WO2023234421A1 - Cartilage-repairing cell sheet and method for producing same - Google Patents

Cartilage-repairing cell sheet and method for producing same 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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French (fr)
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

The present invention addresses the problem of providing a cartilage cell sheet obtained using pluripotent stem cells as a cell source. Said problem is solved by a cartilage-repairing cell sheet that is formed from a cultured product of induced cartilage cells that have been induced from pluripotent stem cells. The cultured product is obtained by culturing the induced cartilage cells in a low serum medium.

Description

軟骨修復用細胞シートおよびその製造方法Cell sheet for cartilage repair and its manufacturing method
　本発明は、多能性幹細胞由来軟骨細胞より作製した軟骨修復用細胞シートに関する。 The present invention relates to a cell sheet for cartilage repair produced from pluripotent stem cell-derived chondrocytes.
　非特許文献１および特許文献１には、多能性幹細胞より軟骨パーティクルを作製し、関節軟骨の欠損部位へ移植することで軟骨パーティクルが周囲の組織と結合し、欠損した関節軟骨を補う方法が記載されている。軟骨パーティクルを関節軟骨の欠損部位へ移植する場合はフィブリン糊などで周囲に固定する必要があるが、固定力が弱いため移植後に軟骨パーティクルが脱落する可能性がある。また、固定が困難なため変形性関節症の軟骨欠損のような広範ですり減った軟骨欠損や変性に対しての移植は難しい。そのため、広範な欠損や変性に対しての移植が可能な、接着性に優れる軟骨細胞シートに対する需要が存在する。また、軟骨パーティクルは欠損した関節軟骨を補い、軟骨パーティクルそのものが関節軟骨としての機能を果たすことが期待される一方、軟骨細胞シートの作用機序としては欠損部を覆う効果と同時にパラクライン効果がメインであると考えられる。軟骨細胞シートは移植時には関節軟骨の特性を有していないが、ホスト側の細胞を活性化させ従来の関節軟骨に近い硝子軟骨様組織の修復再生を促すと考えられる。 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. Are listed. When transplanting cartilage particles to 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. In addition, because fixation is difficult, it is difficult to transplant for extensive and worn-out cartilage defects or degeneration, such as cartilage defects caused by osteoarthritis. Therefore, there is a need for a chondrocyte sheet with excellent adhesive properties that can be transplanted for a wide range of defects and degenerations. In addition, cartilage particles are expected to compensate for defective articular cartilage, and the cartilage particles themselves are expected to function as articular cartilage.However, 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. Although 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.
　非特許文献２、非特許文献３、特許文献２、および特許文献３には、多指症患者などの生体に由来する軟骨組織を細胞ソースとした軟骨細胞シートおよびその製造方法が示されている。また、そのような軟骨細胞シートが変形性膝関節症に対して適用可能であり、臨床研究においてその安全性と有効性を確認したことが記載されている。しかし、多指症患者などの生体に由来する軟骨組織を細胞ソースとする軟骨細胞シートを同種移植に用いる場合の課題として、ドナー不足、ドナーの細胞の特性に応じて軟骨細胞シートの品質が異なること、商業利用には細胞数を確保するために継代培養により増幅させる必要がありその過程で細胞の品質が低下することなどがある。一方、細胞ソースとして多能性幹細胞を用いることで、軟骨細胞シートの品質を安定させることが可能であり、多能性幹細胞は無制限に増幅させることができることから必要な細胞数を確保することも容易である。 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. In particular, for commercial use, it is necessary to expand by subculturing to ensure the number of cells, and the quality of the cells may deteriorate in the process. On the other hand, by using 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.
国際公開２０１５／０６４７５４号International Publication 2015/064754 国際公開２００６／０９３１５１号International Publication 2006/093151 特開２０２０－００６２０７号公報JP2020-006207A
　本発明は、多能性幹細胞を細胞ソースとする軟骨細胞シートを提供することを課題とする。 An object of the present invention is to provide a chondrocyte sheet using pluripotent stem cells as a cell source.
　本発明者らは、上記のような多指症患者などの生体に由来する軟骨組織を細胞ソースとした従来の軟骨細胞シートの製造方法において、当該細胞ソースとして当該生体由来軟骨組織の代わりに多能性幹細胞を用いた場合に製造される軟骨細胞シートは移植モデルにおいて有効性が確認されない一方で、当該細胞ソースとして多能性幹細胞を用いた場合に、多能性幹細胞から誘導された軟骨細胞（以下、誘導軟骨細胞とも称する）を低血清培地において培養することで、関節軟骨の修復・再生に適した軟骨細胞シートを製造し得ることを見出し、本発明を完成させた。本発明は下記の態様を含む。 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. While the efficacy of chondrocyte sheets produced using potent stem cells has not been confirmed in transplant models, when pluripotent stem cells are used 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.
［１］
　軟骨修復用細胞シートであって、
　該軟骨修復用細胞シートは、多能性幹細胞から誘導された誘導軟骨細胞の培養物から形成され、
　前記培養物は、前記誘導軟骨細胞を低血清培地にて培養して得られたものである、軟骨修復用細胞シート。
［２］
　以下の（１）～（３）のすべてを満たす、［１］に記載の軟骨修復用細胞シート：
（１）培養７２時間における単位面積当たりのＴｒａｎｓｆｏｒｍｉｎｇ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ－β１（ＴＧＦ－β１）の分泌量が３．０ｎｇ／ｃｍ２以上である。
（２）培養７２時間における単位面積当たりのＭｅｌａｎｏｍａ　ｉｎｈｉｂｉｔｏｒｙ　ａｃｔｉｖｉｔｙ（ＭＩＡ）の分泌量が１０．０ｎｇ／ｃｍ２以上である。
（３）細胞シートに含まれる全細胞数当たりのＣＤ５６陽性細胞数の割合が８５％以上である。
［３］
　以下の（１）～（３）のすべてを満たす、［１］または［２］に記載の軟骨修復用細胞シート：
（１）培養７２時間における単位面積当たりのＴｒａｎｓｆｏｒｍｉｎｇ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ－β１（ＴＧＦ－β１）の分泌量が３．５ｎｇ／ｃｍ２以上である。
（２）培養７２時間における単位面積当たりのＭｅｌａｎｏｍａ　ｉｎｈｉｂｉｔｏｒｙ　ａｃｔｉｖｉｔｙ（ＭＩＡ）の分泌量が１２．５ｎｇ／ｃｍ２以上である。
（３）細胞シートに含まれる全細胞数当たりのＣＤ５６陽性細胞数の割合が９０％以上である。
［４］
　以下の（Ａ）～（Ｄ）のすべてを満たす、［１］～［３］のいずれかに記載の軟骨修復用細胞シート：
（Ａ）Ｉ型コラーゲンに対する抗体を用いた免疫染色において陽性である。
（Ｂ）ＩＩ型コラーゲンに対する抗体を用いた免疫染色において陰性である。
（Ｃ）サフラニンＯ染色において陰性である。
（Ｄ）アグリカンに対する抗体を用いた免疫染色において陽性である。
［５］
　前記低血清培地の血清濃度が、５ｖ／ｖ ％以下である、［１］～［４］のいずれかに記載の軟骨修復用細胞シート。
［６］
　前記多能性幹細胞は、ヒト由来のｉＰＳ細胞である、［１］～［５］のいずれかに記載の軟骨修復用細胞シート。
［７］
　ヒト多指症患者軟骨組織由来軟骨細胞シートにおけるＣＯＬ２Ａ１遺伝子の発現量を１としたとき、ＣＯＬ２Ａ１遺伝子の発現量が１００以上である、［１］～［６］のいずれかに記載の軟骨修復用細胞シート。
［８］
　前記誘導軟骨細胞を低血清培地にて培養することが、低酸素条件下で行われるものである、［１］～［７］のいずれかに記載の軟骨修復用細胞シート。
［９］
　前記低酸素条件下は、培養雰囲気中の酸素濃度が５ｖ／ｖ％以下の条件下である、［８］に記載の軟骨修復用細胞シート。 [1]
A cell sheet for cartilage repair,
The 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.
[2]
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-β1 (TGF-β1) 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.
(3) The ratio of the number of CD56 positive cells to the total number of cells contained in the cell sheet is 85% or more.
[3]
The cell sheet for cartilage repair according to [1] or [2], which satisfies all of the following (1) to (3):
(1) The amount of transforming growth factor-β1 (TGF-β1) secreted per unit area during 72 hours of culture is 3.5 ng/cm 2 or more.
(2) The amount of Melanoma inhibitory activity (MIA) secreted per unit area during 72 hours of culture is 12.5 ng/cm 2 or more.
(3) The ratio of the number of CD56 positive cells to the total number of cells contained in the cell sheet is 90% 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.
[7]
For cartilage repair according to any one of [1] to [6], wherein the expression level of the COL2A1 gene is 100 or more when the expression level of the COL2A1 gene in a chondrocyte sheet derived from cartilage tissue of a human polydactyly patient is 1. cell sheet.
[8]
The cell sheet for cartilage repair according to any one of [1] to [7], wherein the induced chondrocytes are cultured in a low serum medium under hypoxic conditions.
[9]
The cell sheet for cartilage repair according to [8], wherein the hypoxic conditions are conditions in which the oxygen concentration in the culture atmosphere is 5 v/v% or less.
［１０］
　軟骨修復用細胞シートの製造方法であって、
　多能性幹細胞から誘導された誘導軟骨細胞を低血清培地にて培養する工程を含む、方法。
［１１］
　前記低血清培地の血清濃度が、５ｖ／ｖ％以下である、［１０］に記載の方法。
［１２］
　前記誘導軟骨細胞を低血清培地にて培養する工程が、培養雰囲気中の酸素濃度が５ｖ／ｖ％以下の条件下で前記誘導軟骨細胞を培養する工程を含む、［１０］または［１１］に記載の方法。
［１３］
　多能性幹細胞から誘導された誘導軟骨細胞を培養する方法であって、
　該誘導軟骨細胞を低血清培地にて培養する工程を含む、方法。
［１４］
　前記低血清培地の血清濃度が、５ｖ／ｖ％以下である、［１３］に記載の方法。
［１５］
　前記誘導軟骨細胞を低血清培地にて培養する工程が、培養雰囲気中の酸素濃度が５ｖ／ｖ％以下の条件下で前記誘導軟骨細胞を培養する工程を含む、［１３］または［１４］に記載の方法。 [10]
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.
[13]
A method for culturing induced chondrocytes derived from pluripotent stem cells, the method comprising:
A method comprising culturing the induced chondrocytes in a low serum medium.
[14]
The method according to [13], wherein the serum concentration of the low serum medium is 5 v/v% or less.
[15]
[13] or [14], 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.
　本発明により、細胞ソースとして多能性幹細胞を用いた、関節軟骨の修復・再生に適した軟骨細胞シートを製造できる。また、細胞ソースとして多能性幹細胞を用いることで、品質の安定した軟骨細胞シートを作製することが可能となり、確立されたドナー細胞より無制限に多能性幹細胞を増やすことができるため、必要な細胞数を容易に確保できる。さらに、多指症患者由来軟骨組織などの同種細胞のドナー選定には継続してコストがかかることから、細胞ソースとして多能性幹細胞を用いることでコストを削減し得る。
　加えて、一態様においては、本発明の軟骨細胞シートは接着性に優れ、広範ですり減った軟骨欠損や変性部位に対しての移植が容易となる。例えば、超高齢化社会を迎え、日本での有症状者が800万人ともいわれている変形性膝関節症に対して、人工関節に依拠しない根治的な治療法となる可能性もあり、医療費削減や健康寿命の延伸の観点からも極めて有用である。 According to the present invention, a chondrocyte sheet suitable for repairing and regenerating articular cartilage can be produced using pluripotent stem cells as a cell source. In addition, by using pluripotent stem cells as a cell source, it is possible to create chondrocyte sheets with stable quality, and pluripotent stem cells can be increased without limit from established donor cells. Cell numbers can be easily secured. Furthermore, since selecting a donor for allogeneic cells such as cartilage tissue derived from polydactyly patients continues to be costly, using pluripotent stem cells as a cell source may reduce costs.
In addition, in one embodiment, 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. For example, with the arrival of a super-aging society, it is said that 8 million people in Japan have symptoms of knee osteoarthritis, and this could become a radical treatment that does not rely on artificial joints. It is also extremely useful from the perspective of cost reduction and extending healthy life expectancy.
２０ｖ／ｖ％ＦＢＳを含むＤＭＥＭ／Ｆ１２培地を用いて作製した軟骨細胞シート（ｉＰＳＣシートＤ）および本発明の方法で作製した軟骨細胞シート（ｉＰＳＣシートＭ）における、ヘマトキシリン・エオジン染色（ＨＥ）、サフラニンＯ染色（ＳａｆＯ）、トルイジンブルー染色（ＴＢ）、Ｉ型コラーゲン免疫染色（ＣＯＬ１）、ＩＩ型コラーゲン免疫染色（ＣＯＬＩＩ）、アグリカン染色（ＡＣＡＮ）、およびフィブロネクチン染色（ＦＮ）の写真（図面代用写真）。図中、スケールバーは１００μｍを示す。Hematoxylin and eosin staining (HE) in a chondrocyte sheet (iPSC sheet D) 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) (photos in place of drawings) ). In the figure, the scale bar indicates 100 μm. ｉＰＳＣシートＤおよびｉＰＳＣシートＭを用いた異種同所性移植実験における、移植から４週後の膝関節組織の染色後の写真（図面代用写真）。ＨＥ：ヘマトキシリン・エオジン染色、ＴＢ：トルイジンブルー染色、ＳａｆＯ：サフラニンＯ染色、ＣＯＬ１：Ｉ型コラーゲン免疫染色、ＣＯＬ２：ＩＩ型コラーゲン免疫染色、ｈＶｉｍ：ヒト特異的ビメンチン染色。ｉＰＳＣシートＤおよびｉＰＳＣシートＭの最下段は、ｈＶｉｍの写真の枠線部分を拡大。A photograph after staining of knee joint tissue 4 weeks after transplantation in a heterogeneous orthotopic transplantation experiment using iPSC sheet D and iPSC sheet M (photograph substituted for a drawing). 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. ｉＰＳＣシートＤおよびｉＰＳＣシートＭを用いた異種同所性移植実験における、移植から１２週後の膝関節組織の染色後の写真（図面代用写真）。ＨＥ：ヘマトキシリン・エオジン染色、ＴＢ：トルイジンブルー染色、ＳａｆＯ：サフラニンＯ染色、ＣＯＬ１：Ｉ型コラーゲン免疫染色、ＣＯＬ２：ＩＩ型コラーゲン免疫染色、ｈＶｉｍ：ヒト特異的ビメンチン染色。ｉＰＳＣシートＤおよびｉＰＳＣシートＭの最下段は、ｈＶｉｍの写真の枠線部分を拡大。Photographs after staining of knee joint tissue 12 weeks after transplantation (photographs substituted for drawings) 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. フローサイトメトリーによるＣＤ５６蛍光強度の測定結果を示すグラフ（上図および左下）と、ＣＤ５６陽性細胞率（％）を示すグラフ（右下）。ｎ＝６。Graphs showing the measurement results of CD56 fluorescence intensity by flow cytometry (upper panel and lower left) and graphs showing the CD56-positive cell rate (%) (lower right). n=6. ＴＧＦ－β１（左図）およびＭＩＡ（右図）の培養７２時間における１シート（４．２ｃｍ２）あたりの分泌量（ｎｇ）を示すグラフ。ＴＧＦ－β１：ｎ＝７、ＭＩＡ：ｎ＝８。各カラム上の数字は１シート（４．２ｃｍ２）あたりの分泌量（ｎｇ）の平均値を示す。Graph showing the secretion amount (ng) of TGF-β1 (left figure) and MIA (right figure) per sheet (4.2 cm 2 ) during 72 hours of culture. TGF-β1: n=7, MIA: n=8. The numbers on each column indicate the average secreted amount (ng) per sheet (4.2 cm 2 ). ＣＯＬ２Ａ１の遺伝子発現量を、ＰＤＣのＣＯＬ２Ａ１の遺伝子発現量の平均値を１とした相対値で示すグラフ。ＰＤＣはｎ＝２、ｉＰＳＣシートＤおよびｉＰＳＣシートＭはｎ＝３。A graph showing the gene expression level of COL2A1 as a relative value with the average value of the gene expression level of COL2A1 in PDC as 1. n=2 for PDC, n=3 for iPSC sheet D and iPSC sheet M. 多指症患者軟骨組織由来の軟骨細胞シート（ＰＤＣシート）およびｉＰＳＣシートＤを用いた異種同所性移植実験における、移植から４週後の膝関節組織の染色後の写真（図面代用写真）。ＨＥ：ヘマトキシリン・エオジン染色、ＴＢ：トルイジンブルー染色、ＳａｆＯ：サフラニンＯ染色、ＣＯＬ１：Ｉ型コラーゲン免疫染色、ＣＯＬ２：ＩＩ型コラーゲン免疫染色、ｈＶｉｍ：ヒト特異的ビメンチン染色。ＰＤＣシートおよびｉＰＳＣシートＤの最下段は、ｈＶｉｍの写真の枠線部分を拡大。A photograph (photograph substituted for a drawing) of knee joint tissue 4 weeks after transplantation in a heterogeneous orthotopic transplantation experiment using a chondrocyte sheet derived from polydactyly patient cartilage tissue (PDC sheet) and iPSC sheet D. 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. ｉＰＳＣシートＭとｉＰＳＣシートＤとの間で、差次的発現タンパク質（Differentially expressed proteins : DEPs）をデータ非依存的解析（DIA）－質量分析（MS）プロテオミクスにより解析した結果を示す図。具体的には、４１個のタンパク質がｉＰＳＣシートＭで発現増加するタンパク質として同定され、２０個のタンパク質がｉＰＳＣシートＭで発現減少するタンパク質として同定されたため、これら計６１個のタンパク質名およびその量的変化を、ヒートマップで示した図である。A diagram showing the results of analyzing differentially expressed proteins (DEPs) between iPSC sheet M and iPSC sheet D by data independent analysis (DIA)-mass spectrometry (MS) proteomics. Specifically, 41 proteins were identified as proteins whose expression increases in iPSC sheet M, and 20 proteins were identified as proteins whose expression decreases in iPSC sheet M, so the names and amounts of these 61 proteins in total were determined. FIG. 2 is a diagram showing the changes in temperature as a heat map. ｉＰＳＣシートＭで発現増加する４１個のタンパク質と発現減少する２０個のタンパク質について、Metascapeにより解析し、遺伝子オントロジー（GO）タームを示した図。A diagram showing Gene Ontology (GO) terms analyzed by Metascape for 41 proteins whose expression increases and 20 proteins whose expression decreases in iPSC sheet M.
　以下、本発明を詳細に説明する。
　本発明において、ｖ／ｖ％とは「体積パーセント」を意味する。
　また、本発明における各数値の範囲は、実質的にその数値範囲内にあることを排除するものではなく、例えば、当該数値の±１０％までを意味しうる。 The present invention will be explained in detail below.
In the present invention, v/v% means "volume percent".
Moreover, the 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.
＜１＞本発明の方法
　本発明の方法の一態様は、多能性幹細胞から誘導された誘導軟骨細胞を低血清培地にて培養する工程を含む、軟骨修復用細胞シートの製造方法である。 <1> Method of the present invention 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.
　本発明の方法により製造される軟骨細胞シートは、軟骨の修復・再生に適した軟骨細胞シートであってよい。そのような軟骨細胞シートを、軟骨修復用細胞シートともいう。また、本発明の方法により製造される軟骨細胞シートは、具体的には、後述する本発明の軟骨細胞シートであってよい。 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.
　本発明の方法の他の態様は、多能性幹細胞から誘導された誘導軟骨細胞を培養する方法であって、該誘導軟骨細胞を低血清培地にて培養する工程を含む、方法である。 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.
　「多能性幹細胞」とは、生体に存在するすべての細胞に分化可能である多能性を有し、かつ、増殖能をも併せもつ幹細胞を指す。多能性幹細胞は、特に限定されないが、例えば胚性幹（ＥＳ）細胞、核移植により得られるクローン胚由来の胚性幹（ｎｔＥＳ）細胞、***幹細胞（ＧＳ細胞）、胚性生殖細胞（ＥＧ細胞）、人工多能性幹（ｉＰＳ）細胞、培養線維芽細胞や骨髄幹細胞由来の多能性細胞（Ｍｕｓｅ細胞）などが含まれる。多能性幹細胞として、好ましくは、ＥＳ細胞、ｎｔＥＳ細胞、および／またはｉＰＳ細胞であり、特に好ましくは、ｉＰＳ細胞である。多能性幹細胞の由来は本発明の効果が得られるならば特に限定されないが、哺乳類であることが好ましく、ヒトなどの霊長類、マウスもしくはラットなどのげっ歯類、イヌ、ネコ、ウサギ、ウシ、ウマ、ヤギ、ヒツジ、ブタ、またはニワトリであることがより好ましく、ヒトであることがさらにより好ましい。 "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), induced pluripotent stem (iPS) cells, cultured fibroblasts, and pluripotent cells derived from bone marrow stem cells (Muse cells). The pluripotent stem cells are preferably ES cells, ntES cells, and/or iPS cells, particularly preferably iPS cells. The origin of pluripotent stem cells is 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.
　多能性幹細胞は、上記の多能性幹細胞の例および由来の任意の組み合わせであってよい。多能性幹細胞は、好ましくは、哺乳類由来のＥＳ細胞、ｎｔＥＳ細胞、および／またはｉＰＳ細胞であり、より好ましくは、ヒトなどの霊長類、マウスもしくはラットなどのげっ歯類、イヌ、ネコ、ウサギ、ウシ、ウマ、ヤギ、ヒツジ、ブタ、またはニワトリ由来のＥＳ細胞、ｎｔＥＳ細胞、および／またはｉＰＳ細胞であり、さらにより好ましくは、ヒト由来のＥＳ細胞、ｎｔＥＳ細胞、および／またはｉＰＳ細胞である。また、多能性幹細胞がｉＰＳ細胞である場合は、好ましくは、哺乳類由来のｉＰＳ細胞であり、より好ましくは、ヒトなどの霊長類、マウスもしくはラットなどのげっ歯類、イヌ、ネコ、ウサギ、ウシ、ウマ、ヤギ、ヒツジ、ブタ、またはニワトリ由来のｉＰＳ細胞であり、さらにより好ましくは、ヒト由来のｉＰＳ細胞である。 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. . When 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.
　ｉＰＳ細胞の製造方法は当該分野で公知であり、任意の体細胞へ初期化因子を導入することなどによって製造され得る。ここで、初期化因子とは、例えば、Ｏｃｔ３／４、Ｓｏｘ２、Ｓｏｘ１、Ｓｏｘ３、Ｓｏｘ１５、Ｓｏｘ１７、Ｋｌｆ４、Ｋｌｆ２、ｃ－Ｍｙｃ、Ｎ－Ｍｙｃ、Ｌ－Ｍｙｃ、Ｎａｎｏｇ、Ｌｉｎ２８、Ｆｂｘ１５、ＥＲａｓ、ＥＣＡＴ１５－２、Ｔｃｌ１、ｂｅｔａ－ｃａｔｅｎｉｎ、Ｌｉｎ２８ｂ、Ｓａｌｌ１、Ｓａｌｌ４、Ｅｓｒｒｂ、Ｎｒ５ａ２、Ｔｂｘ３またはＧｌｉｓ１等の遺伝子または遺伝子産物が例示され、これらの初期化因子は、単独で用いてもよく、組み合わせて用いてもよい。初期化因子の組み合わせとしては、ＷＯ２００７／０６９６６６、ＷＯ２００８／１１８８２０、ＷＯ２００９／００７８５２、ＷＯ２００９／０３２１９４、ＷＯ２００９／０５８４１３、ＷＯ２００９／０５７８３１、ＷＯ２００９／０７５１１９、ＷＯ２００９／０７９００７、ＷＯ２００９／０９１６５９、ＷＯ２００９／１０１０８４、ＷＯ２００９／１０１４０７、ＷＯ２００９／１０２９８３、ＷＯ２００９／１１４９４９、ＷＯ２００９／１１７４３９、ＷＯ２００９／１２６２５０、ＷＯ２００９／１２６２５１、ＷＯ２００９／１２６６５５、ＷＯ２００９／１５７５９３、ＷＯ２０１０／００９０１５、ＷＯ２０１０／０３３９０６、ＷＯ２０１０／０３３９２０、ＷＯ２０１０／０４２８００、ＷＯ２０１０／０５０６２６、ＷＯ２０１０／０５６８３１、ＷＯ２０１０／０６８９５５、ＷＯ２０１０／０９８４１９、ＷＯ２０１０／１０２２６７、ＷＯ２０１０／１１１４０９、ＷＯ２０１０／１１１４２２、ＷＯ２０１０／１１５０５０、ＷＯ２０１０／１２４２９０、ＷＯ２０１０／１４７３９５、ＷＯ２０１０／１４７６１２、Ｈｕａｎｇｆｕ Ｄ，ｅｔ ａｌ．（２００８），Ｎａｔ．Ｂｉｏｔｅｃｈｎｏｌ．，２６：７９５－７９７、Ｓｈｉ Ｙ，ｅｔ ａｌ．（２００８），Ｃｅｌｌ Ｓｔｅｍ Ｃｅｌｌ，２：５２５－５２８、Ｅｍｉｎｌｉ Ｓ，ｅｔ ａｌ．（２００８），Ｓｔｅｍ Ｃｅｌｌｓ．２６：２４６７－２４７４、Ｈｕａｎｇｆｕ Ｄ，ｅｔ ａｌ．（２００８），Ｎａｔ．Ｂｉｏｔｅｃｈｎｏｌ．２６：１２６９－１２７５、Ｓｈｉ Ｙ，ｅｔ ａｌ．（２００８），Ｃｅｌｌ Ｓｔｅｍ Ｃｅｌｌ，３，５６８－５７４、Ｚｈａｏ Ｙ，ｅｔ ａｌ．（２００８），Ｃｅｌｌ Ｓｔｅｍ Ｃｅｌｌ，３：４７５－４７９、Ｍａｒｓｏｎ Ａ，（２００８），Ｃｅｌｌ Ｓｔｅｍ Ｃｅｌｌ，３，１３２－１３５、Ｆｅｎｇ Ｂ，ｅｔ ａｌ．（２００９），Ｎａｔ．Ｃｅｌｌ Ｂｉｏｌ．１１：１９７－２０３、Ｒ．Ｌ．Ｊｕｄｓｏｎ ｅｔ ａｌ．，（２００９），Ｎａｔ．Ｂｉｏｔｅｃｈｎｏｌ．，２７：４５９－４６１、Ｌｙｓｓｉｏｔｉｓ ＣＡ，ｅｔ ａｌ．（２００９），Ｐｒｏｃ Ｎａｔｌ Ａｃａｄ Ｓｃｉ Ｕ Ｓ Ａ．１０６：８９１２－８９１７、Ｋｉｍ ＪＢ，ｅｔ ａｌ．（２００９），Ｎａｔｕｒｅ．４６１：６４９－６４３、Ｉｃｈｉｄａ ＪＫ，ｅｔ ａｌ．（２００９），Ｃｅｌｌ Ｓｔｅｍ Ｃｅｌｌ．５：４９１－５０３、Ｈｅｎｇ ＪＣ，ｅｔ ａｌ．（２０１０），Ｃｅｌｌ Ｓｔｅｍ  Ｃｅｌｌ．６：１６７－７４、Ｈａｎ Ｊ，ｅｔ ａｌ．（２０１０），Ｎａｔｕｒｅ．４６３：１０９６－１００、Ｍａｌｉ Ｐ，ｅｔ ａｌ．（２０１０），Ｓｔｅｍ Ｃｅｌｌｓ．２８：７１３－７２０、Ｍａｅｋａｗａ Ｍ，ｅｔ ａｌ．（２０１１），Ｎａｔｕｒｅ．４７４：２２５－９に記載の組み合わせが例示される。 Methods for producing iPS cells are known in the art, and can be produced by introducing reprogramming factors into any somatic cells. Here, 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, Huangfu D, et al. (2008), Nat. Biotechnol. , 26:795-797, Shi Y, et al. (2008), Cell Stem Cell, 2:525-528, Eminli S, et al. (2008), Stem Cells. 26:2467-2474, Huangfu D, et al. (2008), Nat. Biotechnol. 26:1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008), Cell Stem Cell, 3:475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat. Cell Biol. 11:197-203, R. L. Judson et al. , (2009), Nat. Biotechnol. , 27:459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci USA. 106:8912-8917, Kim JB, et al. (2009), Nature. 461:649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503, Heng JC, et al. (2010), Cell Stem Cell. 6:167-74, Han J, et al. (2010), Nature. 463:1096-100, Mali P, et al. (2010), Stem Cells. 28:713-720, Maekawa M, et al. (2011), Nature. 474:225-9 is exemplified.
　ｉＰＳ細胞を得るために使用される体細胞は特に限定されず、例えば、胎児（仔）の体細胞、新生児（仔）の体細胞、および成熟した健全なもしくは疾患性の体細胞のいずれも含んでよく、また、初代培養細胞、継代細胞、および株化細胞のいずれも含んでよい。体細胞は、具体的には、例えば（１）神経幹細胞、造血幹細胞、間葉系幹細胞、歯髄幹細胞等の組織幹細胞（体性幹細胞）、（２）組織前駆細胞、（３）血液細胞（末梢血細胞、臍帯血細胞等）、リンパ球、上皮細胞、内皮細胞、筋肉細胞、線維芽細胞（皮膚細胞等）、毛細胞、肝細胞、胃粘膜細胞、腸細胞、脾細胞、膵細胞（膵外分泌細胞等）、脳細胞、肺細胞、腎細胞および脂肪細胞等の分化した細胞などであってもよいが、これらに限定されるものではない。 The 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. Specifically, 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). blood cells, umbilical cord blood cells, etc.), 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.
　「軟骨細胞」とは、コラーゲンなど、軟骨を構成する細胞外マトリックスを産生する細胞、または、このような細胞となる前駆細胞を意味する。また、このような軟骨細胞は、軟骨細胞マーカーを発現する細胞であってもよく、軟骨細胞マーカーとしてＩＩ型コラーゲン（ＣＯＬ２Ａ１）またはＳＯＸ９が例示される。「誘導軟骨細胞」とは、未分化な細胞から誘導されることで得られた軟骨細胞を意味する。この場合において、「未分化な細胞」とは、軟骨細胞に分化しうる細胞を意味し、多能性幹細胞を細胞ソースとすることが好ましい。また、「誘導される」とは、任意の方法で分化を誘導されることであってよい。 "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.
　よって、「多能性幹細胞から誘導された誘導軟骨細胞」とは、多能性幹細胞を細胞ソースとして任意の方法で分化誘導することで得られた軟骨細胞であってよく、具体的には、例えば、ｉｎ　ｖｉｔｒｏで多能性幹細胞から分化誘導することで得られた軟骨細胞であってよい。多能性幹細胞から誘導軟骨細胞を誘導する方法は、任意の既知の方法を用いることができ、例えば、Yamashita, A. et al. Generation of Scaffoldless Hyaline Cartilaginous Tissue from Human iPSCs. Stem Cell Reports 4, 404-418 (2015)、ＷＯ２０１５／０６４７５４、またはＷＯ２０１６／１３３２０８に記載の方法が挙げられる。 Therefore, "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.
　本発明の方法は、多能性幹細胞から誘導された誘導軟骨細胞を低血清培地にて培養する工程を含む。本発明における「低血清培地」とは、従来の軟骨細胞シートの製造方法、例えば、Maehara, M. et al. Characterization of polydactyly-derived chondrocyte sheets versus adult chondrocyte sheets for articular cartilage repair. Inflammation and Regeneration 37, 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、または特開２０２０－００６２０７に記載の軟骨細胞を培養するための培地と比較して、血清濃度が低い培地を指す。従来の軟骨細胞シートの製造方法における培地の血清濃度は、典型的には、２０ｖ／ｖ％でありうる。
　低血清培地の血清濃度は、例えば、１０ｖ／ｖ％以下、５ｖ／ｖ％以下、４ｖ／ｖ％以下、３ｖ／ｖ％以下、２．５ｖ／ｖ％以下、２．２ｖ／ｖ％以下、２ｖ／ｖ％以下であってもよく、０ｖ／ｖ％以上、０．５ｖ／ｖ％以上、１ｖ／ｖ％以上、１．５ｖ／ｖ％以上、１．８ｖ／ｖ％以上、２ｖ／ｖ％以上あってもよく、それらの矛盾しない組み合わせであってもよい。本発明における低血清培地の血清濃度は、具体的には、例えば、０ｖ／ｖ％以上１０ｖ／ｖ％以下、０．５ｖ／ｖ％以上５ｖ／ｖ％以下、０．５ｖ／ｖ％以上２．５ｖ／ｖ％以下、０．５ｖ／ｖ％以上２ｖ／ｖ％以下、１．５ｖ／ｖ％以上２．５ｖ／ｖ％以下、１．８ｖ／ｖ％以上２．２ｖ／ｖ％以下、または２ｖ／ｖ％であってよい。ただし、本発明における低血清培地の血清濃度が２ｖ／ｖ％であるとは、血清濃度が実質的に２ｖ／ｖ％である均等な範囲を排除するものではない。このような範囲としては、一例として、１．８ｖ／ｖ％以上２．２ｖ／ｖ％以下が考えられるが、これに限定されるものではない。 The method of the present invention includes the step of culturing induced chondrocytes derived from pluripotent stem cells in a low serum medium. In the present invention, 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. Specifically, 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%. However, 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.
　血清として用いられるものは、例えば、ウシ胎子血清（ＦＢＳ）、患者自己血清などのヒト血清、ウマ血清、ヒツジ血清、ヤギ血清、ブタ血清、ニワトリ血清、ラット血清、マウス血清などが挙げられ、これらに限定されるものではないが、ＦＢＳまたは患者自己血清が好ましい。 Examples of serum that can be used 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.
　本発明の低血清培地は、血清濃度以外は特に限定されず、任意の既知の培地であってよいが、間葉系幹細胞に適した培地が好ましい。間葉系幹細胞に適した培地としては、例えば、ＭｅｓｅｎＰＲＯやＳｔｅｍＰＲＯが挙げられるが、これらに限定されるものではない。 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. Examples of media suitable for mesenchymal stem cells include, but are not limited to, MesenPRO and StemPRO.
　本発明の多能性幹細胞から誘導された誘導軟骨細胞を低血清培地にて培養する工程は、当該誘導軟骨細胞を、温度応答性ポリマーで表面が被覆された培養器を用いて培養する工程を含んでよい。温度応答性ポリマーとしては、特に限定されないが、例えば、ポリ-N-イソプロピルアクリルアミドが挙げられる。 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.
　本発明の多能性幹細胞から誘導された誘導軟骨細胞を低血清培地にて培養する工程は、当該誘導軟骨細胞を、培養雰囲気の酸素濃度が低い条件下（すなわち、低酸素条件下）で培養する工程を含んでよい。「低酸素条件下で培養」とは、細胞、特に軟骨細胞を培養する際の雰囲気中の酸素濃度が、当該細胞が通常培養される酸素濃度未満であることを意味する。「細胞が通常培養される酸素濃度」は、細胞や由来する種によって異なるために一義的に決定されるものではないが、一例として、一般的な哺乳動物軟骨細胞であれば２０ｖ/ｖ％である。よって、低酸素条件下とは、培養雰囲気中の酸素濃度が２０ｖ/ｖ％未満の条件下であってよい。低酸素条件下は、好ましくは、培養雰囲気中の酸素濃度が１０ｖ／ｖ％以下の条件下であり、より好ましくは、７ｖ／ｖ％以下の条件下であり、さらにより好ましくは５ｖ／ｖ％以下の条件下であり、特により好ましくは３ｖ／ｖ％以下の条件下であり、特にさらにより好ましくは２ｖ／ｖ％以下の条件下である。なお培養雰囲気中の酸素濃度がＸＸｖ/ｖ％未満またはＸＸｖ/ｖ％以下の条件下とは、その下限において、特に断りのない限り、培養雰囲気中に酸素を含まない、すなわち培養雰囲気中の酸素濃度が０％の場合を含む、培養雰囲気中の酸素濃度が０ｖ／ｖ％以上の条件下を指す。また、低酸素条件下における培養雰囲気中の酸素濃度の下限は、上記範囲に矛盾しない限り特に限定しないが、例えば、培養雰囲気中の酸素濃度が０ｖ／ｖ％以上、０．５ｖ／ｖ％以上、または１ｖ／ｖ％以上の条件下であってもよい。また、低酸素条件下は、上記範囲の矛盾しない組み合わせであってよく、具体的には、例えば、０．５ｖ／ｖ％以上１０ｖ／ｖ％以下、０．５ｖ／ｖ％以上５ｖ／ｖ％以下、０．５ｖ／ｖ％以上３ｖ／ｖ％以下、０．５ｖ／ｖ％以上２ｖ／ｖ％以下、１ｖ／ｖ％以上５ｖ／ｖ％以下であってもよい。 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%. be. Therefore, 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. Note that 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%. Furthermore, 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. Further, the 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.
　本発明の多能性幹細胞から誘導された誘導軟骨細胞を低血清培地にて培養する工程において、培養する期間は、本発明の軟骨細胞シートが得られるならば特に限定しないが、例えば、７日以上２１日以下であってよい。 In the step of culturing the induced chondrocytes derived from the pluripotent stem cells of the present invention in a low serum medium, 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.
　本発明の多能性幹細胞から誘導された誘導軟骨細胞を低血清培地にて培養する工程において、培養開始時に播種する細胞数は、本発明の軟骨細胞シートが得られるならば特に限定しないが、例えば、１０００細胞／ｃｍ２以上、１００００細胞／ｃｍ２以上、３００００細胞／ｃｍ２以上、または５００００細胞／ｃｍ２以上であってもよく、１００００００細胞／ｃｍ２以下、１０００００細胞／ｃｍ２以下、７００００細胞／ｃｍ２以下、または５００００細胞／ｃｍ２以下であってもよい。 In the step of culturing induced chondrocytes derived from the pluripotent stem cells of the present invention in a low serum medium, 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. For example, 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.
＜２＞本発明の軟骨細胞シート
　本発明の軟骨細胞シートは、多能性幹細胞から誘導された誘導軟骨細胞の培養物から形成され、当該培養物は、前記誘導軟骨細胞を低血清培地にて培養して得られたものである、軟骨細胞シートである。 <2> Chondrocyte sheet of the present invention 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.
　本発明の軟骨細胞シートは、軟骨の修復・再生に適する。よって、本発明の軟骨細胞シートは、軟骨修復用細胞シートであってよい。 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.
　多能性幹細胞および多能性幹細胞から誘導された誘導軟骨細胞は、上述した通りである。 The pluripotent stem cells and the induced chondrocytes derived from the pluripotent stem cells are as described above.
　本発明の軟骨細胞シートは、「Ｔｒａｎｓｆｏｒｍｉｎｇ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ－β１（ＴＧＦ－β１）の分泌量」、「Ｍｅｌａｎｏｍａ　ｉｎｈｉｂｉｔｏｒｙ　ａｃｔｉｖｉｔｙ（ＭＩＡ）の分泌量」、及び、「細胞シートに含まれる全細胞数当たりのＣＤ５６陽性細胞数の割合」からなる群から選択される一又は複数によって特徴づけられてもよい。ＴＧＦ－β１は関節軟骨の増殖、分化に関わる成長因子であり、軟骨細胞シートがもたらす治療効果に影響している可能性がある。また、ＭＩＡは軟骨由来のレチノイン酸感受性タンパク質としても知られており、軟骨細胞が特異的にＭＩＡを発現することや軟骨細胞に対してＭＩＡの同化作用が報告されている。ＣＤ５６はｎｅｕｒａｌ　ｃｅｌｌ　ａｄｈｅｓｉｏｎ　ｍｏｌｅｃｕｌｅ（Ｎ－ＣＡＭ）や神経細胞接着分子として知られている細胞表面にある糖タンパク質である。ナチュラルキラー細胞、神経細胞、骨格筋細胞などが発現していることが知られているが、骨髄由来間葉系幹細胞の一部でも発現していることが報告されている。 The chondrocyte sheet of the present invention has the following characteristics: "transforming growth factor-β1 (TGF-β1) 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-β1 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.
　ＴＧＦ－β１の分泌量、ＭＩＡの分泌量は、例えば、軟骨細胞シートを培養し、一定の培養期間中に当該軟骨細胞シートが培地に分泌する単位面積当たりのＴＧＦ－β１タンパク質の量、ＭＩＡタンパク質の量であってよい。タンパク質の量の測定は、任意の既知の手法を用いることができ、特に限定されないが、例えば、ＥＬＩＳＡ法やウェスタンブロッティング法などの抗体を用いた手法やポリアクリルアミド電気泳動（ＳＤＳ－ＰＡＧＥ）などの電気泳動を用いた手法、質量分析法などが挙げられる。 The secretion amount of TGF-β1 and the secretion amount of MIA are, for example, the amount of TGF-β1 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. may be the amount of Any known method can be used to measure the amount of protein, including, but not limited to, methods using antibodies such as ELISA and Western blotting, and polyacrylamide electrophoresis (SDS-PAGE). Examples include methods using electrophoresis and mass spectrometry.
　前記分泌量を測定するために軟骨細胞シートを培養する場合において、培養条件は、前記分泌量を測定する際に通常用いられる培養条件であれば特に限定されず、培地としては、例えば、低血清培地を用いてもよいが、１％ＦＢＳを含むＤＭＥＭ／Ｆ１２培地を用いることが好ましい。その他の培養条件も、前記分泌量を測定する際に通常用いられる培養条件であってよい。例えば、温度が３４℃以上４０℃以下、好ましくは３５℃以上３９℃以下であり、より好ましくは３６℃以上３８℃以下であり、二酸化炭素（ＣＯ２）濃度が０ｖ／ｖ％以上１０ｖ／ｖ以下、好ましくは３ｖ／ｖ％以上７ｖ／ｖ％以下であり、酸素濃度が０ｖ／ｖ％以上２５ｖ／ｖ％以下、好ましくは１５ｖ／ｖ％以上２５ｖ／ｖ％以下である。培養時間は、例えば、１時間、３時間、６時間、１２時間、２４時間、３６時間、４８時間、７２時間、または９６時間であってよく、また、これらの中から上限と下限として矛盾しない組み合わせを選択してもよい。尚、当該培養時間は、前記分泌量を測定するための培地を軟骨細胞シートに適用した時刻を０とした場合の培養時間であってよく、例えば、本明細書における「培養７２時間」とは、当該時刻を０とした場合の「培養７２時間」である。 When culturing a chondrocyte sheet to measure the secretion amount, the culture conditions are not particularly limited as long as they are the culture conditions normally used when measuring the secretion amount. Although 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. For example, 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. Hereinafter, 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.
　本発明の軟骨細胞シートは、具体的には、例えば、培養７２時間における単位面積当たりのＴＧＦ－β１の分泌量が、２．０ｎｇ／ｃｍ２以上であってもよく、３．０ｎｇ／ｃｍ２以上であることが好ましく、３．５ｎｇ／ｃｍ２以上であることがより好ましい。また、本発明の軟骨細胞シートは、具体的には、例えば、培養７２時間における単位面積当たりのＭＩＡの分泌量が、６．０ｎｇ／ｃｍ２以上であってもよく、１０．０ｎｇ／ｃｍ２以上であることが好ましく、１２．５ｎｇ／ｃｍ２であることがより好ましい。 Specifically, the chondrocyte sheet of the present invention may have a secretion amount of TGF-β1 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 . Moreover, specifically, 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 .
　「ＣＤ５６陽性細胞」とは、ＣＤ５６を発現する細胞であり、例えば、細胞表面にＣＤ５６を発現する細胞、細胞内部にＣＤ５６を発現する細胞、または細胞表面と内部の両方にＣＤ５６を発現する細胞などが挙げられ、好適には、少なくとも細胞表面にＣＤ５６を発現する細胞である。「ＣＤ５６を発現する」とは、細胞表面にＣＤ５６タンパク質が存在すること、あるいは細胞内部にＣＤ５６タンパク質またはｍＲＮＡが存在することを意味してもよい。よって、ＣＤ５６陽性細胞を検出する方法は、細胞表面のＣＤ５６タンパク質、あるいは細胞内部のＣＤ５６タンパク質またはｍＲＮＡを検出することで、これらが存在する細胞を検出する方法であってよく、好ましくは、細胞表面のＣＤ５６タンパク質を検出することで、細胞表面にＣＤ５６タンパク質が存在する細胞を検出する方法であってよい。細胞表面のＣＤ５６タンパク質を検出する方法としては、例えば、ＣＤ５６タンパク質の特異的に結合する抗体を用いることができる。
　ＣＤ５６陽性細胞を測定する手法として、具体的には、一例として、ＣＤ５６タンパク質の特異的に結合する、蛍光標識された抗体を用いて、フローサイトメトリーにより細胞表面にＣＤ５６タンパク質が存在する細胞数を測定する手法が挙げられる。 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. Preferably, 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. As a method for detecting CD56 protein on the cell surface, for example, an antibody that specifically binds to CD56 protein can be used.
Specifically, one example of 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.
　本発明の軟骨細胞シートは、具体的には、例えば、細胞シートに含まれる全細胞数当たりのＣＤ５６陽性細胞数の割合が、８０％以上であってよく、８５％以上であることが好ましく、９０％以上であることがより好ましい。 Specifically, in the chondrocyte sheet of the present invention, for example, 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.
　本発明の軟骨細胞シートは、「ＴＧＦ－β１の分泌量」、「ＭＩＡの分泌量」、及び、「細胞シートに含まれる全細胞数当たりのＣＤ５６陽性細胞数の割合」が、上述した範囲の任意の組み合わせであってもよい。
　本発明の軟骨細胞シートは、具体的には、例えば、以下の（１）～（３）のすべてを満たしてもよい：
（１）培養７２時間における単位面積当たりのＴｒａｎｓｆｏｒｍｉｎｇ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ－β１（ＴＧＦ－β１）の分泌量が２．０ｎｇ／ｃｍ２以上であり、好ましくは３．０ｎｇ／ｃｍ２以上であり、より好ましくは３．５ｎｇ／ｃｍ２以上である。
（２）培養７２時間における単位面積当たりのＭｅｌａｎｏｍａ　ｉｎｈｉｂｉｔｏｒｙ　ａｃｔｉｖｉｔｙ（ＭＩＡ）の分泌量が６．０ｎｇ／ｃｍ２以上であり、好ましくは１０．０ｎｇ／ｃｍ２以上であり、より好ましくは１２．５ｎｇ／ｃｍ２である。
（３）細胞シートに含まれる全細胞当たりのＣＤ５６陽性細胞の割合が８０％以上であり、好ましくは８５％以上であり、より好ましくは９０％以上である。 The chondrocyte sheet of the present invention has "TGF-β1 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-β1 (TGF-β1) 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.
(2) 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 .
(3) 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.
　本発明の軟骨細胞シートは、さらに、従来の軟骨細胞シートと比較して有意に高いＣＯＬ２Ａ１遺伝子発現を示してもよい。好ましくは、本発明の軟骨細胞シートは、従来の軟骨細胞シートにおけるＣＯＬ２Ａ１遺伝子の発現量を１としたとき、ＣＯＬ２Ａ１遺伝子の発現量が１００以上であり、より好ましくはＣＯＬ２Ａ１遺伝子の発現量が１０００以上である。従来の軟骨細胞シートは、例えば、ヒト多指症患者軟骨組織由来軟骨細胞シートが挙げられ、その一例としては、特開２０２０－００６２０７号公報に記載のヒト多指症患者軟骨組織由来軟骨細胞シートが挙げられる。具体的には、例えば、本発明の軟骨細胞シートは、ヒト多指症患者軟骨組織由来軟骨細胞シートと比較して有意に高いＣＯＬ２Ａ１遺伝子発現を示してもよく、好ましくは、ヒト多指症患者軟骨組織由来軟骨細胞シートにおけるＣＯＬ２Ａ１遺伝子の発現量を１としたとき、ＣＯＬ２Ａ１遺伝子の発現量が１００以上であり、より好ましくは１０００以上である。 The chondrocyte sheet of the present invention may further exhibit significantly higher COL2A1 gene expression compared to conventional chondrocyte sheets. Preferably, 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. Specifically, for example, 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 When 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.
　本発明の軟骨細胞シートは、さらに、以下の（Ａ）～（Ｄ）から選ばれる１以上を満たしてもよく、以下の（Ａ）～（Ｄ）のすべてを満たすことが好ましい：
（Ａ）Ｉ型コラーゲンに対する抗体を用いた免疫染色において陽性である。
（Ｂ）ＩＩ型コラーゲンに対する抗体を用いた免疫染色において陰性である。
（Ｃ）サフラニンＯ染色において陰性である。
（Ｄ）アグリカンに対する抗体を用いた免疫染色において陽性である。 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.
　「抗体を用いた免疫染色」とは、標的タンパク質に対する抗体、例えばＩ型コラーゲンに対する抗体、ＩＩ型コラーゲンに対する抗体、またはアグリカンに対する抗体などと、必要に応じて抗体を認識する２次抗体、抗体（２次抗体など含む）を標識する分子などを用いて、細胞シートや組織などに含まれ得る標的タンパク質を含む細胞を検出することを指す。本発明における、「免疫染色において陽性である」とは、細胞シートや組織に標的タンパク質を含む細胞が検出されることであり、具体的には、例えば、細胞シートや組織などに含まれる全細胞中の９０％以上が標的タンパク質を含む細胞として検出されることであってよい。本発明における、「免疫染色において陰性である」とは、細胞シートや組織に標的タンパク質を含む細胞が一定以下のみ検出される、または検出されないことを指し、具体的には、例えば、細胞シートや組織などに含まれる全細胞中の１０％以下が標的タンパク質を含む細胞として検出されることであってよく、ただし、０％の場合、すなわち、標的タンパク質を含む細胞が検出されない場合を含む。 "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.). In the present invention, "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. In the present invention, "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.
　Ｉ型コラーゲンは、瘢痕組織、腱、靭帯、筋原線維の筋内膜、骨、真皮、象牙質、および変性した軟骨などで見られるコラーゲンであり、ＣＯＬ１Ａ遺伝子から発現する。本発明の軟骨細胞シートは、特に培養時に、Ｉ型コラーゲンに対する抗体を用いた免疫染色において陽性であることが好ましいが、生体への移植後に、移植したシートおよびその周辺組織の一部または全体で消失してもよい。 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.
　ＩＩ型コラーゲンは、角膜、硝子体、および軟骨で見られ、関節軟骨を構成する主要なコラーゲンであり、ＣＯＬ２Ａ遺伝子から発現する。本発明の軟骨細胞シートは、特に培養時に、ＩＩ型コラーゲンに対する抗体を用いた免疫染色において陰性であることが好ましいが、生体への移植後に、移植したシートおよびその周辺組織の一部または全体で出現してもよい。 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.
　サフラニンＯは、細胞核を染色する染料であり、軟骨も染色することが知られている。サフラニンＯ染色は、任意の既知の方法により実施され得るが、具体的には、例えば、まずヘマトキシン処理をし、ファストグリーン水溶液中に浸漬し、酢酸水溶液によって処理した後、サフラニンＯ水溶液中に浸漬することで実施されてもよい。本発明の軟骨細胞シートは、サフラニンＯ染色において陰性であることが好ましい。 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.
　アグリカンは、３つの球状ドメイン（Ｇ１、Ｇ２、およびＧ３）と、Ｇ２とＧ３との間のグリコサミノグリカン（ケラタン硫酸やコンドロイチン硫酸）結合領域からなるコアタンパク質を有する、分子量約２５００ｋＤａのケラタン硫酸／コンドロイチン硫酸プロテオグリカンである。アグリカンは軟骨組織に存在し、軟骨組織の物理的強度や弾力性などに寄与することが知られている。本発明の軟骨細胞シートは、アグリカンに対する抗体を用いた免疫染色において陽性であることが好ましい。 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.
　本発明の軟骨細胞シートは、CHI3L1、STEAP4、CEMIP、METTL7A、DPP4、CLGN、NDUFV3、ISM1、GPNMB、SCARA3、NUBPL、TNFAIP8、ABCC3、RGCC、DAPK2、LRRN4CL、EEPD1、C1QTNF1、PPM1H、SLC12A7、GMPR、FHIT、PLXDC1、GLB1L、MMP13、TMEM119、SLC9A9、MATN2、CHST2、TLR2、CLU、VSNL1、TWIST1、FMNL2、CHCHD5、FABP4、CTSK、MCC、ABCA1、MID1、およびSTRIP2から選択される１以上のタンパク質の高発現、ならびに／またはNEFM、IGFBP7、TNXB、AHSG、MLPH、KCNN4、GRPEL2、COL4A1、HTRA3、EFHD1、KRT7、DPT、MEGF10、CCN2、INHBA、ICAM5、MMP23B、PAPPA、STC2、およびIGF2から選択される１以上のタンパク質の低発現を特徴とすることが好ましい。 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. expression and/or one selected from NEFM, IGFBP7, TNXB, AHSG, MLPH, KCNN4, GRPEL2, COL4A1, HTRA3, EFHD1, KRT7, DPT, MEGF10, CCN2, INHBA, ICAM5, MMP23B, PAPPA, STC2, and IGF2 Preferably, the protein is characterized by low expression of the above proteins.
　本発明の軟骨細胞シートでは、大腸がんの上皮間葉転換、脂質輸送、ホルモンレベルの制御、細胞死の正の制御、腫瘍壊死因子への細胞応答、細胞による分泌の正の制御、血管新生の制御、アミノグリカン代謝プロセス、脂質輸送の正の制御、低酸素レベルへの応答、および／または細胞間接着の制御に関連するタンパク質の高発現を特徴としてもよく、ホルモン応答、インスリン様成長因子(IGF)輸送制御およびインスリン様成長因子結合タンパク(IGFBPs)による取り込み、膜貫通受容体タンパク質セリン／スレオニンキナーゼシグナル経路の制御、超分子繊維組織、および／または表皮形成に関連するタンパク質の低発現を特徴としてもよく、細胞外マトリクス組織、NAVAマトリソーム関連タンパク質、タンパク質リン酸化の正の制御、および／または骨形成に関連するタンパク質において高発現と低発現の双方の特徴を有してもよく、これらの組み合わせでタンパク質の高発現および／または低発現の特徴を有してもよい。本発明の軟骨細胞シートでは、IGF輸送制御およびIGFBPsによる取り込み、および／もしくはホルモン応答に関連するタンパク質の低発現を特徴とする、ならびに／または細胞外マトリクス組織、NABAマトリソーム関連タンパク質、骨形成、および／もしくはタンパク質リン酸化の正の制御に関連するタンパク質において高発現と低発現の双方の特徴を有することが好ましい。 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. may be characterized by high expression of proteins associated with the regulation of aminoglycan metabolic processes, positive regulation of lipid transport, response to low oxygen levels, and/or regulation of cell-cell adhesion, hormonal responses, insulin-like growth factors (IGF) transport regulation and uptake by insulin-like growth factor binding proteins (IGFBPs), regulation of transmembrane receptor protein serine/threonine kinase signaling pathways, supramolecular fibrous organization, and/or underexpression of proteins associated with epidermal formation. may be characterized by both high and low expression in extracellular matrix tissues, NAVA matrisome-associated proteins, positive regulation of protein phosphorylation, and/or proteins associated with bone formation; A combination of these may have the characteristics of high expression and/or low protein expression. 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.
　本発明の軟骨細胞シートは、損傷または変性した軟骨の治療に用いてもよい。軟骨の治療法としては、例えば、損傷または変性した軟骨への移植が考えられる。本発明の軟骨細胞シートは、シート状の形状を有し、優れた接着性および軟骨組織の修復・再生を示すため、例えば変形性関節症のような広範にすり減った軟骨への移植が容易であり、またそのような軟骨の治療も期待できる。本発明の軟骨細胞シートによる軟骨の治療は、軟骨細胞シート自身が損傷部位を埋めるだけでなく、周辺の軟骨組織や骨組織の修復再生を促すことによるものであってもよい。 The chondrocyte sheet of the present invention may be used to treat damaged or degenerated cartilage. As 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.
　よって、本発明の軟骨細胞シートは、外傷性の軟骨損傷、骨軟骨損傷、離断性骨軟骨炎、半月板損傷、椎間板変性、および変形性関節症からなる群から選択される一又は複数の治療に用いてもよい。特に、根治的な治療法が確立されていない変形性膝関節症に対しての治療法として、本発明の意義は大きい。 Therefore, 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. In particular, the present invention has great significance as a treatment method for knee osteoarthritis for which no radical treatment has been established.
　以下、実施例を挙げて本発明をより具体的に説明するが、本発明は以下の実施態様には限定されない。 Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to the following embodiments.
（実施例１）
軟骨細胞の分化誘導
　多能性幹細胞からの軟骨細胞の分化誘導は、京都大学ｉＰＳ細胞研究所のｉＰＳ細胞ストックプロジェクトにて確立された、ヒト末梢血に由来するｉＰＳ細胞であるＱＨＪＩ　０１ｓ０４株を用いて、ＷＯ２０１５／０６４７５４に記載の方法に従い行った。具体的には、前記ｉＰＳ細胞をマトリゲルをコーティングした培養皿でフィーダーフリーの状態で培養し、Ｅｓｓｅｎｔｉａｌ　８（Ｉｎｖｉｔｒｏｇｅｎ）培地で維持した。その後、培地を１０ｎｇ／ｍｌのＷｎｔ３ａ（Ｒ＆Ｄ）、１０ｎｇ／ｍｌのＡｃｔｉｖｉｎ　Ａ（Ｒ＆Ｄ）、１％ＩＴＳ（Ｉｎｖｉｔｒｏｇｅｎ）、１％ＦＢＳを含むＤＭＥＭ／Ｆ１２培地に交換した。７２時間後、１０ｎｇ／ｍｌのＴＧＦ－β１（Ｐｅｐｒｏｔｅｃｈ）、１０ｎｇ／ｍｌのＢＭＰ２（Ｏｓｔｅｏｐｈａｒｍａ）、１０ｎｇ／ｍｌのＧＤＦ５（ＰＴＴ）、５０μｇ／ｍｌのアスコルビン酸、１％ＩＴＳ、１％ＦＢＳ、２ｍＭのＬ－ｇｌｕｔａｍｉｎｅ（Ｉｎｖｉｔｒｏｇｅｎ）、１ｘ１０－４Ｍのｎｏｎｅｓｓｅｎｔｉａｌ　ａｍｉｎｏ　ａｃｉｄｓ（Ｉｎｖｉｔｒｏｇｅｎ），１ｍＭのピルビン酸ナトリウム（Ｉｎｖｉｔｒｏｇｅｎ）を含むＤＭＥＭ培地に交換した。３日目から１４日目では１０ｎｇ／ｍｌのｂＦＧＦ（Ｗａｋｏ）も添加した。１４日目に接着している軟骨の塊を培養皿から物理的に剥がし、その後は浮遊培養で培養を行った。上記の培養は全て３７℃、５％ＣＯ２状態のインキュベーター内で行った。培地交換は２日から７日間ごとに行った。浮遊培養中の軟骨の塊を軟骨パーティクルとも呼ぶ。また、当該軟骨パーティクルに含まれる、多能性幹細胞から誘導された誘導軟骨細胞を、単に誘導軟骨細胞とも呼ぶ。 (Example 1)
Induction of differentiation of chondrocytes 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. Thereafter, the medium was replaced with DMEM/F12 medium containing 10 ng/ml Wnt3a (R&D), 10 ng/ml Activin A (R&D), 1% ITS (Invitrogen), and 1% FBS. After 72 hours, 10 ng/ml TGF-β1 (Peprotech), 10 ng/ml BMP2 (Osteopharma), 10 ng/ml GDF5 (PTT), 50 μg/ml ascorbic acid, 1% ITS, 1% FBS, 2 mM The medium was replaced with a DMEM medium containing L-glutamine (Invitrogen), 1×10 −4 M nonessential amino acids (Invitrogen), and 1 mM sodium pyruvate (Invitrogen). From day 3 to day 14, 10 ng/ml bFGF (Wako) was also added. On the 14th day, the adhered cartilage mass was physically peeled off from the culture dish, and thereafter cultured in suspension culture. All of the above cultures were performed in an incubator at 37° C. and 5% CO 2 . Medium exchange was performed every 2 to 7 days. Cartilage masses in suspension culture are also called cartilage particles. In addition, the induced chondrocytes derived from pluripotent stem cells contained in the cartilage particles are also simply referred to as induced chondrocytes.
（実施例２）
軟骨細胞シートの作製
　１９週間培養した前記軟骨パーティクル（湿重量１～４ｇ）を、５ｍｇのＬｉｂｅｒａｓｅ（Ｒｏｃｈｅ）を含む３０ｍｌの２０％ＦＢＳ、１％の抗生剤を含むＤＭＥＭ／Ｆ１２培地で分散した。スターラーバーを用いた拡散状態で回転数６０ｒｐｍの条件で３７℃、５％ＣＯ２状態のインキュベーター内で２時間半分散した。分散後の軟骨細胞を含む培地を４０μｍのセルストレーナーを通し、ＤＰＢＳで洗浄し、室温で１５００ｒｐｍで５分遠心した。アスピレーターで上清を取り除き、３５ｍｌの基礎培地で洗浄し、室温で１５００ｒｐｍで５分遠心した。アスピレーターで上清を取り除き、１０ｍｌの基礎培地で洗浄し、室温で１５００ｒｐｍで５分遠心した。アスピレーターで上清を取り除き、５ｍｌのＭｅｓｅｎＰＲＯ培地（２ｖ／ｖ％のウシ胎児血清（ＦＢＳ）を含有；付属Ｓｕｐｐｌｅｍｅｎｔ、１：１００　ＧｌｕｔａＭＡＸ、１％の抗生剤を添加）で懸濁した。細胞懸濁液の一部を１：１でトリパンブルーと混合し、Ｃｏｕｎｔｅｓｓで細胞数を測定した。細胞シートを作製するための培養機材としてインサート用の６－ｗｅｌｌプレート（Ｆａｌｃｏｎ）と温度応答性培養インサート（ＵｐＣｅｌｌインサート、セルシード社）を準備し、３ｍｌのＭｅｓｅｎＰＲＯ培地を６－ｗｅｌｌプレートの各ウェルに添加し、インサートを設置した。測定した細胞数をもとに、インサート内に約５００００細胞／ｃｍ２になるように２ｍｌのＭｅｓｅｎＰＲＯ培地に懸濁し、播種した。すなわち、約２１万個の細胞を２ｍｌのＭｅｓｅｎＰＲＯ培地に懸濁し、インサートに播種した。その後、３７℃、５％ＣＯ２、２％Ｏ２状態のインキュベーター内で１４日間培養を行い、隔日で培地交換を行った。これにより得られた軟骨細胞シートを、ｉＰＳＣシートＭと呼ぶ。さらに、ＭｅｓｅｎＰＲＯ培地の代わりに２０ｖ／ｖ％ＦＢＳを含むＤＭＥＭ／Ｆ１２培地を用いたことを除いて、同様の手法により軟骨細胞シートを作製し、ｉＰＳＣシートＤと名付けた。 (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 μm 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. Prepare 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.
（実施例３）
試薬の調製
　組織学的分析に用いられた試薬類は以下の手順で調製された。
　ヘマトキシリン水溶液：フラスコにイオン交換水を入れて、沸騰させる。沸騰したイオン交換水にヘマトキシリン（品番：１．１５９３８、製造会社名：ＭＥＲＣＫ）１．５ｇを添加し、スターラーでヘマトキシリンを溶解させる。攪拌しながら、溶解液を室温に下げる。当該溶解液に酢酸（０１７－００２５６、和光純薬株式会社）を加えてｐＨ３．０に調整する。当該溶解液に、ヨウ素酸ナトリウム（１９０－０２２５２、和光純薬株式会社）０．３ｇを加え攪拌し、そして、アンモニウムみょうばん（０１８－０１８２５、和光純薬）７５ｇを加え攪拌する。撹拌後、イオン交換水を加えて１Ｌとし、そして、濾過してヘマトキシリン水溶液を得る。
　エオシン溶液：ピュアエオシン液（３２０４－２、武藤化学）３０ｍＬと９５％エタノール１２０ｍＬとを混合してエオシン液を作製する。染色において用いるときに時に、当該エオシン液は９５％エタノールで５倍希釈して、希釈された溶液がエオシン溶液として用いられる。
　ファストグリーン水溶液：ファストグリーン（１Ａ３０４、ＣＨＲＯＭＡ）８０ｍｇをイオン交換水１００ｍＬで溶解し、そして、当該溶解液を濾過して、０．０８質量％ファストグリーン水溶液を得る。
　サフラニンＯ水溶液：サフラニンＯ（１Ｂ４６３、ＣＨＲＯＭＡ）１００ｍｇをイオン交換水１００ｍＬで溶解し０．１質量％サフラニンＯ水溶液を得る。
　クエン酸緩衝液：クエン酸一水和物をイオン交換水で溶解し、０．０１Ｍクエン酸水溶液（以下Ａ液という）を得る。クエン酸三ナトリウム２水和物をイオン交換水で溶解し、０．１Ｍクエン酸ナトリウム水溶液（以下Ｂ液という）を得る。Ａ液９５ｍＬ及びＢ液４１５ｍＬを合わせ攪拌し、そして、１ＮのＮａＯＨでｐＨ６．０に調整して、０．０１Ｍクエン酸緩衝液を得る。 (Example 3)
Preparation of reagents Reagents used for histological analysis were prepared according to the following procedure.
Hematoxylin aqueous solution: Pour ion exchange water into a flask and bring to a boil. Add 1.5 g of hematoxylin (product number: 1.15938, manufacturer name: MERCK) to boiled ion-exchanged water, and dissolve the hematoxylin with a stirrer. While stirring, the solution is cooled to room temperature. Acetic acid (017-00256, Wako Pure Chemical Industries, Ltd.) is added to the solution to adjust the pH to 3.0. To the solution, 0.3 g of sodium iodate (190-02252, Wako Pure Chemical Industries, Ltd.) is added and stirred, and then 75 g of ammonium alum (018-01825, Wako Pure Chemical Industries, Ltd.) is added and stirred. After stirring, add ion-exchanged water to make up to 1 L, and filter to obtain a hematoxylin aqueous solution.
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). 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.
（実施例４）
組織学的分析に付される凍結切片の調製方法
　細胞シートは４％パラホルムアルデヒド液で固定され、ティシュー・テックＯ．Ｃ．Ｔ．コンパウンド（４５８３、サクラファインテックジャパン株式会社）に包埋されて、凍結切片が作製された。当該細胞シートの凍結切片は、温度応答性培養皿に接していた面に対して垂直に薄切された。薄切された切片の厚みは、当該切片を免疫染色に付す場合は２０μｍであり、及び、免疫染色以外の染色に付す場合は１０μｍであった。薄切された凍結切片は、以下で述べる染色に付された。 (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 μm when subjected to immunostaining, and 10 μm when subjected to staining other than immunostaining. The frozen sections were subjected to the staining described below.
（実施例５）
細胞シートのヘマトキシリン及びエオシン染色
１．前処理
　前記切片の１００％エタノールへの１０分間の浸漬が１回行われた。
２．水洗処理
　上記１．で処理された切片が、水で洗浄された。
３．イオン交換水への浸漬
　上記２．の水洗後、当該切片が、イオン交換水に５分間浸漬された。
４．ヘマトキシリン処理
　上記３．の浸漬後、当該切片が、前記ヘマトキシリン水溶液中に３～４分間浸漬された。
５．水洗処理
　上記４．の処理後、当該切片が、お湯（５０℃）で３～４分間洗浄された。
６．エオシン処理
　上記５．の洗浄後、当該切片が、前記エオシン溶液中に１０分間浸漬された。
７．脱水
　上記６．の処理後、当該切片が、エタノール列を用いて脱水された。当該エタノール列は、７つの１００体積％エタノールであった。
８．透徹
　上記７．の脱水後、当該切片が、キシレン列を用いて透徹された。すなわち、キシレンが入った容器を７つ用意し、当該切片は、各容器中のキシレンに浸漬された。
９．封入
　上記８．の透徹後、当該切片を封入した。当該封入において、封入剤としてＭａｌｉｎｏｌ（品番：２００９－３、会社名：武藤化学株式会社）を用いた。 (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. Dehydration 6 above. After treatment, the sections were dehydrated using an ethanol column. The ethanol column was seven 100% ethanol by volume.
8. Transparent 7. After dehydration, the sections were cleared using a xylene column. That is, seven containers containing xylene were prepared, and the section was immersed in the xylene in each container.
9. Enclosure 8 above. After clearing, the sections were mounted. In the mounting, Malinol (product number: 2009-3, company name: Muto Kagaku Co., Ltd.) was used as a mounting medium.
（実施例６）
細胞シートのサフラニンＯ染色
１．前処理
　前記切片の１００％エタノールへの１０分間の浸漬が１回行われた。
２．水洗処理
　上記１．で処理された切片が、水で洗浄された。
３．イオン交換水への浸漬
　上記２．の水洗後、当該切片が、イオン交換水に５分間浸漬された。
４．ヘマトキシリン処理
　上記３．の浸漬後、当該切片が、前記ヘマトキシリン水溶液中に４～５秒間浸漬された。
５．水洗処理
　上記４．の処理後、当該切片が、お湯（５０℃）で３～４分間洗浄された。
６．ファストグリーン処理
　上記５．の洗浄後、当該切片が、前記０．０８質量％ファストグリーン水溶液中に１０分間浸漬された。
７．酢酸処理
　上記６．の処理後、当該切片が、１体積％酢酸水溶液によって１又は２回処理された。
８．サフラニン処理
　上記７．の処理後、当該切片が、前記０．１質量％サフラニンＯ水溶液中に１０分間浸漬された。
９．脱水、透徹、及び封入
　上記「細胞シートのヘマトキシリン及びエオシン染色」において述べたとおりに、これらの処理が行われた。 (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.
（実施例７）
細胞シートのトルイジンブルー染色
１．前処理
　前記切片の１００％エタノールへの１０分間の浸漬が１回行われた。
２．水洗処理
　上記１．で処理された切片が、水で洗浄された。
３．イオン交換水への浸漬
　上記２．の水洗後、当該切片が、イオン交換水に５分間浸漬された。
４．ヘマトキシリン処理
　上記３．の浸漬後、当該切片が、前記ヘマトキシリン水溶液中に３～４分間浸漬された。
５．水洗処理
　上記４．の処理後、当該切片が、お湯（５０℃）で３～４分間洗浄された。
６．トルイジンブルー処理
　上記５．の処理後、当該切片が、０．０５％　トルイジンブルーｐＨ４．１（武藤化学　＃４０９７１）中に１５分間浸漬された。
７．水洗処理
　上記６．で処理された切片が、水で洗浄された。
８．脱水、透徹、及び封入
　上記「細胞シートのヘマトキシリン及びエオシン染色」において述べたとおりに、これらの処理が行われた。 (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. After treatment, the sections were immersed in 0.05% toluidine blue pH 4.1 (Muto Kagaku #40971) for 15 minutes.
7. Water washing treatment 6 above. The treated sections were washed with water.
8. Dehydration, Clearing, and Encapsulation These treatments were performed as described in "Hematoxylin and eosin staining of cell sheets" above.
（実施例８）
細胞シートのアグリカン染色
１．前処理
　切片の１００％エタノールへの５分間の浸漬が３回行われた。次に、当該切片の７０％エタノールへの５分間の浸漬が１回行われた。
２．水洗処理
　上記１．で処理された切片が、水で洗浄された。
３．イオン交換水への浸漬
　上記２．の水洗後、当該切片が、イオン交換水に５分間浸漬された。
４．抗原賦活化処理
　上記３．の浸漬後、当該切片が、前記クエン酸緩衝液中に、９８℃で１０分間浸漬された。
５．イオン交換水への浸漬
　上記４．の処理後、当該切片のイオン交換水への５分間の浸漬が３回行われた。
６．過酸化水素含有メタノール溶液への浸漬
　上記５．の浸漬後、当該切片が、過酸化水素含有メタノール溶液（０．３質量％Ｈ２Ｏ２）中に１５分間浸漬された。
７．イオン交換水への浸漬
　上記６．の浸漬後、当該切片のイオン交換水への５分間の浸漬が３回行われた。
８．ＰＢＳ洗浄
　上記７．の浸漬後、当該切片の０．０１Ｍ　ＰＢＳへの５分間の浸漬が３回行われた。
９．血清によるブロッキング
　上記８．の洗浄後、当該切片が、２．５％　ｎｏｒｍａｌ　ｈｏｒｓｅ　ｓｅｒｕｍ（ＶＥＣＴＯＲ社のＩｍｍＰＲＥＳＳ　ＨＲＰ　ＲＥＡＧＥＮＴ　ＫＩＴ　Ａｎｔｉ－ＧＯＡＴ　ＩｇＧ（＃ＭＰ－７４０５）に添付されている血清）中に１０分間浸漬された。
１０．一次抗体反応
　上記９．のブロッキング後、当該切片が、Ｇｏａｔ　ａｎｔｉ－ｈｕｍａｎ－ａｇｇｒｅｃａｎ　（ＳＣ００６、Ｒ＆Ｄ、ＢｌｏｃｋＡｉｄＴＭ　Ｂｌｏｃｋｉｎｇ　Ｓｏｌｕｔｉｏｎ（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ　Ｉｎｃ．、Ｃａｔａｌｏｇ　ｎｏ．　Ｂ１０７１０）で１０　ｍｇ／ｍＬに希釈）を用いた一次抗体反応に２時間付された。
１１．ＰＢＳ洗浄
　上記１０．の反応後、当該切片の０．０１Ｍ　ＰＢＳへの５分間の浸漬が３回行われた。
１２．二次抗体反応
　上記１１．の洗浄後、当該切片が、Ｄｏｎｋｅｙ　ａｎｔｉ－Ｇｏａｔ　ＩｇＧ（Ｈ＋Ｌ）　Ｃｒｏｓｓ－Ａｄｓｏｒｂｅｄ　Ｓｅｃｏｎｄａｒｙ　Ａｎｔｉｂｏｄｙ，　Ａｌｅｘａ　Ｆｌｕｏｒ　５４６（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ　Ｉｎｃ．、Ａ－１１０５６）ＩｍｍＰＲＥＳＳ　Ｐｏｌｙｍｅｒ　Ａｎｔｉ－Ｇｏａｔ　ＩｇＧ　Ｒｅａｇｅｎｔ　（ＭＰ－７４０５、Ｖｅｃｔｏｒ　Ｌａｂｏｒａｔｏｒｉｅｓ）を用いた二次抗体反応に１時間付された。
１３．ＰＢＳ洗浄
　上記１２．の反応後、当該切片の０．０１Ｍ　ＰＢＳへの５分間の浸漬が３回行われた。
１４．発色
　上記１３．の洗浄後、当該切片が、ＤＡＢ（０．０５Ｍ　Ｔｒｉｓ－ＨＣｌ　（２００ｍｌ）、ＤＡＢ（４０　ｍｇ）、３０％　Ｈ２Ｏ２　（３４μｌ））による発色反応に２分間付された。
１５．イオン交換水への浸漬
　上記１４．の発色反応後、当該切片が、イオン交換水に５分間浸漬された。
１６．核染色
　上記１５．の浸漬後、当該切片が、前記ヘマトキシリン水溶液によって２秒間処理された。
１７．水洗処理
　上記１６．の処理後、当該切片が水で洗浄された。
１８．脱水、透徹、及び封入
　上記「細胞シートのヘマトキシリン及びエオシン染色」において述べたとおりに、これらの処理が行われた。 (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. Immersion in ion-exchanged water 4. above. After the treatment, the sections were immersed in ion-exchanged water for 5 minutes three times.
6. Immersion in methanol solution containing hydrogen peroxide 5. After immersion, the section was immersed in a hydrogen peroxide-containing methanol solution (0.3% by mass H 2 O 2 ) for 15 minutes.
7. Immersion in ion-exchanged water 6 above. After immersion, the section was immersed in ion-exchanged water for 5 minutes three times.
8. PBS washing 7. After immersion, the sections were immersed in 0.01M PBS for 5 minutes three times.
9. Blocking with serum 8. After washing, the sections were immersed for 10 minutes in 2.5% normal horse serum (serum attached to VECTOR's ImmPRESS HRP REAGENT KIT Anti-GOAT IgG (#MP-7405)).
10. Primary antibody reaction 9 above. After blocking, the sections were treated with Goat anti-human-aggrecan (SC006, R&D, BlockAidTM Blocking Solution (Thermo Fisher Scientific Inc., Catalog no. B1071). 0) for the primary antibody reaction using 10 mg/mL). Timed.
11. PBS washing 10 above. After the reaction, the sections were immersed in 0.01M PBS for 5 minutes three times.
12. Secondary antibody reaction 11 above. After washing, the sections were treated with Donkey anti-Goat IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 (Thermo Fisher Scientific Inc., A-110 56) ImmPRESS Polymer Anti-Goat IgG Reagent (MP-7405, Vector Laboratories ) was subjected to a secondary antibody reaction for 1 hour.
13. PBS washing 12 above. After the reaction, the sections were immersed in 0.01M PBS for 5 minutes three times.
14. Color development 13 above. After washing, the sections were subjected to a color reaction with DAB (0.05 M Tris-HCl (200 ml), DAB (40 mg), 30% H 2 O 2 (34 μl)) for 2 minutes.
15. Immersion in ion-exchanged water 14 above. After the color reaction, the section was immersed in ion-exchanged water for 5 minutes.
16. Nuclear staining 15 above. After immersion, the section was treated with the hematoxylin aqueous solution for 2 seconds.
17. Water washing treatment 16 above. After treatment, the sections were washed with water.
18. Dehydration, Clearing, and Encapsulation These treatments were performed as described in "Hematoxylin and eosin staining of cell sheets" above.
（実施例９）
細胞シートのフィブロネクチン染色
　上記「細胞シートのアグリカン染色」の「１０．一次抗体反応」及び「１２．二次抗体反応」において用いた試薬を変更した以外は、同じ方法でアグリカン染色が行われた。一次抗体反応において用いた抗体は、Ａｎｔｉ－Ｆｉｂｒｏｎｅｃｔｉｎ　ｍＩｇＧ　（ＭＡＢ１９４０、Ｃｈｅｍｉｃｏｎ、１％　Ｇｏａｔ　Ｎｏｒｍａｌ　Ｓｅｒｕｍで０．２μｇ／ｍＬに希釈）であった。二次抗体反応において用いた試薬は、Ｆ（ａｂ’）２－Ｇｏａｔ　ａｎｔｉ－Ｍｏｕｓｅ　ＩｇＧ（Ｈ＋Ｌ）Ｃｒｏｓｓ－Ａｄｓｏｒｂｅｄ　Ｓｅｃｏｎｄａｒｙ　Ａｎｔｉｂｏｄｙ，　Ａｌｅｘａ　Ｆｌｕｏｒ　４８８（Ｔｈｅｒｍｏ　Ｆｉｓｈｅｒ　Ｓｃｉｅｎｔｉｆｉｃ　Ｉｎｃ．、Ａ－１１０１７）であった。 (Example 9)
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 μg/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).
（実施例１０）
細胞シートのＩ型コラーゲン染色
　上記「細胞シートのアグリカン染色」の「１０．一次抗体反応」及び「１２．二次抗体反応」において用いた試薬を変更した以外は、同じ方法でＩＩ型コラーゲン染色が行われた。一次抗体反応において用いた抗体は、Ｉ型コラーゲンに対する抗体（１３１０－０１、ＳｏｕｔｈｅｒｎＢｉｏｔｅｃｈ社、０．０１ＭのＰＢＳによる１：１００希釈）であった。二次抗体反応において用いた試薬は、ＩｍｍＰＲＥＳＳ　Ｐｏｌｙｍｅｒ　Ａｎｔｉ－Ｇｏａｔ　ＩｇＧ　Ｒｅａｇｅｎｔ　（ＭＰ－７４０５、Ｖｅｃｔｏｒ　Ｌａｂｏｒａｔｏｒｉｅｓ）であった。 (Example 10)
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).
（実施例１１）
細胞シートのＩＩ型コラーゲン染色
　上記「細胞シートのアグリカン染色」の「１０．一次抗体反応」及び「１２．二次抗体反応」において用いた試薬を変更した以外は、同じ方法でＩＩ型コラーゲン染色が行われた。一次抗体反応において用いた抗体は、ＩＩ型コラーゲン１次抗体（協和ファーマケミカル株式会社、０．０１ＭのＰＢＳにより１：１００希釈したもの）であった。二次抗体反応において用いた試薬は、ＩｍｍＰＲＥＳＳ　Ｐｏｌｙｍｅｒ　Ａｎｔｉ－Ｍｏｕｓｅ　ＩｇＧ　Ｒｅａｇｅｎｔ（ＭＰ－７４０２、Ｖｅｃｔｏｒ　Ｌａｂｏｒａｔｏｒｉｅｓ社）であった。 (Example 11)
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).
（実施例１２）
異種同所性移植実験
　細胞シートは室温へ３０分間静置してからポリフッ化ビニリデン（ＰＶＤＦ）メンブレンを用いて剥離し、異種同所性移植実験に用いた。異種同所性移植実験には１２週令のヌードラット（Ｆ３３４／ＮＪｃｌ－ｒｎｕ／ｒｎｕ、クレア）を用いた。片膝の関節軟骨に軟骨全層欠損（直径２ｍｍ、深さ１ｍｍ）を作製し、細胞シート半分を欠損部に覆う形で移植した。移植から４週後と１２週後に膝関節組織を採取し、関節軟骨組織の組織学的評価を行った。組織学的評価は、ヘマトキシリン・エオジン染色、トルイジンブルー染色、サフラニンＯ染色、Ｉ型コラーゲン免疫染色、ＩＩ型コラーゲン免疫染色、およびヒト特異的ビメンチン染色により行った。 (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. Twelve-week-old nude rats (F334/NJcl-rnu/rnu, Claire) were used for the xeno-orthotopic transplantation experiment. 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.
（実施例１３）
組織学的評価のための切片の調製方法
　軟骨部分は２０％ホルマリンに浸漬することで固定化され、そして、パラフィン中に包埋された。包埋は、包埋剤としてヒストプレップ５８６（４１５－２５７９１、和光純薬株式会社）を用い且つエンベディングコンソールシステム（Ｔｉｓｓｕｅ－Ｔｅｋ、サクラファインテックジャパン株式会社）により行われた。包埋された試料を立位の状態としたときに垂直方向に薄切された。薄切された切片の厚みは３μｍであった。薄切された切片は、スライドガラスに載せられ、以下で述べる染色に付された。 (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 μm. The thinly sliced sections were placed on glass slides and subjected to the staining described below.
（実施例１４）
軟骨のヘマトキシリン及びエオシン染色
１．脱パラフィン処理
　切片のキシレンへの５分間の浸漬が３回行われた。次に、当該切片の１００％エタノールへの５分間の浸漬が３回行われた。次に、当該切片の７０％エタノールへの５分間の浸漬が１回行われた。
２．水洗処理、イオン交換水への浸漬、ヘマトキシリン処理、水洗処理、エオシン処理、脱水、透徹、及び封入
　上記「細胞シートのヘマトキシリン及びエオシン染色」において述べたとおりに、これらの処理が行われた。 (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.
軟骨のサフラニンＯ染色
　上記「細胞シートのサフラニンＯ染色」における「１．前処理」の代わりに上記「軟骨のヘマトキシリン及びエオシン染色」で述べた「１．脱パラフィン処理」が行われたこと以外は、上記「細胞シートのサフラニンＯ染色」と同じ方法で染色が行われた。 Safranin O 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 "Safranin O staining of cell sheets" above. The staining was performed in the same manner as in the above-mentioned "Safranin O staining of cell sheets".
軟骨のトルイジンブルー染色
　上記「細胞シートのトルイジンブルー染色」における「１．前処理」の代わりに上記「軟骨のヘマトキシリン及びエオシン染色」で述べた「１．脱パラフィン処理」が行われたこと以外は、上記「細胞シートのトルイジンブルー染色」と同じ方法で染色が行われた。 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”.
軟骨のＩ型コラーゲン染色
　上記「細胞シートのＩ型コラーゲン染色」における「１．前処理」の代わりに上記「軟骨のヘマトキシリン及びエオシン染色」で述べた「１．脱パラフィン処理」が行われたこと以外は、上記「細胞シートのＩ型コラーゲン染色」と同じ方法で染色が行われた。 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.
軟骨のＩＩ型コラーゲン染色
　上記「細胞シートのＩＩ型コラーゲン染色」における「１．前処理」の代わりに上記「軟骨のヘマトキシリン及びエオシン染色」で述べた「１．脱パラフィン処理」が行われたこと以外は、上記「細胞シートのＩＩ型コラーゲン染色」と同じ方法で染色が行われた。 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.
（実施例１５）
軟骨のｈＶｉｍｅｎｔｉｎ染色
１．脱パラフィン処理
　上記「軟骨のヘマトキシリン及びエオシン染色」で述べたとおりに、脱パラフィン処理が行われた。
２．水洗処理
　上記１．の脱パラフィン処理後、当該切片が水で洗浄された。
３．イオン交換水への浸漬
　上記２．の水洗後、当該切片が、イオン交換水に５分間浸漬された。
４．抗原賦活化
　上記３．の浸漬後、当該切片が、前記クエン酸緩衝液中に、９８℃で１０分間浸漬された。
５．冷却
　上記４．の処理後、当該切片が、実験台で３０分間冷却された。
６．血清によるブロッキング
　上記５．の冷却後、当該切片が、５％のｎｏｒｍａｌ　　ｇｏａｔ　　ｓｅｒｕｍ（品番：Ｄ２０４－００－０１００、会社名：ＲＯＣＫＬＡＮＤ）を含む０．０１Ｍ　　ＰＢＳ（０．２％のＴｗｅｅｎを含む）により１時間処理された。
７．抗体反応
　上記６．のブロッキング後、当該切片が、ａｎｔｉ－ｈｕｍａｎ　　Ｖｉｍｅｎｔｉｎ抗体Ａｌｅｘａ　　Ｆｌｕｏｒ　６４７　　Ｃｏｎｊｕｇａｔｅ　（＃９８５６、ＣＳＴ社、０．０１ＭのＰＢＳで１：１００　希釈）を用いた抗体反応に４℃で１６時間付された。
８．ＰＢＳ洗浄
　上記７．の反応後、当該切片の０．０１Ｍ　　ＰＢＳへの５分間の浸漬が３回行われた。
９．封入
　上記８．の洗浄後、当該切片が、ＶＥＣＴＡＳＨＩＥＬＤ　　ＨａｒｄＳｅｔ　　Ａｎｔｉｆａｄｅ　　Ｍｏｕｎｔｉｎｇ　　Ｍｅｄｉｕｍ　　ｗｉｔｈ　　ＤＡＰＩ（Ｖｅｃｔｏｒ　Ｌａｂｏｒａｔｏｒｉｅｓ社）を用いて封入された。 (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. After cooling, the sections were treated with 0.01M PBS (containing 0.2% Tween) containing 5% normal goat serum (product number: D204-00-0100, company name: ROCKLAND) for 1 hour. .
7. Antibody reaction 6 above. After blocking, the sections were subjected to an antibody reaction using anti-human Vimentin antibody Alexa Fluor 647 Conjugate (#9856, CST, diluted 1:100 in 0.01M PBS) for 16 hours at 4°C.
8. PBS washing 7. After the reaction, the sections were immersed in 0.01M PBS for 5 minutes three times.
9. Enclosure 8 above. After washing, the sections were mounted using VECTASHIELD HardSet Antifade Mounting Medium with DAPI (Vector Laboratories).
（実施例１６）
細胞表面マーカーＣＤ５６の測定
　前記軟骨細胞シートの作製により得られた軟骨細胞シートから細胞を単離し、細胞表面マーカーＣＤ５６の単染色を行い、フローサイトメトリーにより測定してＣＤ５６陽性細胞率を明らかにした。 (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. .
（実施例１７）
タンパク質分泌量の測定
　前記軟骨細胞シートの作製により得られた軟骨細胞シートに対して、培養しているインサート内に２ｍｌ、インサートを設置している培養皿に３ｍｌの１％ＦＢＳと１％抗生剤を含むＤＭＥＭ／Ｆ１２培地を添加し、３７℃、５％ＣＯ２、通常酸素状態で７２時間培養し（すなわち、培養７２時間）、その後インサート内の２ｍｌの培地を回収し、ＥＬＩＳＡ法によりＴＧＦ－β１とＭＩＡのタンパク質濃度を定量した。 (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- β1 and MIA protein concentrations were quantified.
（結果）
　作製した軟骨細胞シートｉＰＳＣシートＭおよびｉＰＳＣシートＤについて、ヘマトキシリン・エオジン染色（ＨＥ）、サフラニンＯ染色（ＳａｆＯ）、トルイジンブルー染色（ＴＢ）、Ｉ型コラーゲン免疫染色（ＣＯＬ１）、ＩＩ型コラーゲン免疫染色（ＣＯＬＩＩ）、アグリカン染色（ＡＣＡＮ）、およびフィブロネクチン染色（ＦＮ）を行った。結果を図１に示す。ｉＰＳＣシートＭおよびｉＰＳＣシートＤは双方とも、Ｉ型コラーゲンに対する抗体を用いた免疫染色において陽性、ＩＩ型コラーゲンに対する抗体を用いた免疫染色において陰性、サフラニンＯ染色に対して陰性、アグリカンに対する抗体を用いた免疫染色において陽性を示した。これは特開２０２０－００６２０７号公報に開示の通り、多能性幹細胞に由来する軟骨細胞シートも軟骨修復作用を有し得ることを示唆する。 (result)
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). The results are shown in Figure 1. Both 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.
　次に、ｉＰＳＣシートＭおよびｉＰＳＣシートＤを用いて異種同所性移植実験を行った。移植から４週後の結果を図２に、移植から１２週後の結果を図３に示す。
　非治療群においてはサフラニンＯの染色性およびトルイジンブルーの異染色性は確認されず、わずかに再生した組織では主にＩ型コラーゲンの染色性が確認され、ＩＩ型コラーゲンの染色性は確認されなかった。従来の製造方法にて作製したｉＰＳＣシートＤを移植した群では、修復再生した関節軟骨組織での弱いサフラニンＯの染色性、弱いトルイジンブルーの異染色性が確認された。また、Ｉ型コラーゲンの強い染色性が確認され、わずかにＩＩ型コラーゲンの染色性が確認された。 Next, 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.
In the non-treated group, 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.
　本発明の製造方法にて作製したｉＰＳＣシートＭを移植した群では、修復再生した関節軟骨組織での強いサフラニンＯの染色性、強いトルイジンブルーの異染色性が確認された。また、Ｉ型コラーゲンの染色性は表層部分のみで確認され、強いＩＩ型コラーゲンの染色性が確認された。ヒト特異的ビメンチン染色の結果、修復再生した関節軟骨部位は主に移植したヒト細胞で構成されていることが確認され、ｉＰＳＣシートＤ群は４週後では軟骨細胞の密度が比較的高く、１２週後でも軟骨細胞の密度が比較的高い部分と低い部分が混在しているのに対して、ｉＰＳＣシートＭ群においては軟骨細胞の密度が比較的低く、細胞ラクナが形成され始めていることが確認された。これは、本発明の製造方法にて作製したｉＰＳＣシートＭが軟骨の修復・再生に適していることを示唆している。 In the group in which 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. Even after a week, 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.
　多能性幹細胞由来のｉＰＳＣシートＭおよびｉＰＳＣシートＤ、ならびに特開２０２０－００６２０７号公報に記載の方法で作製した多指症患者軟骨組織由来の軟骨細胞シート（以下、ＰＤＣシートとも称する）の特徴を明らかにするため、細胞表面マーカーＣＤ５６の測定、ＴＧＦ－β１とＭＩＡのタンパク質分泌量の測定、および定量的逆転写ＰＣＲ法によるＣＯＬ２Ａ１の遺伝子発現解析を行った。結果を図４、５および６に示す。 Characteristics of 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 In order to clarify this, we measured the cell surface marker CD56, measured the protein secretion levels of TGF-β1 and MIA, and analyzed the gene expression of COL2A1 using quantitative reverse transcription PCR. The results are shown in Figures 4, 5 and 6.
　ｉＰＳＣシートＭは、ＣＤ５６陽性細胞率が９０．３～９６．３％、ＴＧＦ－β１の分泌量は1シート（４．２ｃｍ２）あたり１５．２～４０ｎｇ以上（すなわち、３．６２～９．５２ｎｇ／ｃｍ２以上：ｎ＝７）、ＭＩＡの分泌量は１シート（４．２ｃｍ２）あたり５４．３４～１００ｎｇ以上（すなわち、１２．９４～２３．８１ｎｇ／ｃｍ２以上：ｎ＝８）、ＣＯＬ２Ａ１遺伝子の発現量はＰＤＣの１０００倍程度であり、ｉＰＳＣシートＤやＰＤＣと比較してＣＤ５６陽性細胞率、ＴＧＦ－β１とＭＩＡの分泌量、およびＣＯＬ２Ａ１の遺伝子発現で有意に高い値を示した。 The iPSC sheet M has a CD56 positive cell rate of 90.3 to 96.3%, and a secreted amount of TGF-β1 of 15.2 to 40 ng or more per sheet (4.2 cm 2 ) (that is, 3.62 to 9.2 ng). 52 ng/cm 2 or more: n = 7), the secretion amount of MIA is 54.34 to 100 ng or more per sheet (4.2 cm 2 ) (i.e., 12.94 to 23.81 ng/cm 2 or more: n = 8) , 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-β1 and MIA, and the gene expression of COL2A1 were significantly higher. Ta.
（参考例）
　ｉＰＳＣシートＤおよびＰＤＣシートについて、異種同所性移植実験で移植から４週後のみの評価を行った。結果を図７に示す。ＰＤＣシート移植群においては、修復再生した関節軟骨組織での強いサフラニンＯの染色性、強いトルイジンブルーの異染色性が確認された。また、Ｉ型コラーゲンの染色性は表層部分のみで確認され、強いＩＩ型コラーゲンの染色性が確認された。ヒト特異的ビメンチン染色の結果、修復再生した関節軟骨部位は主に移植したヒト細胞で構成されていることが確認され、軟骨細胞の密度は比較的低く細胞ラクナの形成が多く確認された。従来法で作製したｉＰＳＣシートＤの移植群では、修復再生した関節軟骨組織でのサフラニンＯの染色性は確認されず、トルイジンブルーの異染色性はわずかにしか確認されなかった。また、修復再生した関節軟骨組織ではＩ型コラーゲンの染色性が確認され、ＩＩ型コラーゲンの染色性はほぼ確認されなかった。ヒト特異的ビメンチン染色の結果、再生した関節軟骨部位は主に移植したヒト細胞で構成されていることが確認され、軟骨細胞の密度は比較的高く細胞ラクナの形成はほぼ確認されなかった。この結果により、従来法で作製された多指症患者軟骨組織に由来する軟骨細胞シートは軟骨の修復・再生が可能であることに対し、従来法で作製された多能性幹細胞に由来する軟骨細胞シートは軟骨の修復・再生に適さないことが明らかとなった。 (Reference example)
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. In the PDC sheet transplantation group, strong safranin O staining and strong toluidine blue metachromaticity 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 density of chondrocytes was relatively low, and the formation of many cell lacunae was confirmed. In the transplant group of iPSC sheet D produced by the conventional method, no staining of Safranin O was observed in the repaired and regenerated articular cartilage tissue, and only slight metachromaticity of Toluidine Blue was observed. Further, in the repaired and regenerated articular cartilage tissue, type I collagen staining was confirmed, and type II collagen staining was almost not observed. As a result of human-specific vimentin staining, it was confirmed that the regenerated articular cartilage site was mainly composed of transplanted human cells, and the density of chondrocytes was relatively high, and the formation of cell lacunae was hardly observed. These results show that chondrocyte sheets derived from polydactyly patient cartilage tissues produced using conventional methods can repair and regenerate cartilage, whereas cartilage cells derived from pluripotent stem cells produced using conventional methods can repair and regenerate cartilage. It has become clear that cell sheets are not suitable for cartilage repair and regeneration.
（実施例１８）
　さらに、ｉＰＳＣシートＭとｉＰＳＣシートＤとの間で、差次的発現タンパク質（Differentially expressed proteins : DEPs）をデータ非依存的解析（DIA）－質量分析（MS）プロテオミクスにより解析した。４１個のタンパク質がｉＰＳＣシートＭにおいて、ｉＰＳＣシートＤと比較して発現増加するタンパク質として同定され、２０個のタンパク質がｉＰＳＣシートＭにおいて、ｉＰＳＣシートＤと比較して発現減少するタンパク質として同定された。これら計６１個のタンパク質名およびその量的変化を、図８にヒートマップで示した。これらタンパク質の発現量の増減は、本発明の軟骨修復用細胞シートの一例であるｉＰＳＣシートＭに特徴的であることが示唆される。 (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.
　次いで、当該、発現増加する４１個のタンパク質と発現減少する２０個のタンパク質に関し、遺伝子オントロジー（GO）タームを図９に示した。GOタームの解析にはMetascapeを用いた。また、主なGOタームに分類されるDEPsの一覧を表１に示す。これらで示されたGOタームは、ｉＰＳＣシートＭに特徴的であると考えられ、本発明の軟骨修復用細胞シートが有する特徴と関連する生物学的プロセスを示唆していると考えられる。
Figure JPOXMLDOC01-appb-T000001
  Next, gene ontology (GO) terms are shown in FIG. 9 for the 41 proteins whose expression increases and the 20 proteins whose expression decreases. Metascape was used to analyze GO terms. In addition, Table 1 shows a list of DEPs classified into major GO terms. The GO terms shown above are considered to be characteristic of iPSC sheet M, and are considered to suggest biological processes related to the characteristics of the cell sheet for cartilage repair of the present invention.
Figure JPOXMLDOC01-appb-T000001
　以上より、従来法で作製された多能性幹細胞に由来する軟骨細胞シートは軟骨の修復・再生に適さないが、本発明の方法はこの欠点を克服し、多能性幹細胞に由来する本発明の軟骨細胞シートは軟骨の修復・再生に適切であることが明らかとなった。 From the above, 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.
　本発明により、細胞ソースとして多能性幹細胞を用いた、関節軟骨の修復・再生に適した軟骨細胞シートを製造できる。また、細胞ソースとして多能性幹細胞を用いることで、品質の安定した軟骨細胞シートを作製することが可能となり、確立されたドナー細胞より無制限に多能性幹細胞を増やすことができるため、必要な細胞数を容易に確保できる。さらに、多指症患者由来軟骨組織などの同種細胞のドナー選定には継続してコストがかかることから、細胞ソースとして多能性幹細胞を用いることでコストを削減し得る。
　加えて、一態様においては、本発明の軟骨細胞シートは接着性に優れ、広範ですり減った軟骨欠損や変性部位に対しての移植が容易となる。例えば、超高齢化社会を迎え、日本での有症状者が800万人ともいわれている変形性膝関節症に対して、人工関節に依拠しない根治的な治療法となる可能性もあり、医療費削減や健康寿命の延伸の観点からも極めて有用である。 According to the present invention, a chondrocyte sheet suitable for repairing and regenerating articular cartilage can be produced using pluripotent stem cells as a cell source. In addition, by using pluripotent stem cells as a cell source, it is possible to create chondrocyte sheets with stable quality, and pluripotent stem cells can be increased without limit from established donor cells. Cell numbers can be easily secured. Furthermore, since selecting a donor for allogeneic cells such as cartilage tissue derived from polydactyly patients continues to be costly, using pluripotent stem cells as a cell source may reduce costs.
In addition, in one embodiment, 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. For example, with the arrival of a super-aging society, it is said that 8 million people in Japan have symptoms of knee osteoarthritis, and this could become a radical treatment that does not rely on artificial joints. It is also extremely useful from the perspective of cost reduction and extending healthy life expectancy.

Claims (15)

  1. 　軟骨修復用細胞シートであって、
    　該軟骨修復用細胞シートは、多能性幹細胞から誘導された誘導軟骨細胞の培養物から形成され、
    　前記培養物は、前記誘導軟骨細胞を低血清培地にて培養して得られたものである、軟骨修復用細胞シート。     A cell sheet for cartilage repair,
    The 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.
  2. 　以下の（１）～（３）のすべてを満たす、請求項１に記載の軟骨修復用細胞シート：
    （１）培養７２時間における単位面積当たりのＴｒａｎｓｆｏｒｍｉｎｇ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ－β１（ＴＧＦ－β１）の分泌量が３．０ｎｇ／ｃｍ２以上である。
    （２）培養７２時間における単位面積当たりのＭｅｌａｎｏｍａ　ｉｎｈｉｂｉｔｏｒｙ　ａｃｔｉｖｉｔｙ（ＭＩＡ）の分泌量が１０．０ｎｇ／ｃｍ２以上である。
    （３）細胞シートに含まれる全細胞数当たりのＣＤ５６陽性細胞数の割合が８５％以上である。     The cell sheet for cartilage repair according to claim 1, which satisfies all of the following (1) to (3):
    (1) The amount of transforming growth factor-β1 (TGF-β1) 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.
    (3) The ratio of the number of CD56 positive cells to the total number of cells contained in the cell sheet is 85% or more.
  3. 　以下の（１）～（３）のすべてを満たす、請求項１または２に記載の軟骨修復用細胞シート：
    （１）培養７２時間における単位面積当たりのＴｒａｎｓｆｏｒｍｉｎｇ　ｇｒｏｗｔｈ　ｆａｃｔｏｒ－β１（ＴＧＦ－β１）の分泌量が３．５ｎｇ／ｃｍ２以上である。
    （２）培養７２時間における単位面積当たりのＭｅｌａｎｏｍａ　ｉｎｈｉｂｉｔｏｒｙ　ａｃｔｉｖｉｔｙ（ＭＩＡ）の分泌量が１２．５ｎｇ／ｃｍ２以上である。
    （３）細胞シートに含まれる全細胞数当たりのＣＤ５６陽性細胞数の割合が９０％以上である。     The cell sheet for cartilage repair according to claim 1 or 2, which satisfies all of the following (1) to (3):
    (1) The amount of transforming growth factor-β1 (TGF-β1) secreted per unit area during 72 hours of culture is 3.5 ng/cm 2 or more.
    (2) The amount of Melanoma inhibitory activity (MIA) secreted per unit area during 72 hours of culture is 12.5 ng/cm 2 or more.
    (3) The ratio of the number of CD56 positive cells to the total number of cells contained in the cell sheet is 90% or more.
  4. 　以下の（Ａ）～（Ｄ）のすべてを満たす、請求項１または２に記載の軟骨修復用細胞シート：
    （Ａ）Ｉ型コラーゲンに対する抗体を用いた免疫染色において陽性である。
    （Ｂ）ＩＩ型コラーゲンに対する抗体を用いた免疫染色において陰性である。
    （Ｃ）サフラニンＯ染色において陰性である。
    （Ｄ）アグリカンに対する抗体を用いた免疫染色において陽性である。     The cell sheet for cartilage repair according to claim 1 or 2, 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. 　前記低血清培地の血清濃度が、５ｖ／ｖ％以下である、請求項１または２に記載の軟骨修復用細胞シート。 The cell sheet for cartilage repair according to claim 1 or 2, wherein the serum concentration of the low serum medium is 5 v/v% or less.
  6. 　前記多能性幹細胞は、ヒト由来のｉＰＳ細胞である、請求項１または２に記載の軟骨修復用細胞シート。 The cell sheet for cartilage repair according to claim 1 or 2, wherein the pluripotent stem cells are human-derived iPS cells.
  7. 　ヒト多指症患者軟骨組織由来軟骨細胞シートにおけるＣＯＬ２Ａ１遺伝子の発現量を１としたとき、ＣＯＬ２Ａ１遺伝子の発現量が１００以上である、請求項１または２に記載の軟骨修復用細胞シート。 The cell sheet for cartilage repair according to claim 1 or 2, wherein the expression level of the COL2A1 gene is 100 or more when the expression level of the COL2A1 gene in the chondrocyte sheet derived from cartilage tissue of a human polydactyly patient is 1.
  8. 　前記誘導軟骨細胞を低血清培地にて培養することが、低酸素条件下で行われるものである、請求項１または２に記載の軟骨修復用細胞シート。 The cell sheet for cartilage repair according to claim 1 or 2, wherein the induced chondrocytes are cultured in a low serum medium under hypoxic conditions.
  9. 　前記低酸素条件下は、培養雰囲気中の酸素濃度が５ｖ／ｖ％以下の条件下である、請求項８に記載の軟骨修復用細胞シート。 The cell sheet for cartilage repair according to claim 8, wherein the hypoxic conditions are conditions in which the oxygen concentration in the culture atmosphere is 5 v/v% or less.
  10. 　軟骨修復用細胞シートの製造方法であって、
    　多能性幹細胞から誘導された誘導軟骨細胞を低血清培地にて培養する工程を含む、方法。     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. 　前記低血清培地の血清濃度が、５ｖ／ｖ％以下である、請求項１０に記載の方法。 The method according to claim 10, wherein the serum concentration of the low serum medium is 5 v/v% or less.
  12. 　前記誘導軟骨細胞を低血清培地にて培養する工程が、培養雰囲気中の酸素濃度が５ｖ／ｖ％以下の条件下で前記誘導軟骨細胞を培養する工程を含む、請求項１０または１１に記載の方法。 12. The induced chondrocytes according to claim 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.
  13. 　多能性幹細胞から誘導された誘導軟骨細胞を培養する方法であって、
    　該誘導軟骨細胞を低血清培地にて培養する工程を含む、方法。     A method for culturing induced chondrocytes derived from pluripotent stem cells, the method comprising:
    A method comprising culturing the induced chondrocytes in a low serum medium.
  14. 　前記低血清培地の血清濃度が、５ｖ／ｖ％以下である、請求項１３に記載の方法。 The method according to claim 13, wherein the serum concentration of the low serum medium is 5 v/v% or less.
  15. 　前記誘導軟骨細胞を低血清培地にて培養する工程が、培養雰囲気中の酸素濃度が５ｖ／ｖ％以下の条件下で前記誘導軟骨細胞を培養する工程を含む、請求項１３または１４に記載の方法。 15. The induced chondrocytes according to claim 13 or 14, 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.
PCT/JP2023/020854 2022-06-03 2023-06-05 Cartilage-repairing cell sheet and method for producing same WO2023234421A1 (en)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006093151A1 (en) * 2005-02-28 2006-09-08 Cellseed Inc. Cultured cell sheet, production method thereof, and application method thereof
WO2018154813A1 (en) * 2017-02-24 2018-08-30 株式会社セルシード Tissue regeneration cultured cell sheet, method for producing same, and use of same
WO2021182633A1 (en) * 2020-03-13 2021-09-16 学校法人東海大学 Cell culture, method for evaluating cell culture, method for producing cell culture, and marker for use in evaluation of chondroid tissue formation property
JP2022042998A (en) * 2020-09-03 2022-03-15 学校法人東海大学 Cell sheet pieces, syringe containing cell sheet pieces, method for producing cell sheet pieces, and method for using the same

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006093151A1 (en) * 2005-02-28 2006-09-08 Cellseed Inc. Cultured cell sheet, production method thereof, and application method thereof
WO2018154813A1 (en) * 2017-02-24 2018-08-30 株式会社セルシード Tissue regeneration cultured cell sheet, method for producing same, and use of same
WO2021182633A1 (en) * 2020-03-13 2021-09-16 学校法人東海大学 Cell culture, method for evaluating cell culture, method for producing cell culture, and marker for use in evaluation of chondroid tissue formation property
JP2022042998A (en) * 2020-09-03 2022-03-15 学校法人東海大学 Cell sheet pieces, syringe containing cell sheet pieces, method for producing cell sheet pieces, and method for using the same

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ITOKAZU MAKI, WAKITANI SHIGEYUKI, MERA HISASHI, TAMAMURA YOSHIHIRO, SATO YASUSHI, TAKAGI MUTSUMI, NAKAMURA HIROAKI: "Transplantation of Scaffold-Free Cartilage-Like Cell-Sheets Made from Human Bone Marrow Mesenchymal Stem Cells for Cartilage Repair : A Preclinical Study", CARTILAGE, vol. 7, no. 4, 1 October 2016 (2016-10-01), pages 361 - 372, XP093081650, ISSN: 1947-6035, DOI: 10.1177/1947603515627342 *
UCHIYAMA, RYOKA EL AL.: "1-6-2: Improvement of hyaline cartilage repair ability of iPS cell-derived chondrocyte sheet under low serum and hypoxic conditions", THE JOURNAL OF JAPANESE ORTHOPAEDIC SURGICAL SOCIETY, NIPPON SEIKEI GEKA GAKKAI, TOKYO, JP, vol. 96, no. 8, 1 August 2022 (2022-08-01), JP , pages S1556, XP009551399, ISSN: 0021-5325 *

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