cell sheets” except for this.
  • Example 15 hVimentin staining of cartilage1.
  • Deparaffinization treatment Deparaffinization treatment was performed as described in "Hematoxylin and eosin staining of cartilage" above. 2. Water washing treatment 1. After deparaffinization, the sections were washed with water. 3. Immersion in ion-exchanged water 2. above. After washing with water, the sections were immersed in ion-exchanged water for 5 minutes. 4. Antigen retrieval 3. above. After immersion, the section was immersed in the citrate buffer at 98° C. for 10 minutes. 5. Cooling 4 above. After processing, the sections were cooled on the bench for 30 minutes. 6. Blocking with serum 5. above.
  • Example 16 Measurement of cell surface marker CD56 Cells were isolated from the chondrocyte sheet obtained by the production of the chondrocyte sheet, single staining of the cell surface marker CD56 was performed, and the CD56-positive cell rate was determined by flow cytometry. .
  • Example 17 Measurement of protein secretion amount To the chondrocyte sheet obtained by producing the chondrocyte sheet, add 2 ml of 1% FBS and 1% antibiotic to the cultured insert and 3 ml of 1% FBS and 1% antibiotic to the culture dish in which the insert is placed. DMEM/F12 medium containing TGF-F12 was added and cultured for 72 hours at 37°C, 5% CO 2 and normoxic conditions (i.e., 72 hours of culture), and then 2 ml of the medium in the insert was collected and TGF- ⁇ 1 and MIA protein concentrations were quantified.
  • the prepared chondrocyte cell sheets iPSC sheet M and iPSC sheet D were subjected to hematoxylin and eosin staining (HE), safranin O staining (SafO), toluidine blue staining (TB), type I collagen immunostaining (COL1), and type II collagen immunostaining. (COLII), aggrecan staining (ACAN), and fibronectin staining (FN).
  • HE hematoxylin and eosin staining
  • SafO safranin O staining
  • TB toluidine blue staining
  • COL1 type I collagen immunostaining
  • FN fibronectin staining
  • iPSC sheet M and iPSC sheet D were positive in immunostaining using an antibody against type I collagen, negative in immunostaining using an antibody against type II collagen, negative in safranin O staining, and negative in immunostaining using an antibody against aggrecan. It was positive in immunostaining. This suggests that a chondrocyte sheet derived from pluripotent stem cells may also have a cartilage repair effect, as disclosed in JP-A-2020-006207.
  • a heterogeneous orthotopic transplantation experiment was performed using iPSC sheet M and iPSC sheet D.
  • the results 4 weeks after transplantation are shown in FIG. 2, and the results 12 weeks after transplantation are shown in FIG. 3.
  • safranin O staining and toluidine blue metachromaticity were not confirmed, and in the slightly regenerated tissue, type I collagen staining was mainly confirmed, and type II collagen staining was not confirmed.
  • Ta In the group in which iPSC sheet D produced by the conventional manufacturing method was transplanted, weak safranin O staining and weak toluidine blue discoloration were confirmed in the repaired and regenerated articular cartilage tissue. In addition, strong staining of type I collagen was confirmed, and slight staining of type II collagen was confirmed.
  • the iPSC sheet M produced by the production method of the present invention was transplanted, strong safranin O staining and strong toluidine blue discoloration were confirmed in the repaired and regenerated articular cartilage tissue. Furthermore, type I collagen staining was confirmed only in the surface layer, and strong type II collagen staining was confirmed. As a result of human-specific vimentin staining, it was confirmed that the repaired and regenerated articular cartilage site was mainly composed of transplanted human cells, and the iPSC sheet group D had a relatively high density of chondrocytes after 4 weeks.
  • the density of chondrocytes was mixed in some areas with relatively high and low densities, whereas in the iPSC sheet M group, the density of chondrocytes was relatively low, confirming that cell lacunae were beginning to form. It was done. This suggests that the iPSC sheet M produced by the production method of the present invention is suitable for repairing and regenerating cartilage.
  • iPSC sheet M and iPSC sheet D derived from pluripotent stem cells, and chondrocyte sheet derived from polydactyly patient cartilage tissue (hereinafter also referred to as PDC sheet) produced by the method described in JP 2020-006207A
  • PDC sheet polydactyly patient cartilage tissue
  • the iPSC sheet M has a CD56 positive cell rate of 90.3 to 96.3%, and a secreted amount of TGF- ⁇ 1 of 15.2 to 40 ng or more per sheet (4.2 cm 2 ) (that is, 3.62 to 9.2 ng).
  • the expression level of the COL2A1 gene was about 1000 times that of PDC, and compared with iPSC sheet D and PDC, the CD56-positive cell rate, the secretion amount of TGF- ⁇ 1 and MIA, and the gene expression of COL2A1 were significantly higher.
  • the iPSC sheet D and PDC sheet were evaluated only 4 weeks after transplantation in a heterogeneous orthotopic transplantation experiment. The results are shown in FIG.
  • the results are shown in FIG.
  • strong safranin O staining and strong toluidine blue metachromaticity were confirmed in the repaired and regenerated articular cartilage tissue.
  • type I collagen staining was confirmed only in the surface layer, and strong type II collagen staining was confirmed.
  • human-specific vimentin staining it was confirmed that the repaired and regenerated articular cartilage site was mainly composed of transplanted human cells, and the density of chondrocytes was relatively low, and the formation of many cell lacunae was confirmed.
  • Example 18 Furthermore, differentially expressed proteins (DEPs) between iPSC sheet M and iPSC sheet D were analyzed by data independent analysis (DIA)-mass spectrometry (MS) proteomics. 41 proteins were identified as proteins with increased expression in iPSC sheet M compared to iPSC sheet D, and 20 proteins were identified as proteins with decreased expression in iPSC sheet M compared to iPSC sheet D. . The names of these 61 proteins and their quantitative changes are shown in a heat map in FIG. It is suggested that the increase or decrease in the expression levels of these proteins is characteristic of iPSC sheet M, which is an example of the cell sheet for cartilage repair of the present invention.
  • DIA data independent analysis
  • MS mass spectrometry
  • GO gene ontology
  • the chondrocyte sheet derived from pluripotent stem cells produced by the conventional method is not suitable for cartilage repair/regeneration, but the method of the present invention overcomes this drawback, and the chondrocyte sheet derived from pluripotent stem cells It has been revealed that the chondrocyte cell sheet is suitable for cartilage repair and regeneration.
  • a chondrocyte sheet suitable for repairing and regenerating articular cartilage can be produced using pluripotent stem cells as a cell source.
  • pluripotent stem cells as a cell source
  • using pluripotent stem cells as a cell source may reduce costs.
  • the chondrocyte sheet of the present invention has excellent adhesive properties and can be easily transplanted into extensive and worn cartilage defects or degeneration sites.

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Abstract

La présente invention a pour but de procurer un feuillet cellulaire de cartilage obtenu à l'aide de cellules souches pluripotentes en tant que source cellulaire. La solution selon l'invention consiste en un feuillet cellulaire réparateur de cartilage constitué d'un produit de culture de cellules cartilagineuses induites à partir de cellules souches pluripotentes. Le produit cultivé est obtenu en cultivant les cellules cartilagineuses induites dans un milieu à faible teneur en sérum.
PCT/JP2023/020854 2022-06-03 2023-06-05 Feuillet cellulaire réparateur de cartilage et son procédé de fabrication WO2023234421A1 (fr)

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Citations (4)

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WO2006093151A1 (fr) * 2005-02-28 2006-09-08 Cellseed Inc. Feuille de cellules cultivees, son procede de production et son procede d’application
WO2018154813A1 (fr) * 2017-02-24 2018-08-30 株式会社セルシード Feuille cellulaire cultivée pour régénération tissulaire, son procédé de production et son utilisation
WO2021182633A1 (fr) * 2020-03-13 2021-09-16 学校法人東海大学 Culture de cellules, procédé d'évaluation de la culture de cellules, procédé de production de la culture de cellules, et marqueur à utiliser dans l'évaluation de la propriété de formation du tissu chondroïde
JP2022042998A (ja) * 2020-09-03 2022-03-15 学校法人東海大学 細胞シート小片、該細胞シート小片を収容した注射器、該細胞シート小片の製造方法及びその使用方法

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WO2006093151A1 (fr) * 2005-02-28 2006-09-08 Cellseed Inc. Feuille de cellules cultivees, son procede de production et son procede d’application
WO2018154813A1 (fr) * 2017-02-24 2018-08-30 株式会社セルシード Feuille cellulaire cultivée pour régénération tissulaire, son procédé de production et son utilisation
WO2021182633A1 (fr) * 2020-03-13 2021-09-16 学校法人東海大学 Culture de cellules, procédé d'évaluation de la culture de cellules, procédé de production de la culture de cellules, et marqueur à utiliser dans l'évaluation de la propriété de formation du tissu chondroïde
JP2022042998A (ja) * 2020-09-03 2022-03-15 学校法人東海大学 細胞シート小片、該細胞シート小片を収容した注射器、該細胞シート小片の製造方法及びその使用方法

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