Cartilage-repairing cell sheet and method for producing the same

Abstract

To provide a cartilage cell sheet obtained using pluripotent stem cells as a cell source.SOLUTION: Provided is 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.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] The present invention relates to a cell sheet for cartilage repair produced from pluripotent stem cell-derived chondrocytes.

Background Art

[0002] Non-Patent Document 1 and Patent Document 1 describe a method in which cartilage particles are prepared from pluripotent stem cells and transplanted into a defect site of articular cartilage, and the cartilage particles bind to surrounding tissues to supplement the defective articular cartilage. When transplanting cartilage particles into a defect site of articular cartilage, it is necessary to fix them to the surroundings with fibrin glue or the like. However, since the fixing force is weak, the cartilage particles may fall off after transplantation. In addition, since fixation is difficult, transplantation for extensive and worn cartilage defects or degeneration such as cartilage defects in osteoarthritis is difficult. Therefore, there is a need for a cartilage cell sheet that has excellent adhesiveness and enables transplantation for extensive defects and degeneration. In addition, while cartilage particles are expected to supplement defective articular cartilage and the cartilage particles themselves to function as articular cartilage, the mechanism of action of the cartilage cell sheet is considered to mainly have an effect of covering the defect site and a paracrine effect at the same time. The cartilage cell sheet does not have the characteristics of articular cartilage at the time of transplantation, but it is considered to activate host cells and promote the repair and regeneration of hyaline cartilage-like tissue close to conventional articular cartilage.

[0003] Non-Patent Document 2, Non-Patent Document 3, Patent Document 2, and Patent Document 3 disclose chondrocyte sheets using cartilage tissue derived from a living body such as a polydactyly patient as a cell source and a method for producing the same. It is also described that such chondrocyte sheets are applicable to osteoarthritis of the knee and that their safety and efficacy have been confirmed in clinical studies. However, as problems in using chondrocyte sheets using cartilage tissue derived from a living body such as a polydactyly patient as a cell source for allogeneic transplantation, there are donor shortage, the quality of the chondrocyte sheet varies depending on the characteristics of the donor cells, and it is necessary to amplify by subculture to secure the number of cells for commercial use, and the quality of the cells deteriorates during 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 amplified without limit, it is also easy to secure the necessary number of cells.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

[0006] An object of the present invention is to provide a chondrocyte sheet using pluripotent stem cells as a cell source. [Means for Solving the Problems]

[0007] The present inventors have found that in a conventional method for producing a chondrocyte sheet using a cartilage tissue derived from a living body such as a polydactyly patient as a cell source, when pluripotent stem cells are used instead of the cartilage tissue derived from the living body as the cell source, the chondrocyte sheet produced is not confirmed to be effective in a transplantation model. On the other hand, when pluripotent stem cells are used as the cell source, chondrocytes derived from pluripotent stem cells (hereinafter also referred to as induced chondrocytes) are cultured in a low serum medium, and it has been found that a chondrocyte sheet suitable for the repair and regeneration of articular cartilage can be produced, and the present invention has been completed. The present invention includes the following aspects.

[0008] [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, wherein the culture is obtained by culturing the induced chondrocytes in a low serum medium, and is a cell sheet for cartilage repair. [2] The cell sheet for cartilage repair according to [1], satisfying all of the following (1) to (3): (1) The secretion amount of Transforming growth factor-β1 (TGF-β1) per unit area at 72 hours of culture is 3.0 ng / cm or more. 2 (2) The secretion amount of Melanoma inhibitory activity (MIA) per unit area at 72 hours of culture is 10.0 ng / cm or more. 2 (3) The ratio of the number of CD56-positive cells per 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], satisfying all of the following (1) to (3): (1) The secretion amount of Transforming growth factor-β1 (TGF-β1) per unit area at 72 hours of culture is 3.5 ng / cm or more. 2 (2) The secretion amount of Melanoma inhibitory activity (MIA) per unit area at 72 hours of culture is 12.5 ng / cm or more. 2 (3) The ratio of the number of CD56-positive cells per 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], satisfying all of the following (A) to (D): (A) Positive in immunostaining using an antibody against type I collagen. (B) Negative in immunostaining using an antibody against type II collagen. It is negative in safranin O staining. It is 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. Cell sheet for cartilage repair as described above. [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] When the expression level of the COL2A1 gene in the chondrocyte sheet derived from the cartilage tissue of a human polydactyly patient is set to 1, the cell sheet for cartilage repair according to any one of [1] to [6], wherein the expression level of the COL2A1 gene is 100 or more. Cell sheet for cartilage repair as described above. [8] The cell sheet for cartilage repair according to any one of [1] to [7], wherein the step of culturing the induced chondrocytes in a low serum medium is performed under hypoxic conditions. [9] The cell sheet for cartilage repair according to [8], wherein the hypoxic conditions are conditions where the oxygen concentration in the culture atmosphere is 5 v / v% or less.

[0009]

[10] A method for producing a cell sheet for cartilage repair, comprising: a step of 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] The method according to

[10] or

[11] , wherein the step of culturing the induced chondrocytes in a low serum medium includes a step of culturing the induced chondrocytes under conditions where the oxygen concentration in the culture atmosphere is 5 v / v% or less.

[13] A method for culturing induced chondrocytes derived from pluripotent stem cells, comprising: a step of 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] The method according to

[13] or

[14] , wherein the step of culturing the induced chondrocytes in a low-serum medium includes culturing the induced chondrocytes under the condition that the oxygen concentration in the culture atmosphere is 5 v / v% or less. [Advantages of the Invention]

[0010] According to the present invention, it is possible to produce a chondrocyte sheet suitable for the repair and regeneration of articular cartilage using pluripotent stem cells as a cell source. In addition, by using pluripotent stem cells as a cell source, it becomes possible to produce a chondrocyte sheet with stable quality, and since pluripotent stem cells can be increased without limit compared to established donor cells, the required number of cells can be easily secured. Furthermore, since continuous costs are required for the selection of donors of allogeneic cells such as chondral tissues derived from polydactyly patients, the cost can be reduced by using pluripotent stem cells as a cell source. In addition, in one aspect, the chondrocyte sheet of the present invention has excellent adhesiveness and is easy to transplant to a wide range of abraded cartilage defects and degenerated sites. For example, for osteoarthritis, which has reached an ultra-aging society and the number of symptomatic patients in Japan is said to be 8 million, it may become a radical treatment method that does not rely on artificial joints and is extremely useful from the viewpoints of reducing medical costs and extending the healthy life span. [Brief Description of the Drawings]

[0011]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Embodiments for Carrying Out the Invention

[0012] The present invention will be described in detail below. In the present invention, v / v% means "volume percent". Also, the range of each numerical value in the present invention does not exclude the fact that it is substantially within that numerical range, and for example, it may mean up to ±10% of that numerical value.

[0013] <1> Method of the Present Invention One aspect 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.

[0014] 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 cell sheet for cartilage repair. Further, the chondrocyte sheet produced by the method of the present invention may specifically be the chondrocyte sheet of the present invention described below.

[0015] Another aspect of the method of the present invention is a method for culturing induced chondrocytes derived from pluripotent stem cells, which includes a step of culturing the induced chondrocytes in a low-serum medium.

[0016] The term "pluripotent stem cell" refers to a stem cell that has the pluripotency to differentiate into all cells existing in a living body and also has the ability to proliferate. Although not particularly limited, pluripotent stem cells include, for example, embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transfer, spermatogonial stem cells (GS cells), embryonic germ cells (EG cells), induced pluripotent stem (iPS) cells, pluripotent cells (Muse cells) derived from cultured fibroblasts or bone marrow stem cells, and the like. As the pluripotent stem cell, preferably, it is an ES cell, an ntES cell, and / or an iPS cell, and particularly preferably, it is an iPS cell. The origin of the pluripotent stem cell is not particularly limited as long as the effects of the present invention can be obtained, but it is preferably a mammal, more preferably a primate such as a human, a rodent such as a mouse or a rat, a dog, a cat, a rabbit, a cow, a horse, a goat, a sheep, a pig, or a chicken, and even more preferably a human.

[0017] The pluripotent stem cell may be any combination of the above examples of pluripotent stem cells and origins. The pluripotent stem cell is preferably an ES cell, an ntES cell, and / or an iPS cell derived from a mammal, more preferably an ES cell, an ntES cell, and / or an iPS cell derived from a primate such as a human, a rodent such as a mouse or a rat, a dog, a cat, a rabbit, a cow, a horse, a goat, a sheep, a pig, or a chicken, and even more preferably an ES cell, an ntES cell, and / or an iPS cell derived from a human. Further, when the pluripotent stem cell is an iPS cell, preferably, it is an iPS cell derived from a mammal, more preferably an iPS cell derived from a primate such as a human, a rodent such as a mouse or a rat, a dog, a cat, a rabbit, a cow, a horse, a goat, a sheep, a pig, or a chicken, and even more preferably an iPS cell derived from a human.

[0018] Methods for producing iPS cells are known in the art and can be produced, for example, by introducing reprogramming factors into any somatic cells. Here, reprogramming factors include, for example, genes or gene products such as 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 or Glis1, etc. These reprogramming factors may be used alone or in combination. Combinations of reprogramming factors include 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., 26:7 95-797, Shi Y, et al. (2008), Cell StemCell, 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 StemCell, 3, 568-574, Zhao Y, et al.( (2008), Cell StemCell, 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 NatlA cad SciU S A. 106:8912-8917, KimJB, et al. (2 009), Nature. 461:649-643, Ichida JK, et al. (2009), Cell StemCell. 5:491-503, Heng JC, et al. (2010), Cell Stem Cell. 6:167-74, Han J, e t al. (2010), Nature. 463:1096-100, Mali P, et al. (2010), Stem Cells. 28:713-720, MaekawaM, et al. (2011), Nature. 474:225-9 are exemplified. Exemplified.

[0019] The somatic cells used to obtain iPS cells are not particularly limited and may include, for example, somatic cells of a fetus (offspring), somatic cells of a newborn (offspring), and somatic cells of a mature, healthy, or diseased individual, and may also include any of primary cultured cells, subcultured cells, and established cell lines. Specifically, somatic cells may be, for example, (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, dental pulp stem cells, etc., (2) tissue progenitor cells, (3) blood cells (peripheral blood cells, cord blood cells, etc.), lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (such as skin cells), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (such as pancreatic exocrine cells), brain cells, lung cells, kidney cells, and adipocytes, etc., but are not limited thereto.

[0020] "Chondrocyte" means a cell that produces an extracellular matrix constituting cartilage, such as collagen, or a progenitor cell that becomes such a cell. Such chondrocytes may also be cells that express chondrocyte markers, and type II collagen (COL2A1) or SOX9 is exemplified as a chondrocyte marker. "Induced chondrocyte" means a chondrocyte obtained by being induced from undifferentiated cells. In this case, "undifferentiated cells" means cells that can differentiate into chondrocytes, and it is preferable to use pluripotent stem cells as the cell source. Also, "induced" may mean being induced to differentiate by any method.

[0021] Therefore, the "induced chondrocytes derived from pluripotent stem cells" may be chondrocytes obtained by inducing differentiation of pluripotent stem cells by any method using pluripotent stem cells as a cell source. Specifically, for example, they may be chondrocytes obtained by inducing differentiation from pluripotent stem cells in vitro. As a method for inducing induced chondrocytes from pluripotent stem cells, any known method can be used. For example, the methods described in 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 can be mentioned.

[0022] The method of the present invention includes a step of culturing induced chondrocytes derived from pluripotent stem cells in a low-serum medium. The "low-serum medium" in the present invention refers to a medium having a lower serum concentration compared to the media for culturing chondrocytes described in conventional methods for manufacturing chondrocyte sheets, for example, 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 / 0931 51, or JP-A-2020-006207. The serum concentration of the medium in the conventional method for manufacturing chondrocyte sheets can typically be 20 v / v%. The serum concentration of the low serum medium may be, for example, 10 v / v% or less, 5 v / v% or less, 4 v / v% or less, 3 v / v% or less, 2.5 v / v% or less, 2.2 v / v% or less, 2 v / v% or less, and may also be 0 v / v% or more, 0.5 v / v% or more, 1 v / v% or more, 1.5 v / v% or more, 1.8 v / v% or more, 2 v / v% or more, and may be any non - conflicting combination thereof. Specifically, the serum concentration of the low serum medium in the present invention may be, 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, 0.5 v / v% or more and 2.5 v / v% or less, 0.5 v / v% or more and 2 v / v% or less, 1.5 v / v% or more and 2.5 v / v% or less, 1.8 v / v% or more and 2.2 v / v% or less, or 2 v / v%. However, when the serum concentration of the low serum medium in the present invention is 2 v / v%, it does not exclude an equivalent range where the serum concentration is substantially 2 v / v%. As such a range, for example, 1.8 v / v% or more and 2.2 v / v% or less can be considered, but it is not limited thereto.

[0023] Examples of the serum used include, but are not limited to, 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., and FBS or patient's own serum is preferred.

[0024] The low serum medium of the present invention is not particularly limited other than the serum concentration and may be any known medium, but a medium suitable for mesenchymal stem cells is preferred. Examples of the medium suitable for mesenchymal stem cells include, but are not limited to, MesenPRO and StemPRO.

[0025] The step of culturing the induced chondrocytes derived from the pluripotent stem cells of the present invention in a low serum medium may include the step of culturing the induced chondrocytes using a culture vessel whose surface is coated with a temperature - responsive polymer. The temperature - responsive polymer is not particularly limited, and examples thereof include, for example, poly - N - isopropylacrylamide.

[0026] The step of culturing the induced chondrocytes derived from the pluripotent stem cells of the present invention in a low-serum medium may include the step of culturing the induced chondrocytes under conditions where the oxygen concentration in the culture atmosphere is low (i.e., under hypoxic conditions). "Culturing under hypoxic conditions" means that the oxygen concentration in the atmosphere when culturing cells, particularly chondrocytes, is less than the oxygen concentration under which the cells are normally cultured. The "oxygen concentration under which cells are normally cultured" is not uniquely determined because it varies depending on the cells and the species from which they are derived. As an example, for general mammalian chondrocytes, it is 20 v / v%. Therefore, hypoxic conditions may be conditions where the oxygen concentration in the culture atmosphere is less than 20 v / v%. Hypoxic conditions are preferably conditions where the oxygen concentration in the culture atmosphere is 10 v / v% or less, more preferably 7 v / v% or less, even more preferably 5 v / v% or less, particularly more preferably 3 v / v% or less, and particularly even more preferably 2 v / v% or less. Note that the condition where the oxygen concentration in the culture atmosphere is less than XX v / v% or XX v / v% or less means, at its lower limit, the condition where the culture atmosphere contains oxygen, i.e., the oxygen concentration in the culture atmosphere is 0% or more, including the case where the oxygen concentration in the culture atmosphere is 0 v / v%, unless otherwise specified. Also, the lower limit of the oxygen concentration in the culture atmosphere under hypoxic conditions is not particularly limited as long as it does not conflict with the above range. For example, it may be conditions where the oxygen concentration in the culture atmosphere is 0 v / v% or more, 0.5 v / v% or more, or 1 v / v% or more. Also, hypoxic conditions may be a non-conflicting combination within the above range. Specifically, for example, it may be 0.5 v / v% or more and 10 v / v% or less, 0.5 v / v% or more and 5 v / v% or less, 0.5 v / v% or more and 3 v / v% or less, 0.5 v / v% or more and 2 v / v% or less, 1 v / v% or more and 5 v / v% or less.

[0027] 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. For example, it may be 7 days or more and 21 days or less.

[0028] In the step of culturing the induced chondrocytes derived from the pluripotent stem cells of the present invention in a low-serum medium, the number of cells seeded at the start of the culture is not particularly limited as long as the chondrocyte sheet of the present invention can be obtained. For example, 1000 cells / cm 2 or more, 10000 cells / cm 2 or more, 30000 cells / cm 2 or more, or 50000 cells / cm 2 or more may be used, and 1000000 cells / cm 2 or less, 100000 cells / cm 2 or less, 70000 cells / cm 2 or less, or 50000 cells / cm 2 or less may also be used.

[0029] <2>The 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 a chondrocyte sheet obtained by culturing the induced chondrocytes in a low-serum medium.

[0030] 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.

[0031] The pluripotent stem cells and the induced chondrocytes derived from the pluripotent stem cells are as described above.

[0032] The chondrocyte sheet of the present invention may be characterized by one or more selected from the group consisting of "the secretion amount of Transforming growth factor-β1 (TGF-β1)", "the secretion amount of Melanoma inhibitory activity (MIA)", and "the ratio of the number of CD56-positive cells per total number of cells contained in the cell sheet". TGF-β1 is a growth factor involved in the growth and differentiation of articular cartilage and may affect the therapeutic effect brought about by the chondrocyte sheet. In addition, MIA is also known as a retinoic acid-sensitive protein derived from cartilage, and it has been reported that chondrocytes specifically express MIA and that MIA has an assimilating effect on chondrocytes. CD56 is a glycoprotein on the cell surface known as neural cell adhesion molecule (N-CAM) or nerve cell adhesion molecule. It is known to be expressed in natural killer cells, nerve cells, skeletal muscle cells, etc., and it has also been reported that it is expressed in some bone marrow-derived mesenchymal stem cells.

[0033] The secretion amount of TGF-β1 and the secretion amount of MIA may be, for example, the amount of TGF-β1 protein and the amount of MIA protein per unit area secreted by the chondrocyte sheet into the culture medium during a certain culture period when the chondrocyte sheet is cultured. For measuring the amount of protein, any known method can be used and is not particularly limited. Examples include methods using antibodies such as ELISA method and Western blotting method, methods using electrophoresis such as polyacrylamide electrophoresis (SDS-PAGE), and mass spectrometry method.

[0034] When culturing the chondrocyte sheet to measure the secretion amount, the culture conditions are not particularly limited as long as they are the culture conditions commonly used when measuring the secretion amount. As the culture medium, for example, a low serum medium may be used, but it is preferable to use a DMEM / F12 medium containing 1% FBS. Other culture conditions may also be the culture conditions commonly used when measuring the secretion amount. For example, the temperature is 34°C or higher and 40°C or lower, preferably 35°C or higher and 39°C or lower, more preferably 36°C or higher and 38°C or lower; the carbon dioxide (CO2) concentration is 0 v / v% or higher and 10 v / v% or lower, preferably 3 v / v% or higher and 7 v / v% or lower; the oxygen concentration is 0 v / v% or higher and 25 v / v% or lower, preferably 15 v / v% or higher and 25 v / v% or lower. 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 a combination that does not conflict as the upper and lower limits may be selected from these. Incidentally, 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, "culturing for 72 hours" in this specification means "culturing for 72 hours" when the time is set to 0.

[0035] Specifically, for example, the secretion amount of TGF-β1 per unit area at 72 hours of culture of the chondrocyte sheet of the present invention may be 2.0 ng / cm 2 or more, preferably 3.0 ng / c m 2 or more, and more preferably 3.5 ng / cm 2 or more. Further, specifically, for example, the secretion amount of MIA per unit area at 72 hours of culture of the chondrocyte sheet of the present invention may be 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.

[0036] The "CD56-positive cell" is a cell that expresses CD56. For example, it includes cells that express CD56 on the cell surface, cells that express CD56 inside the cell, or cells that express CD56 on both the cell surface and inside. Preferably, it is a cell that expresses CD56 at least on the cell surface. "Expressing CD56" may mean that the CD56 protein exists on the cell surface, or that the CD56 protein or mRNA exists inside the cell. Therefore, the method for detecting CD56-positive cells may be a method for detecting cells in which these exist by detecting the CD56 protein on the cell surface or the CD56 protein or mRNA inside the cell. Preferably, it may be a method for detecting cells in which the CD56 protein exists on the cell surface by detecting the CD56 protein on the cell surface. As a method for detecting the CD56 protein on the cell surface, for example, an antibody that specifically binds to the CD56 protein can be used. As a method for measuring CD56-positive cells, specifically, as an example, a method for measuring the number of cells in which the CD56 protein exists on the cell surface by flow cytometry using a fluorescently labeled antibody that specifically binds to the CD56 protein can be mentioned.

[0037] Specifically, for example, the ratio of the number of CD56-positive cells per total number of cells contained in the cell sheet in the chondrocyte sheet of the present invention may be 80% or more, preferably 85% or more, and more preferably 90% or more.

[0038] In the chondrocyte sheet of the present invention, the "secretion amount of TGF-β1", the "secretion amount of MIA", and the "ratio of the number of CD56-positive cells per total number of cells contained in the cell sheet" may be any combination within the above-described ranges. Specifically, for example, the chondrocyte sheet of the present invention may satisfy all of the following (1) to (3): (1) The secretion amount of Transforming growth factor-β1 (TGF-β1) per unit area at 72 hours of culture is 2.0 ng / cm 2or more, preferably 3.0 ng / cm 2 or more, more preferably 3.5 ng / cm 2 or more. (2) The secretion amount of Melanoma inhibitory activity (MIA) per unit area at 72 hours of culture is 6.0 ng / cm or more, preferably 10.0 ng / cm 2 or more, more preferably 12.5 ng / cm 2 or more. 2 (3) The ratio of CD56-positive cells per total cells contained in the cell sheet is 80% or more, preferably 85% or more, more preferably 90% or more.

[0039] The chondrocyte sheet of the present invention may further show significantly higher COL2A1 gene expression compared to a conventional chondrocyte sheet. Preferably, when the expression level of the COL2A1 gene in the conventional chondrocyte sheet is set to 1, the expression level of the COL2A1 gene is 100 or more, more preferably 1000 or more. Examples of the conventional chondrocyte sheet include, for example, a chondrocyte sheet derived from cartilage tissue of a human polydactyly patient, and as an example thereof, a chondrocyte sheet derived from cartilage tissue of a human polydactyly patient described in JP-A-2020-006207 can be mentioned. Specifically, for example, the chondrocyte sheet of the present invention may show significantly higher COL2A1 gene expression compared to a chondrocyte sheet derived from cartilage tissue of a human polydactyly patient. Preferably, when the expression level of the COL2A1 gene in the chondrocyte sheet derived from cartilage tissue of a human polydactyly patient is set to 1, the expression level of the COL2A1 gene is 100 or more, more preferably 1000 or more.

[0040] The chondrocyte sheet of the present invention may further satisfy one or more selected from 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) Negative in immunostaining using an antibody against type II collagen. ​(C) It is negative in safranin O staining. (D) It is positive in immunostaining using an antibody against aggrecan.

[0041] "Immunostaining using an antibody" refers to detecting cells containing a target protein that may be contained in a cell sheet, tissue, etc., using an antibody against the 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, a molecule that labels the antibody (including the secondary antibody, etc.). In the present invention, "being positive in immunostaining" means that cells containing the target protein are detected in the cell sheet or tissue. Specifically, for example, 90% or more of all the cells contained in the cell sheet, tissue, etc. may be detected as cells containing the target protein. In the present invention, "being negative in immunostaining" means that only a certain amount or less of the cells containing the target protein are detected in the cell sheet or tissue, or that the cells are not detected. Specifically, for example, 10% or less of all the cells contained in the cell sheet, tissue, etc. may be detected as cells containing the target protein, provided that the case of 0%, that is, the case where the cells containing the target protein are not detected, is included.

[0042] Type I collagen is the collagen found in scar tissue, tendon, ligament, 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, particularly during culture, but may disappear in part or in whole of the transplanted sheet and its surrounding tissue after transplantation into the living body.

[0043] Type II collagen is found in the cornea, vitreous body, and cartilage, and is the main collagen constituting 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, particularly during culture, but may appear in part or in whole of the transplanted sheet and its surrounding tissue after transplantation into the living body.

[0044] Safranin O is a dye that stains cell nuclei and is known to stain cartilage as well. Safranin O staining can be carried out by any known method. Specifically, for example, it may be carried out by first treating with hematoxylin, immersing in an aqueous solution of fast green, treating with an aqueous acetic acid solution, and then immersing in an aqueous solution of safranin O. The chondrocyte sheet of the present invention is preferably negative in safranin O staining.

[0045] Aggrecan is a keratan sulfate / chondroitin sulfate proteoglycan with a molecular weight of about 2500 kDa, having 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. 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.

[0046] The chondrocyte sheet of the present invention may be used for the treatment of damaged or degenerated cartilage. As a treatment method for cartilage, for example, transplantation to damaged or degenerated cartilage can be considered. Since the chondrocyte sheet of the present invention has a sheet-like shape and exhibits excellent adhesiveness and repair / regeneration of cartilage tissue, transplantation to widely worn cartilage such as osteoarthritis is easy, and treatment of such cartilage can also be expected. The treatment of cartilage by the chondrocyte sheet of the present invention may be due to the fact that the sheet itself not only fills the damaged site but also promotes the repair and regeneration of the surrounding cartilage tissue and bone tissue.

[0047] Therefore, the chondrocyte sheet of the present invention may be used for one or more treatments selected from the group consisting of traumatic cartilage injury, osteochondral injury, osteochondritis dissecans, meniscus injury, intervertebral disc degeneration, and osteoarthritis. In particular, the significance of the present invention is great as a treatment method for osteoarthritis of the knee for which a radical treatment method has not been established.

Example

[0048] Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the following embodiments.

[0049] (Example 1) Induction of chondrocyte differentiation Induction of chondrocyte differentiation from pluripotent stem cells was carried out according to the method described in WO2015 / 064754 using the QHJI 01s04 strain, which is an iPS cell derived from human peripheral blood and established in the iPS cell stock project of the Institute for iPS Cell Research, Kyoto University. 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. Then, the medium was replaced with DMEM / F12 medium containing 10 ng / ml of Wnt3a (R&D), 10 ng / ml of Activin A (R&D), 1% ITS (Invitrogen), and 1% FBS. After 72 hours, the medium was replaced with DMEM medium containing 10 ng / ml of TGF-β1 (Peprotech), 10 ng / ml of BMP2 (Osteopharma), 10 ng / ml of GDF5 (PTT), 50 μg / ml of ascorbic acid, 1% ITS, 1% FBS, 2 mM of L-glutamine (Invitrogen), 1x10 -4 M of nonessential amino acids (Invitrogen), and 1 mM of sodium pyruvate (Invitrogen). From day 3 to day 14, 10 ng / ml of bFGF (Wako) was also added. On day 14, the adhered cartilage mass was physically detached from the culture dish, and then cultured in suspension. All of the above cultures were carried out in an incubator at 37°C and 5% CO2. The medium was changed every 2 to 7 days. The cartilage mass in suspension culture is also called a cartilage particle. In addition, the induced chondrocytes derived from pluripotent stem cells contained in the cartilage particle are also simply called induced chondrocytes.

[0050] (Example 2) Preparation of chondrocyte sheet The cartilage particles (wet weight 1 - 4 g) cultured for 19 weeks were dispersed in 30 ml of DMEM / F12 medium containing 5 mg of Liberase (Roche), 20% FBS, and 1% antibiotic. They were dispersed for 2.5 hours in an incubator at 37°C and 5% CO2 under the condition of 60 rpm rotation in a diffusion state using a stirrer bar. 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); attached supplement, 1:100 GlutaMAX, 1% antibiotic added). A part of the cell suspension was mixed with trypan blue at a ratio of 1:1, and the cell count was measured with a Countess. As culture equipment for preparing cell sheets, a 6 - well plate for inserts (Falcon) and a temperature - responsive culture insert (UpCell insert, CellSeed) were prepared. 3 ml of MesenPRO medium was added to each well of the 6 - well plate, and the insert was placed. Based on the measured cell count, it was suspended in 2 ml of MesenPRO medium to achieve about 50000 cells / cm 2 and seeded. That is, about 210000 cells were suspended in 2 ml of MesenPRO medium and seeded into the insert. Then, it was cultured in an incubator at 37°C, 5% CO2, and 2% O2 for 14 days, and the medium was changed every other day. The chondrocyte sheet thus obtained was named iPSC sheet M. Furthermore, a chondrocyte sheet was prepared by the same method except that DMEM / F12 medium containing 20 v / v% FBS was used instead of MesenPRO medium, and it was named iPSC sheet D.

[0051] (Example 3) Preparation of Reagents The reagents used for histological analysis were prepared by the following procedure. Hematoxylin aqueous solution: Put ion-exchanged water into a flask and bring it to a boil. Add 1.5 g of hematoxylin (product number: 1.15938, manufacturer: MERCK) to the boiling ion-exchanged water and dissolve the hematoxylin with a stirrer. While stirring, lower the temperature of the solution to room temperature. Add acetic acid (017-00256, Wako Pure Chemical Industries, Ltd.) to the solution and adjust the pH to 3.0. Add 0.3 g of sodium iodate (190-02252, Wako Pure Chemical Industries, Ltd.) to the solution and stir, and then add 75 g of ammonium alum (018-01825, Wako Pure Chemicals) and stir. After stirring, add ion-exchanged water to make 1 L, and then filter to obtain a hematoxylin aqueous solution. Eosin solution: Prepare an eosin solution by mixing 30 mL of pure eosin solution (3204-2, Mutoh Chemical) and 120 mL of 95% ethanol. Sometimes when used in staining, the eosin solution is diluted 5 times with 95% ethanol, and the diluted solution is used as the eosin solution. Fast Green aqueous solution: Dissolve 80 mg of Fast Green (1A304, CHROMA) in 100 mL of ion-exchanged water, and then filter the solution to obtain a 0.08 mass% Fast Green aqueous solution. Safranin O aqueous solution: Dissolve 100 mg of Safranin O (1B463, CHROMA) in 100 mL of ion-exchanged water to obtain a 0.1 mass% Safranin O aqueous solution. Citric acid buffer solution: Dissolve citric acid monohydrate in ion-exchanged water to obtain a 0.01 M citric acid aqueous solution (hereinafter referred to as solution A). Dissolve trisodium citrate dihydrate in ion-exchanged water to obtain a 0.1 M sodium citrate aqueous solution (hereinafter referred to as solution B). Combine 95 mL of solution A and 415 mL of solution B and stir, and then adjust the pH to 6.0 with 1 N NaOH to obtain a 0.01 M citric acid buffer solution.

[0052] (Example 4) Method for preparing frozen sections for histological analysis The cell sheet was fixed with 4% paraformaldehyde solution and embedded in Tissue-Tek O.C.T. compound (4583, Sakura Finetek Japan Co., Ltd.) to prepare frozen sections. The frozen sections of the cell sheet were thinly sliced perpendicular to the surface that had been in contact with the temperature-responsive culture dish. The thickness of the thinly sliced sections was 20 μm when the sections were subjected to immunostaining, and 10 μm when subjected to staining other than immunostaining. The thinly sliced frozen sections were subjected to the staining described below.

[0053] (Example 5) Hematoxylin and eosin staining of cell sheet 1. Pretreatment The sections were immersed in 100% ethanol for 10 minutes once. 2. Washing treatment The sections treated in 1. above were washed with water. 3. Immersion in ion-exchanged water After the water washing in 2. above, the sections were immersed in ion-exchanged water for 5 minutes. 4. Hematoxylin treatment After the immersion in 3. above, the sections were immersed in the hematoxylin aqueous solution for 3 - 4 minutes. 5. Washing treatment After the treatment in 4. above, the sections were washed with hot water (50 °C) for 3 - 4 minutes. 6. Eosin treatment After the washing in 5. above, the sections were immersed in the eosin solution for 10 minutes. 7. Dehydration After the treatment in 6. above, the sections were dehydrated using an ethanol series. The ethanol series consisted of seven 100% ethanol. 8. Clearing After the dehydration in 7. above, the sections were cleared using a xylene series. That is, seven containers filled with xylene were prepared, and the sections were immersed in the xylene in each container. 9. Mounting After the clearing in 8. above, the sections were mounted. In this mounting, Malinol (product number: 2009 - 3, company name: Mutoh Chemical Co., Ltd.) was used as the mounting medium.

[0054] (Example 6) Safranin O staining of cell sheet 1. Pretreatment The section was immersed in 100% ethanol for 10 minutes once. 2. Washing treatment The section treated in 1. above was washed with water. 3. Immersion in ion-exchanged water After the water washing in 2. above, the section was immersed in ion-exchanged water for 5 minutes. 4. Hematoxylin treatment After the immersion in 3. above, the section was immersed in the hematoxylin aqueous solution for 4 to 5 seconds. 5. Washing treatment After the treatment in 4. above, the section was washed with hot water (50 °C) for 3 to 4 minutes. 6. Fast Green treatment After the washing in 5. above, the section was immersed in the 0.08 mass% Fast Green aqueous solution for 10 minutes. 7. Acetic acid treatment After the treatment in 6. above, the section was treated one or two times with 1 volume% acetic acid aqueous solution. 8. Safranin treatment After the treatment in 7. above, the section was immersed in the 0.1 mass% Safranin O aqueous solution for 10 minutes. 9. Dehydration, clearing, and embedding These treatments were performed as described in the above-mentioned "Hematoxylin and Eosin Staining of Cell Sheet".

[0055] (Example 7) Toluidine blue staining of cell sheet 1. Pretreatment The section was immersed in 100% ethanol for 10 minutes once. 2. Washing treatment The section treated in 1. above was washed with water. 3. Immersion in ion-exchanged water After the water washing in 2. above, the section was immersed in ion-exchanged water for 5 minutes. 4. Hematoxylin treatment After the immersion in the above 3., the section was immersed in the hematoxylin aqueous solution for 3 to 4 minutes. 5. Washing treatment After the treatment in the above 4., the section was washed with hot water (50 °C) for 3 to 4 minutes. 6. Toluidine blue treatment After the treatment in the above 5., the section was immersed in 0.05% toluidine blue pH 4.1 (Muto Chemical #40971) for 15 minutes. 7. Washing treatment The section treated in the above 6. was washed with water. 8. Dehydration, clearing, and embedding These treatments were performed as described in the above "Hematoxylin and Eosin Staining of Cell Sheet".

[0056] (Example 8) Agarican staining of cell sheet 1. Pretreatment The section was immersed in 100% ethanol for 5 minutes three times. Next, the section was immersed in 70% ethanol for 5 minutes once. 2. Washing treatment The section treated in the above 1. was washed with water. 3. Immersion in ion-exchanged water After the water washing in the above 2., the section was immersed in ion-exchanged water for 5 minutes. 4. Antigen retrieval treatment After the immersion in the above 3., the section was immersed in the citrate buffer at 98 °C for 10 minutes. 5. Immersion in ion-exchanged water After the treatment in the above 4., the section was immersed in ion-exchanged water for 5 minutes three times. 6. Immersion in hydrogen peroxide-containing methanol solution After the immersion in the above 5., the section was immersed in a hydrogen peroxide-containing methanol solution (0.3 mass% H2O2) for 15 minutes. 7. Immersion in ion-exchanged water After the immersion in the above 6., the section was immersed in ion-exchanged water for 5 minutes three times. 8. PBS washing After the immersion in step 7 above, the sections were immersed in 0.01M PBS for 5 minutes three times. 9. Blocking with serum After the washing in step 8 above, the sections were immersed in 2.5% normal horse serum (the serum attached to ImmPRESS HRP REAGENT KIT Anti-GOAT IgG (#MP-7405) from VECTOR) for 10 minutes. 10. Primary antibody reaction After the blocking in step 9 above, the sections were subjected to a primary antibody reaction using Goat anti-human-aggrecan (SC006, R&D, diluted to 10 mg / mL with BlockAidTM Blocking Solution (Thermo Fisher Scientific Inc., Catalog no. B10710)) for 2 hours. 11. PBS washing After the reaction in step 10 above, the sections were immersed in 0.01M PBS for 5 minutes three times. 12. Secondary antibody reaction After the washing in step 11 above, the sections were subjected to a secondary antibody reaction using Donkey anti-Goat IgG(H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 546 (Thermo Fisher Scientific Inc., A-11056) and ImmPRESS Polymer Anti-Goat IgG Reagent (MP-7405, Vector Laboratories) for 1 hour. 13. PBS washing After the reaction in step 12 above, the sections were immersed in 0.01M PBS for 5 minutes three times. 14. Color development After the washing in step 13 above, the sections were subjected to a color development reaction with DAB (0.05M Tris-HCl (200ml), DAB (40 mg), 30% H2O2 (34μl)) for 2 minutes. 15. Immersion in ion-exchanged water After the color development reaction in step 14 above, the sections were immersed in ion-exchanged water for 5 minutes. 16. Nuclear staining After the immersion in step 15, the section was treated with the hematoxylin aqueous solution for 2 seconds. 17. Washing with water After the treatment in step 16, the section was washed with water. 18. Dehydration, clearing, and embedding These treatments were performed as described in the above-mentioned "Hematoxylin and eosin staining of cell sheets".

[0057] (Example 9) Fibronectin staining of cell sheets Except that the reagents used in "10. Primary antibody reaction" and "12. Secondary antibody reaction" of the above-mentioned "Agarican staining of cell sheets" were changed, the agarican staining was performed in the same manner. 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).

[0058] (Example 10) Type I collagen staining of cell sheets Except that the reagents used in "10. Primary antibody reaction" and "12. Secondary antibody reaction" of the above-mentioned "Agarican staining of cell sheets" were changed, the type II collagen staining was performed in the same manner. The antibody used in the primary antibody reaction was an antibody against type I collagen (1310-01, SouthernBiotech, diluted 1:100 with 0.01 M PBS). The reagent used in the secondary antibody reaction was ImmPRESS Polymer Anti-Goat IgG Reagent (MP-7405, Vector Laboratories).

[0059] (Example 11) Type II Collagen Staining of Cell Sheet Type II collagen staining was performed in the same manner except that the reagents used in "10. Primary Antibody Reaction" and "12. Secondary Antibody Reaction" of the above-mentioned "Agarican Staining of Cell Sheet" were changed. The antibody used in the primary antibody reaction was a type II collagen primary antibody (Kyowa Pharmchem 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).

[0060] (Example 12) Heterotopic Transplantation Experiment The cell sheet was allowed to stand at room temperature for 30 minutes and then peeled off using a polyvinylidene fluoride (PVDF) membrane and used for the heterotopic transplantation experiment. A 12-week-old nude rat (F334 / NJcl-rnu / rnu, CLEA) was used for the heterotopic 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 in a manner covering the defect. Knee joint tissues were collected 4 weeks and 12 weeks after transplantation, and histological evaluation of the articular cartilage tissue was performed. Histological evaluation was performed by hematoxylin-eosin staining, toluidine blue staining, safranin O staining, type I collagen immunostaining, type II collagen immunostaining, and human-specific vimentin staining.

[0061] (Example 13) Method for Preparing Sections for Histological Evaluation The cartilage part was fixed by immersion in 20% formalin and then embedded in paraffin. Embedding was performed using Histo Prep 586 (415 - 25791, Wako Pure Chemical Industries, Ltd.) as the embedding agent and an embedding console system (Tissue-Tek, Sakura Finetek Japan Co., Ltd.). When the embedded sample was in an upright state, it was sectioned thinly in the vertical direction. The thickness of the thinly sectioned slices was 3 μm. The thinly sectioned slices were placed on glass slides and subjected to the staining described below.

[0062] (Example 14) Hematoxylin and Eosin Staining of Cartilage 1. Deparaffinization The sections were immersed in xylene for 5 minutes three times. Next, the sections were immersed in 100% ethanol for 5 minutes three times. Next, the sections were immersed in 70% ethanol for 5 minutes once. 2. Washing Treatment, Immersion in Ion-Exchanged Water, Hematoxylin Treatment, Washing Treatment, Eosin Treatment, Dehydration, Clearing, and Mounting These treatments were performed as described in the above-mentioned "Hematoxylin and Eosin Staining of Cell Sheets".

[0063] Safranin O Staining of Cartilage Staining was performed in the same manner as the above-mentioned "Safranin O Staining of Cell Sheets", except that the "1. Deparaffinization" described in the above-mentioned "Hematoxylin and Eosin Staining of Cartilage" was performed instead of the "1. Pretreatment" in the above-mentioned "Safranin O Staining of Cell Sheets".

[0064] Toluidine Blue Staining of Cartilage Staining was performed in the same manner as the above-mentioned "Toluidine Blue Staining of Cell Sheets", except that the "1. Deparaffinization" described in the above-mentioned "Hematoxylin and Eosin Staining of Cartilage" was performed instead of the "1. Pretreatment" in the above-mentioned "Toluidine Blue Staining of Cell Sheets".

[0065] Type I Collagen Staining of Cartilage Staining was performed in the same manner as the "Type I collagen staining of cell sheets" except that the "1. Deparaffinization treatment" described in the "Hematoxylin and eosin staining of cartilage" was performed instead of the "1. Pretreatment" in the "Type I collagen staining of cell sheets".

[0066] Type II collagen staining of cartilage Staining was performed in the same manner as the "Type II collagen staining of cell sheets" except that the "1. Deparaffinization treatment" described in the "Hematoxylin and eosin staining of cartilage" was performed instead of the "1. Pretreatment" in the "Type II collagen staining of cell sheets".

[0067] (Example 15) hVimentin staining of cartilage 1. Deparaffinization treatment Deparaffinization treatment was performed as described in the "Hematoxylin and eosin staining of cartilage". 2. Washing treatment After the deparaffinization treatment in 1., the sections were washed with water. 3. Immersion in ion-exchanged water After the washing in 2., the sections were immersed in ion-exchanged water for 5 minutes. 4. Antigen activation After the immersion in 3., the sections were immersed in the citrate buffer at 98 °C for 10 minutes. 5. Cooling After the treatment in 4., the sections were cooled on the laboratory bench for 30 minutes. 6. Blocking with serum After the cooling in 5., the sections were treated with 0.01 M PBS (containing 0.2% Tween) containing 5% normal goat serum (product number: D204-00-0100, company name: ROCKLAND) for 1 hour. 7. Antibody reaction After the blocking in 6 above, the section was subjected to an antibody reaction using anti-human Vimentin antibody Alexa Fluor 647 Conjugate (#9856, Cell Signaling Technology, diluted 1:100 with 0.01M PBS) at 4°C for 16 hours. 8. PBS washing After the reaction in 7 above, the section was immersed in 0.01M PBS for 5 minutes three times. 9. Mounting After the washing in 8 above, the section was mounted using VECTASHIELD HardSet Antifade Mounting Medium with DAPI (Vector Laboratories).

[0068] (Example 16) Measurement of cell surface marker CD56 Cells were isolated from the chondrocyte sheet obtained by preparing the chondrocyte sheet, and single staining of the cell surface marker CD56 was performed, and the CD56 positive cell rate was clarified by flow cytometry.

[0069] (Example 17) Measurement of protein secretion amount For the chondrocyte sheet obtained by preparing the chondrocyte sheet, 2 ml of DMEM / F12 medium containing 1% FBS and 1% antibiotic was added to the insert during culture, and 3 ml was added to the culture dish in which the insert was placed, and cultured at 37°C, 5% CO2, and normal oxygen state for 72 hours (that is, cultured for 72 hours). Then, 2 ml of the medium in the insert was collected, and the protein concentrations of TGF-β1 and MIA were quantified by ELISA.

[0070] (Results) For the prepared chondrocyte sheets, iPSC sheet M, and iPSC sheet D, hematoxylin and eosin staining (HE), 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) were performed. The results are shown in Fig. 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 positive in immunostaining using an antibody against aggrecan. This suggests that chondrocyte sheets derived from pluripotent stem cells can also have a cartilage repair effect, as disclosed in Japanese Patent Laid-Open No. 2020-006207.

[0071] Next, heterotopic transplantation experiments were 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-treatment group, no safranin O stainability or toluidine blue metachromasia was confirmed. In the slightly regenerated tissue, mainly type I collagen stainability was confirmed, and type II collagen stainability was not confirmed. In the group transplanted with iPSC sheet D prepared by the conventional production method, weak safranin O stainability and weak toluidine blue metachromasia in the repaired and regenerated articular cartilage tissue were confirmed. In addition, strong stainability of type I collagen was confirmed, and slightly type II collagen stainability was confirmed.

[0072] In the group transplanted with the iPSC sheet M produced by the production method of the present invention, strong safranin O staining and strong toluidine blue metachromasia were confirmed in the repaired and regenerated articular cartilage tissue. In addition, the staining of type I collagen was confirmed only in the surface layer portion, and strong staining of type II collagen 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. In the iPSC sheet D group, the density of chondrocytes was relatively high after 4 weeks, and there were mixed parts with relatively high and low chondrocyte densities even after 12 weeks. In contrast, in the iPSC sheet M group, the density of chondrocytes was relatively low, and it was confirmed that cell lacunae were starting to form.

[0073] To clarify the characteristics of the pluripotent stem cell-derived iPSC sheet M and iPSC sheet D, and the chondrocyte sheet derived from the cartilage tissue of a polydactyly patient produced by the method described in JP-A-2020-006207 (hereinafter also referred to as the PDC sheet), measurement of the cell surface marker CD56, measurement of the protein secretion amounts of TGF-β1 and MIA, and gene expression analysis of COL2A1 by quantitative reverse transcription PCR were performed. The results are shown in FIGS. 4, 5, and 6.

[0074] The iPSC sheet M had a CD56-positive cell rate of 90.3 to 96.3%, and the secretion amount of TGF-β1 was 15.2 to 40 ng or more per sheet (4.2 cm 2 ) (that is, 3.62 to 9.52 ng / cm 2 or more: n = 7). The secretion amount of MIA was 54.34 to 100 ng or more per sheet (4.2 cm 2 ) (that is, 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 the PDC, and it showed significantly higher values in terms of the CD56-positive cell rate, the secretion amounts of TGF-β1 and MIA, and the gene expression of COL2A1 compared to the iPSC sheet D and the PDC.

[0075] (Reference Example) Regarding the iPSC sheet D and the PDC sheet, only the evaluation 4 weeks after transplantation was performed in the heterotopic transplantation experiment. The results are shown in Fig. 7. In the PDC sheet transplantation group, strong safranin O stainability and strong metachromasia of toluidine blue were confirmed in the repaired and regenerated articular cartilage tissue. In addition, the stainability of type I collagen was confirmed only in the surface layer part, and strong stainability of type II collagen 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 the transplanted human cells, and the density of chondrocytes was relatively low and the formation of cell lacunae was frequently confirmed. In the transplantation group of the iPSC sheet D prepared by the conventional method, the safranin O stainability in the repaired and regenerated articular cartilage tissue was not confirmed, and only slight metachromasia of toluidine blue was confirmed. In addition, the stainability of type I collagen was confirmed in the repaired and regenerated articular cartilage tissue, and the stainability of type II collagen was hardly confirmed. As a result of human-specific vimentin staining, it was confirmed that the regenerated articular cartilage site was mainly composed of the transplanted human cells, and the density of chondrocytes was relatively high and the formation of cell lacunae was hardly confirmed. From this result, it became clear that the chondrocyte sheet derived from the cartilage tissue of a polydactyly patient prepared by the conventional method is capable of cartilage repair and regeneration, while the chondrocyte sheet derived from pluripotent stem cells prepared by the conventional method is not suitable for cartilage repair and regeneration.

[0076] From the above, it became clear that the chondrocyte sheet derived from pluripotent stem cells prepared by the conventional method is not suitable for cartilage repair and regeneration, but the method of the present invention overcomes this drawback, and the chondrocyte sheet of the present invention derived from pluripotent stem cells is suitable for cartilage repair and regeneration.

Claims

1. A cell sheet for cartilage repair, wherein the cell sheet for cartilage repair is formed from a culture of induced chondrocytes derived from pluripotent stem cells, and the culture is obtained by culturing the induced chondrocytes in a low-serum medium, the cell sheet for cartilage repair.

2. The cell sheet for cartilage repair according to Claim 1, satisfying all of the following (1) to (3): (1) Transforming growth per unit area at 72 hours of culture The secretion amount of factor-β1 (TGF-β1) is 3.0 ng / cm ２ or more. (2) Melanoma inhibitory per unit area at 72 hours of culture The secretion amount of activity (MIA) 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 Claim 1 or 2, satisfying all of the following (1) to (3): (1) Transforming growth per unit area at 72 hours of culture The secretion amount of factor-β1 (TGF-β1) is 3.5 ng / cm ２ or more. (2) Melanoma inhibitory per unit area at 72 hours of culture The secretion amount of activity (MIA) 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 Claim 1 or 2, satisfying all of the following (A) to (D): (A) Positive in immunostaining using an antibody against type I collagen. (B) Negative in immunostaining using an antibody against type II collagen. (C) Negative in safranin O staining. (D) Positive in immunostaining using an antibody against aggrecan.

5. 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. The cell sheet for cartilage repair according to Claim 1 or 2, wherein the pluripotent stem cells are human-derived iPS cells.

7. The cell sheet for cartilage repair according to Claim 1 or 2, wherein when the expression level of the COL2A1 gene in a cartilage cell sheet derived from human polydactyly patient cartilage tissue is set to 1, the expression level of the COL2A1 gene is 100 or more.

8. The cell sheet for cartilage repair according to Claim 1 or 2, wherein culturing the induced chondrocytes in a low-serum medium is performed under low-oxygen conditions.

9. The cell sheet for cartilage repair according to Claim 8, wherein the low-oxygen conditions are conditions where the oxygen concentration in the culture atmosphere is 5 v / v% or less.

10. A method for manufacturing a cell sheet for cartilage repair, A method comprising the step of culturing induced chondrocytes derived from pluripotent stem cells in a low serum medium 。

11. The method according to claim 10, wherein the serum concentration of the low serum medium is 5 v / v% or less.

12. The method according to claim 10 or 11, wherein the step of culturing the induced chondrocytes in a low serum medium comprises culturing the induced chondrocytes under the condition that the oxygen concentration in the culture atmosphere is 5 v / v% or less.

13. A method for culturing induced chondrocytes derived from pluripotent stem cells, comprising: a step of culturing the induced chondrocytes in a low serum medium.

14. The method according to claim 13, wherein the serum concentration of the low serum medium is 5 v / v% or less.

15. The method according to claim 13 or 14, wherein the step of culturing the induced chondrocytes in a low serum medium comprises culturing the induced chondrocytes under the condition that the oxygen concentration in the culture atmosphere is 5 v / v% or less.

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