Marker for evaluating chondroid tissue formation characteristics

Abstract

The present invention provides a technique for providing a cell culture suitable for cartilage repair, particularly a cell culture suitable for hyaline cartilage repair.　Provided is a marker for evaluating chondroid tissue formation characteristics that includes one or more gene markers selected from the group consisting of (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, and (6) mitochondrial-related genes.

Description

Markers for evaluating the characteristics of cartilage-like tissue formation

[0001] The present technology relates to a cell culture, a method for evaluating the cell culture, a method for producing the cell culture, and a marker for evaluating the characteristics of cartilage-like tissue formation.

[0002] For the treatment of musculoskeletal diseases such as osteoarthritis, cartilage tissue has been treated using tissue regeneration engineering techniques. In this treatment, cultured chondrocytes or cartilage tissue produced from chondrocytes can be transplanted into the affected area. Various transplant materials have been proposed.

[0003] For example, Patent Document 1 discloses a method and device for searching for marker genes of cartilage cell sheets, a method and device for evaluating cartilage cell sheets, a cartilage cell sheet, a method for manufacturing a cartilage cell sheet, and a method for regenerating cartilage using a cartilage cell sheet.

[0004] For example, Patent Document 2 discloses a transplantation material to be transplanted into a predetermined transplantation site, in which cells appropriate for the predetermined transplantation site are retained on a cell retention carrier obtained by subjecting a tissue structure of the same type as the predetermined transplantation site, obtained from body tissue, to an antigenicity suppression treatment while maintaining the shape of the tissue structure. Furthermore, Non-Patent Document 1 discloses that genes and proteins with functions such as extracellular matrices, skeletal development, and angiogenesis have been identified as candidate markers for predicting the effectiveness of cartilage cell sheets.

[0005] JP 2021-90418 A JP 2003-180819 A JP 2-211865 A

[0006] Int J Mol Sci. 2019 Dec 24;21(1):149.Brehm et al., 2006, Osteoarthritis and Cartilage, 14, 1214-1226.Mainil‐Varlet et al., 2003, The Journal of Bone and Joint Surgery. American Volume, 85(A Suppl 2), 45-57.O'Driscoll, Keeley, & Salter, 1988, The Journal of Bone and Joint Surgery. American Volume, 70, 595-606Daichi Takizawa et al., J Tissue Eng Regen Med. 2020 Sep;14(9):1296-1306.)

[0007] A primary object of the present invention is to provide a technique for providing a cell culture suitable for cartilage repair, particularly a cell culture suitable for hyaline cartilage repair.

[0008] The present inventors have found a correlation between the expression levels of specific genes, including (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, and (6) mitochondria-related genes, and the ICRS score. Based on this, the present inventors have identified specific gene markers that can predict the effectiveness of cell cultures for tissue regeneration. Furthermore, the present inventors have found that cell cultures with the specific characteristic of expressing these specific genes are suitable for cartilage repair, particularly hyaline cartilage repair. (1) Adhesion-related genes: LAP3, TENM4, PTHLH, THBS3, IL27RA, NTN4, COL1A1, CHRD, and VCAM1. (2) Angiogenesis-related genes: PDGFA, VEGFC, SERPINE1, CSPG4, RASIP1, WARS, AAMP, FGF18, and S1PR1. (3) ECM-related genes: BGN, CLEC3B, COL11A1, COL1A1, COL1A2, COL5A1, COL5A2, CRIP2, HAPLN1, JUP, MMP13, MMP23B, SERPINE1, THBS1, TINAGL1, ADAMTS14, CCT2, CYHR1, and MMP17. (4) Collagen-related genes: COL5A1, COL5A2, COL1A1, COL1A2, and COL11A1. (5) Cell migration-related genes: ARPC5, CD2AP, COL5A1, CSPG4, ITGB3, JUP, PODXL, RHOA, SDC2, SHROOM2, SNAI1, and BDKRB1, CDK5, FUT8, S1PR1, SH3KBP1, and TGFBR3.(6) Mitochondrial-related genes: AS3MT, ATP7B, CA5B, CKB, CMC2, CTPS2, ELN, FBXO7, MARC2, ME3, NOL3, PABPC5, PANK2, PRELID2, SEPT4, SPARC, SPTLC2, and ADCK1, ADH5, AFG3L2, AIFM1, AKAP1, ALDH4A1, APEX1, BCS1L, C1QBP, CARS2, CHCHD3, COA4, COQ9, COX7B, DDAH2, DGAT2, DLD, DNAJA1, ECH1, ECHS1, FDXR, FIBP, FMC1, GATB, GCSH, GPX1, HSD17B10, IDH3B, LAP3, LARS2, MAPK8IP1, MDH2, METAP1D, MRPL10, MRPL24, MRPL42, MRPS12, M RPS18C, MRPS22, MRPS27, MRPS35, MTG1, MTHFD1, NDUFB9, NDUFS2, NDUFV1, OCIAD1, O XNAD1, PARS2, PDF, PHB, PHYH, POLRMT, PTCD1, PYURF, RMDN1, RMND1, SDHD, SFXN4, S LC25A11, SMIM4, TACO1, TIMM10, TIMM50, TK2, TOMM22, TOMM5, TRAP1, TUFM, UQCRC1.

[0009] In view of the above, the present invention has been completed as follows.

[0010] The present invention can provide a marker for evaluating cartilage-like tissue formation characteristics, which includes one or more gene markers selected from the group consisting of (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, and (6) mitochondria-related genes. The present invention can also provide a kit for evaluating cartilage-like tissue formation characteristics, which uses one or more gene markers selected from the group consisting of (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, and (6) mitochondria-related genes.

[0011] The present invention also provides a method for evaluating a cell culture, comprising a determining step of determining whether the expression level of at least one positively correlated gene marker selected from the group consisting of (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, and (6) mitochondria-related genes is equal to or greater than a threshold value for each gene marker, and / or whether the expression level of at least one negatively correlated gene marker selected from the group consisting of (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, and (6) mitochondria-related genes is equal to or less than a threshold value for each gene marker.

[0012] The present invention also provides a method for evaluating a cell culture, comprising a step of using an ICRS score estimated based on the expression level of at least one gene marker selected from the group consisting of (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, and (6) mitochondria-related genes, and determining that the sheet-shaped cell culture has cartilage-like tissue-forming properties when the estimated ICRS score is high. Preferably, the expression levels of the selected predetermined gene markers are applied to a regression equation that models the relationship between the gene marker amounts and the IICRS score, and the sheet-shaped cell culture is determined to have cartilage-like tissue-forming properties when the estimated ICRS score is high.

[0013] The present invention also provides a cell culture having cartilage-like tissue-forming properties, in which the expression level of at least one positively correlated gene marker selected from the group consisting of (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, and (6) mitochondria-related genes is equal to or greater than the threshold value for each gene marker, and / or the expression level of at least one negatively correlated gene marker selected from the group consisting of (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, and (6) mitochondria-related genes is equal to or less than the threshold value for each gene marker.

[0014] The present invention also provides a cell culture having cartilage-like tissue-forming properties, wherein the cell culture has an ICRS score estimated based on the expression level of at least one gene marker selected from the group consisting of: (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, and (6) mitochondria-related genes, and the estimated ICRS score is higher than a predetermined ICRS score.

[0015] The present invention can provide a technique for providing a cell culture suitable for cartilage repair, particularly a cell culture suitable for hyaline cartilage repair.

[0016] 1 is a diagram showing the ICSR score of a chondrocyte sheet transplanted into a nude rat cartilage defect model. FIG. 2 is a diagram showing the correlation between gene expression of 10 factors (horizontal axis) and ICSR score (vertical axis). FIG. 3 is a diagram showing the GeneScore correlation of adhesion factors ((1) adhesion-related genes). FIG. 4 is a diagram showing the GeneScore correlation of "(2) angiogenesis-related genes". FIG. 5 is a diagram showing the GeneScore correlation of "(3) ECM-related genes". FIG. 6 is a diagram showing the GeneScore correlation of "(4) collagen-related genes". FIG. 7 is a diagram showing the GeneScore correlation of "(5) cell migration-related genes". FIG. 8 is a diagram showing the GeneScore correlation of "(6) mitochondria-related genes".

[0017] Preferred embodiments for carrying out the present invention are described below. The embodiments described below are representative examples of the present invention and should not be construed as narrowing the scope of the present invention. In this specification, percentages are expressed by mass unless otherwise specified. The upper limit (or less) and lower limit (or more) of each numerical range (to) can be arbitrarily combined as desired. In the following descriptions of "1." to "5.", overlapping descriptions of technical features, configurations, definitions, terms, and methods, such as cell cultures, various genetic markers, origins, culture methods, evaluation methods, and production methods, may be omitted as appropriate. The descriptions of "1." to "5." can be applied to any of the techniques or embodiments of "1." to "5.", and the technical features can be appropriately adopted in each technique or embodiment.

[0018] 1. Markers for evaluating cartilage-like tissue formation characteristics

[0019] Articular cartilage plays a role in absorbing shock when weight is applied, and can become damaged when a large force is applied during sports or external forces. Because articular cartilage has poor blood circulation, it is difficult for it to heal naturally once damaged. If cartilage is left untreated, it will gradually progress to widespread cartilage damage, eventually resulting in osteoarthritis (OA). OA is a disease whose symptoms progress with age, but no fundamental treatment has yet been established. In recent years, progress has been made in the development of regenerative medicine to treat cartilage damage.

[0020] Typically, nude rat cartilage defect models are used to evaluate the efficacy of cell cultures used for cartilage regeneration, and although this model is useful for predicting the efficacy of cartilage regeneration, it is time-consuming, expensive, and poses animal welfare issues.

[0021] In this study, we extracted potential in vitro markers to predict the efficacy of cartilage regeneration. We prepared chondrocyte sheets of different passages using chondrocytes derived from the same polydactyly patient and implanted them into nude rat models. The gene expression and secreted proteins of the same chondrocyte sheets were analyzed by RNA sequencing (RNA-Seq) and aptamer-based proteomics analysis (SOMA Scan), respectively. We obtained 21,121 gene expression data sets and 7,596 protein expression data sets. We then correlated these data sets with in vivo tissue repair potential as scored by the International Cartilage Repair Society (ICRS) histological grading system.

[0022] From these, we focused on adhesion-related molecules, and from both RNA-Seq and SOMA-Scan data, we found 10 molecules (ELANE, LAP3, TENM4, PTHLH (four positively correlated gene markers), THBS3, IL27RA, NTN4, COL1A1, CHRD, VCAM1 (six negatively correlated gene markers) (also referred to as "(1) adhesion-related genes") that were highly correlated with the clinical score after transplantation in a nude rat cartilage injury model animal. At this time, the ICRS score (Hgtot) and R Extraction was performed when the correlation coefficient of NA-Sequence was 0.5 or higher and the correlation coefficient of ICRS score (Hgtot) and SOMA-Scan was 0.5 or higher. The ICRS score was evaluated as the sum (Hgtot) of the scores for the evaluation items of tissue morphology, matrix staining, structural integrity, cell mass formation, tidemark opening, bone formation, histological evaluation of surface structure, histological evaluation of the degree of defect filling, lateral integration of the defect filling tissue, basal integration of the defect filling tissue, and histological signs of inflammation (Non-Patent Document 2: Brehm et al., 2006, Repair of superficial osteochondral defects with an autologous scaffold-free cartilage construct in a caprine model: Implantation method and short-term results. Osteoarthritis and Cartilage, 14, 1214-1226. 10.1016 / j.joca.2006.05.002; Non-Patent Document 3: Mainil-Varlet et al., 2003, Histological assessment of cartilage repair: A report by the Histology Endpoint Committee of the International Cartilage Repair Society (ICRS). The Journal of Bone and Joint Surgery. American Volume, 85(A Suppl 2), 45-57.; Non-patent document 4: O'Driscoll, Keeley, & Salter, 1988, Durability of regenerated articular cartilage produced by free autogenous periosteal grafts in major full-thickness defects in joint surfaces under the influence of continuous passive motion. A follow-up report at one year. The Journal of Bone and Joint Surgery. American Volume, 70, 595-606; Non-patent document 5: Daichi Takizawa et al., Regenerative effects of human chondrocyte sheets in a xenogeneic transplantation model using immune-deficient rats, J Tissue Eng Regen Med. 2020 Sep;14(9):1296-1306.). .

[0023] Furthermore, we prepared new chondrocyte cell sheets from different donors and passage numbers, and performed cartilage regeneration in a nude rat cartilage injury model. We also obtained comprehensive expression data using RNA-Seq and evaluated the 10 identified markers. We found that nine of the 10 markers, excluding ELANE, were reproducible. Specifically, we found three positively correlated gene markers: LAP3, TENM4, and PTHLH; and six negatively correlated gene markers: THBS3, IL27RA, NTN4, COL1A1, CHRD, and VCAM1.

[0024] Furthermore, as a result of extensive research, the present inventors have also found the following genes: (2) Angiogenesis-related genes; (3) ECM-related genes; (4) Collagen-related genes; (5) Cell migration-related genes; and (6) Mitochondria-related genes.

[0025] Thus, the inventors focused on these adhesion-related molecules and correlated the data with in vivo repair ability scored by the International Cartilage Repair Society (ICRS) histological grading system, and found nine or more genes that have a high correlation with in vivo efficacy. In other words, by conducting experiments using cell sheets with different efficacy, the inventors were able to obtain multiple factors whose expression levels correlate with efficacy.

[0026] In this way, the inventors have succeeded in extracting potential markers for predicting the effectiveness of chondrocyte sheets for cartilage regeneration. The inventors believe that in the future, these markers will be able to replace animal testing for donor selection and will also be useful for evaluating the quality of final products.

[0027] That is, the present invention can provide a marker for evaluating cartilage-like tissue formation characteristics. The marker preferably includes one or more gene markers selected from the group consisting of (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, and (6) mitochondria-related genes. These gene markers are suitable for evaluating the cartilage-like tissue formation characteristics of cell cultures. The present invention may use an appropriate combination of one or more, two or more, three or more, four or more, five or more, or six types of genes selected from the group consisting of (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, and (6) mitochondria-related genes. For example, combinations of two types include (1) and (2), (1) and (3), (1) and (4), (1) and (5), (1) and (6), (2) and (3), (2) and (4), (2) and (5), (2) and (6), (3) and (4), (3) and (5), (3) and (6), (4) and (5), (4) and (6), and (5) and (6). Examples of combinations of three types include (1,2,3), (1,2,4), (1,2,5), (1,2,6), (1,3,4), (1,3,5), (1,3,6), (1,4,5), (1,4,6), (1,5,6), (2,3,4), (2,3,5), (2,3,6), (2,4,5), (2,4,6), (2,5,6), (3,4,5), (3,4,6), (3,5,6), (4,5,6), etc. The combinations of the present invention are not limited to these, and multiple combinations may be used as appropriate.

[0028] The markers used in the present invention may be one or more selected from all of the markers described in (1) to (6) above, regardless of group or number. Among all markers, 1, 2, 3, 4, 5, 6, or more markers with a higher correlation coefficient in the case of a positive correlation, or a lower correlation coefficient in the case of a negative correlation, may be used in combination. Furthermore, from all markers or each group, it is preferable to select and use one or more markers with a higher correlation coefficient in the case of a positive correlation, and / or it is preferable to select and use one or more markers with a lower correlation coefficient in the case of a negative correlation. A combination of markers with a high positive correlation and a low negative correlation may also be used. (1) LAP3, TENM4, PTHLH, and THBS3, IL27RA, NTN4, COL1A1, CHRD, and VCAM1. (2) PDGFA, VEGFC, SERPINE1, CSPG4, and RASIP1, WARS, AAMP, FGF18, and S1PR1. (3) ECM-related genes: BGN, CLEC3B, COL11A1, COL1A1, COL1A2, COL5A1, COL5A2, CRIP2, HAPLN1, JUP, MMP13, MMP23B, SERPINE1, THBS1, TINAGL1, and ADAMTS14, CCT2, CYHR1, and MMP17. (4) COL5A1, COL5A2, COL1A1, COL1A2, and COL11A1. (5) ARPC5, CD2AP, COL5A1, CSPG4, ITGB3, JUP, PODXL, RHOA, SDC2, SHROOM2, SNAI1, and BDKRB1, CDK5, FUT8, S1PR1, SH3KBP1, TGFBR3.(6) AS3MT, ATP7B, CA5B, CKB, CMC2, CTPS2, ELN, FBXO7, MARC2, ME3, NOL3, PABPC5, PANK2, PRELID2, SEPT4, SPARC, SPTLC2, and ADCK1, ADH5, AFG3L2, AIFM1, AKAP1, ALDH4A1, APEX1, BCS1L, C1QBP, CARS2, CHCHD3, COA4, COQ9, COX7B, DDAH2, DGAT2, DLD, DNAJA1, ECH1, ECHS1, FDXR, FIBP, FMC1, GATB, GCSH, GPX1, HSD17B10, IDH3B, LAP3, LARS2, MAPK8IP1, MDH2, METAP1D, MRPL10, MRPL24, MRPL42, MRPS12, MRPS18C, MRPS22, MRPS27, MRPS35, MTG1, MTHFD1, NDUFB9, NDUFS2, NDUFV1, OCIAD1, OX NAD1, PARS2, PDF, PHB, PHYH, POLRMT, PTCD1, PYURF, RMDN1, RMDN1, SDHD, SFXN4, SLC25A11, SMIM4, TACO1, TIMM10, TIMM50, TK2, TOMM22, TOMM5, TRAP1, TUF M, UQCRC1.

[0029] In one embodiment of the present invention, the marker for evaluating cartilage-like tissue formation properties preferably comprises one or more gene markers selected from the group consisting of positively correlated genes among (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, and (6) mitochondria-related genes, and one or more gene markers selected from the group consisting of negatively correlated genes among (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, and (6) mitochondria-related genes. These gene markers are suitable for evaluating the cartilage-like tissue formation properties of cell cultures. Furthermore, since expression of the gene results in expression of a protein related to the gene, the protein, peptide, or fragment thereof expressed by or encoded by the gene may be used as a marker for evaluating cartilage-like tissue formation properties, or as an indicator of expression level or an estimated score based on the expression level (preferably an estimated ICSR in vivo repair capacity score or estimated ICSR score). For example, these gene markers may be used to detect or measure gene expression in a cell culture (e.g., a cell sheet). Efficacy may be determined using a threshold value for the expression level of each gene, or by correlation analysis (e.g., Pearson's correlation analysis (e.g., regression line equation), Spearman's correlation analysis) obtained from the relationship between gene expression level and ICSR score, or by an estimated score based on the expression level. The estimated score may be an estimated ICSR score, an estimated efficacy score, or an estimated score for evaluating cartilage-like tissue formation properties, with an estimated score for a cell culture being preferred. The threshold value may be a predetermined value, a predetermined reference value, or a predetermined standard value.In addition, ICRS scores (estimated values) derived from genetic markers include, for example, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, and 45. A value selected from these may be used as an upper limit, lower limit, or numerical range. For example, in the case of the above-mentioned 11 items, the ICRS score may be 11 to 45. Preferred lower limits are 28 or more, 29 or more, or 30 or more (see, for example, Figures 1 and 2). In this specification, it is preferable to determine that any gene is effective if its ICRS score is within this range.

[0030] Furthermore, the genetic markers used in this embodiment are preferably those with a positive correlation or a negative correlation, and in the case of the absolute value of the correlation coefficient, the stronger the correlation, the more preferable, for example, the absolute value |r| = 0.6 or more, 0.7 or more, 0.8 or more, or 0.9 or more. More specifically, in the case of a positive correlation, a higher correlation coefficient (0 to +1) is preferable, preferably 0.6 or more, more preferably 0.7 or more, even more preferably 0.8 or more, and more preferably 0.9 or more. In the case of a negative correlation, a lower correlation coefficient (0 to -1) is preferable, preferably -0.6 or less, more preferably -0.7 or less, even more preferably -0.8 or less, and more preferably -0.9 or less.

[0031] 1-1. Group 1 Genes The adhesion-related genes (1) are described below, but these genes may be referred to as Group 1 genes rather than adhesion-related. One or more gene markers may be selected from this group. Among these, in the case of a positive correlation, it is preferable to select and use one or more genes with a higher correlation, and / or in the case of a negative correlation, it is preferable to select and use one or more genes with a lower correlation. (1) Group 1 Genes: LAP3, TENM4, PTHLH, and THBS3, IL27RA, NTN4, COL1A1, CHRD, VCAM1.

[0032] <Positively correlated genetic markers (Group 1)>

[0033] <LAP3 (leucine aminopeptidase 3)> LAP3 leucine aminopeptidase 3 [Lagopus muta (rock ptarmigan)] Gene ID: 125691647 (https: / / www.ncbi.nlm.nih.gov / gene / 125691647) When LAP3 (positive correlation) is equal to or greater than a threshold, it can be determined that the efficacy is higher. For example, a suitable threshold for LAP3 (positive correlation) is preferably 5.61 or greater, more preferably 5.68 or greater, and even more preferably 5.75 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from LAP3 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0034] <TENM4 (Teneurin Transmembrane Protein 4)> TENM4 teneurin transmembrane protein 4 [Homo sapiens (human)] Gene ID: 26011 (https: / / www.ncbi.nlm.nih.gov / gene / 26011) TENM4 (positive correlation) can be determined to be more effective when it is equal to or greater than a threshold value. The preferred threshold for TENM4 (positive correlation) is preferably 0.72 or greater, more preferably 0.89 or greater, and even more preferably 1.06 or greater. Alternatively, a regression line obtained from TENM4 and the ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown as examples.

[0035] <PTHLH (parathyroid hormone-like hormone)> PTHLH parathyroid hormone-like hormone [ Homo sapiens (human)] Gene ID: 5744 (https: / / www.ncbi.nlm.nih.gov / gene / 5744) PTHLH (positive correlation) is determined to be more effective when it is equal to or greater than a threshold value. A suitable threshold value for PTHLH (positive correlation) is preferably 1.1 or greater, more preferably 1.9 or greater, and even more preferably 2.6 or greater. Alternatively, a regression line obtained from PTHLH and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0036] <Negatively correlated genetic markers (Group 1): 6 types>

[0037] <THBS3 (Thrombospondin 3)> THBS3 thrombospondin 3 [Homo sapiens (human)] Gene ID: 7059 (https: / / www.ncbi.nlm.nih.gov / gene / 7059) THBS3 (negative correlation) can be determined to be more effective when it is below a threshold. For example, a suitable threshold for THBS3 (negative correlation) is preferably 6.12 or less, more preferably 6.02 or less, and even more preferably 5.92 or less. Alternatively, a regression line obtained from THBS3 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0038] <IL27RA (Interleukin 27 Receptor Subunit Alpha)> IL27RA interleukin 27 receptor subunit alpha [ Homo sapiens (human)] Gene ID: 9466 (https: / / www.ncbi.nlm.nih.gov / gene / 9466) IL27RA (negative correlation) can be determined to be more effective when it is below a threshold. For example, a suitable threshold for IL27RA (negative correlation) is preferably 4.76 or less, more preferably 4.19 or less, and even more preferably 3.63 or less. Alternatively, a regression line obtained from IL27RA and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0039] <NTN4 (Netrin 4)> NTN4 netrin 4 [Homo sapiens (human)] Gene ID: 59277 (https: / / www.ncbi.nlm.nih.gov / gene / 59277) When NTN4 (negative correlation) is below a threshold, it can be determined that the efficacy is higher. For example, a suitable threshold for NTN4 (negative correlation) is preferably 7.71 or less, more preferably 7.53 or less, and even more preferably 7.36 or less. Alternatively, a regression line obtained from NTN4 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0040] <COL1A1 (collagen type 1 alpha 1 chain)> COL1A1 collagen type I alpha 1 chain [ Homo sapiens (human)] Gene ID: 1277 (https: / / www.ncbi.nlm.nih.gov / gene / 1277) COL1A1 (negative correlation) can be determined to be more effective when it is below a threshold. For example, a suitable threshold for COL1A1 (negative correlation) is preferably 12.5 or less, more preferably 12.1 or less, and even more preferably 11.8 or less. Alternatively, a regression line obtained from COL1A1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0041] <CHRD (chordin)> CHRD chordin [ Homo sapiens (human)] Gene ID: 8646 (https: / / www.ncbi.nlm.nih.gov / gene / 8646) When CHRD (negative correlation) is equal to or less than a threshold, it can be determined that the efficacy is higher. For example, a suitable threshold for CHRD (negative correlation) is preferably 0.76 or less, more preferably 0.57 or less, and even more preferably 0.39 or less. Alternatively, a regression line obtained from CHRD and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown as examples.

[0042] <VCAM1 (Vascular Cell Adhesion Molecule 1)> VCAM1 vascular cell adhesion molecule 1 [ Homo sapiens (human)] Gene ID: 7412 (https: / / www.ncbi.nlm.nih.gov / gene / 7412) When VCAM1 (negative correlation) is below a threshold, it can be determined that the efficacy is higher. For example, a suitable threshold for VCAM1 (negative correlation) is preferably 8.89 or less, more preferably 8.41 or less, and even more preferably 7.92 or less. Alternatively, a regression line obtained from VCAM1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0043] 1-2. Group 2 Genes The (2) Angiogenesis-related genes are described below, but these genes may also be referred to as Group 2 genes rather than adhesion-related genes. One or more gene markers may be selected from this group. Among these, in the case of a positive correlation, it is preferable to select and use one or more genes with a higher correlation, and / or in the case of a negative correlation, it is preferable to select and use one or more genes with a lower correlation. (2) Group 2 Genes: PDGFA, VEGFC, SERPINE1, CSPG4, and RASIP1, WARS, AAMP, FGF18, and S1PR1.

[0044] <Negatively correlated genetic markers (Group 2)>

[0045] <PDGFA (Platelet-Derived Growth Factor A Chain)> PDGFA platelet-derived growth factor subunit A [Homo sapiens (human)] Gene ID: 5154 (https: / / www.ncbi.nlm.nih.gov / gene / 5154) PDGFA (negative correlation) can be determined to be more effective when it is below a threshold value. For example, this preferred threshold is preferably 4.97 or less, more preferably 4.72 or less, and even more preferably 4.46 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from PDGFA and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0046] <VEGFC (Vascular Endothelial Growth Factor-C)> VEGFC vascular endothelial growth factor C [Homo sapiens (human)] Gene ID: 7424 (https: / / www.ncbi.nlm.nih.gov / gene / 7424) When VEGFC (negative correlation) is below a threshold, it can be determined that the efficacy is higher. For example, this suitable threshold is preferably 8.07 or less, more preferably 7.85 or less, and even more preferably 7.63 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from VEGFC and ICSR score can be used. Alternatively, values ​​normalized by a housekeeping gene can be used, in which case the values ​​may not necessarily match those shown as examples.

[0047] <SERPINE1 (Plasminogen Activator Inhibitor 1 (PAI-1))> SERPINE1 serpin family E member 1 [Homo sapiens (human)] Gene ID: 5054 (https: / / www.ncbi.nlm.nih.gov / gene / 5054) When SERPINE1 (negative correlation) is below a threshold, it can be determined that the efficacy is higher. For example, this suitable threshold is preferably 8.73 or less, more preferably 8.49 or less, and even more preferably 8.24 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from SERPINE1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the exemplified values.

[0048] <CSPG4 (extracellular matrix protein)> CSPG4 chondroitin sulfate proteoglycan 4 [ Homo sapiens (human)] Gene ID: 1464 (https: / / www.ncbi.nlm.nih.gov / gene / 1464) CSPG4 (negative correlation) is considered to be more effective when it is below a threshold. For example, this preferred threshold is preferably 5.63 or less, more preferably 5.49 or less, and even more preferably 5.35 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from CSPG4 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0049] <Positively correlated genetic markers (Group 2)>

[0050] <RASIP1> RASIP1 Ras interacting protein 1 [Homo sapiens (human)] Gene ID: 54922 (https: / / www.ncbi.nlm.nih.gov / gene / 54922). RASIP1 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold. For example, this threshold is preferably 0.01 or greater, more preferably 0.02 or greater, and even more preferably 0.05 or greater; higher values ​​are preferable. Alternatively, a regression line obtained from RASIP1 and ICSR score may be used.

[0051] <WARS> WARS1 tryptophanyl-tRNA synthetase 1 [ Homo sapiens (human)] Gene ID: 7453 (https: / / www.ncbi.nlm.nih.gov / gene / 7453) The WARS gene encodes tryptophanyl-tRNA synthetase 1. A WARS (positive correlation) greater than or equal to a threshold value indicates higher efficacy. For example, this threshold is preferably 6.27 or greater, more preferably 6.33 or greater, and even more preferably 6.40 or greater, with higher values ​​being more preferable. Alternatively, a regression line derived from WARS and ICSR scores may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0052] <AAMP> AAMP angio associated migratory cell protein [ Homo sapiens (human)] Gene ID: 14 ​​(https: / / www.ncbi.nlm.nih.gov / gene / 14) AAMP (positive correlation) can be determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 6.10 or greater, more preferably 6.15 or greater, and even more preferably 6.21 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from AAMP and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0053] <FGF18> FGF18 Fibroblast Growth Factor 18 [Homo sapiens (human)] Gene ID: 8817 (https: / / www.ncbi.nlm.nih.gov / gene / 8817) When FGF18 (positive correlation) is equal to or greater than a threshold, it can be determined that the efficacy is higher. For example, this preferred threshold is preferably 0.35 or greater, more preferably 0.83 or greater, and even more preferably 1.31 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from FGF18 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0054] <S1PR1 (Sphingosine-1-phosphate receptor 1)> S1PR1 sphingosine-1-phosphate receptor 1 [ Homo sapiens (human)] Gene ID: 1901 (https: / / www.ncbi.nlm.nih.gov / gene / 1901) S1PR1 (positive correlation) can be determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 1.58 or greater, more preferably 1.87 or greater, and even more preferably 2.16 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from S1PR1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0055] 1-3. Group 3 Genes The (3) ECM-related genes are described below. These genes are not necessarily adhesion-related but may be referred to as Group 3 genes. One or more gene markers may be selected from this group. Among these, it is preferable to select and use one or more genes with a higher correlation in the case of a positive correlation, and / or it is preferable to select and use one or more genes with a lower correlation in the case of a negative correlation. (3) Group 3 Genes: BGN, CLEC3B, COL11A1, COL1A1, COL1A2, COL5A1, COL5A2, CRIP2, HAPLN1, JUP, MMP13, MMP23B, SERPINE1, THBS1, TINAGL1, and ADAMTS14, CCT2, CYHR1, and MMP17.

[0056] <Negatively correlated genetic markers (Group 3)>

[0057] <BGN (Biglycan)> BGN biglycan [ Homo sapiens (human)] Gene ID: 633 (https: / / www.ncbi.nlm.nih.gov / gene / 633) When BGN (negative correlation) is below a threshold, it can be determined that the efficacy is higher. For example, this suitable threshold is preferably 11.94 or less, more preferably 11.80 or less, and even more preferably 11.65 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from BGN and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown as examples.

[0058] <CLEC3B (C-type lectin-like domain family 3, member B)> LEC3B C-type lectin domain family 3 member B [ Homo sapiens (human)] Gene ID: 7123, (https: / / www.ncbi.nlm.nih.gov / gene / 7123) CLEC3B (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 6.23 or less, more preferably 5.99 or less, and even more preferably 5.73 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from CLEC3B and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the exemplified values.

[0059] <COL11A1 (encoding collagen 11A1 chain)> COL11A1 collagen type XI alpha 1 chain [Homo sapiens (human)] Gene ID: 1301, (https: / / www.ncbi.nlm.nih.gov / gene / 1301) COL11A1 (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 8.68 or less, more preferably 8.28 or less, and even more preferably 7.88 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from COL11A1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0060] <COL1A1 (type I collagen alpha 1 chain)> COL1A1 collagen type I alpha 1 chain [ Homo sapiens (human)] Gene ID: 1277, (https: / / www.ncbi.nlm.nih.gov / gene / 1277) COL1A1 (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 12.5 or less, more preferably 12.1 or less, and even more preferably 11.8 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from COL1A1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0061] <COL1A2 (type I collagen alpha 2 chain)> COL1A2 collagen type I alpha 2 chain [ Homo sapiens (human)] Gene ID: 1278, (https: / / www.ncbi.nlm.nih.gov / gene / 1278) COL1A2 (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 12.5 or less, more preferably 12.3 or less, and even more preferably 12.1 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from COL1A2 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0062] <COL5A1 (Collagen Type V Alpha 1 Chain)> COL5A1 collagen type V alpha 1 chain [Homo sapiens (human)] Gene ID: 1289, (https: / / www.ncbi.nlm.nih.gov / gene / 1289) COL5A1 (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 8.37, more preferably 8.09 or less, and even more preferably 7.81 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from COL5A1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0063] <COL5A2 (collagen α2(V) chain)> COL5A2 collagen type V alpha 2 chain [ Homo sapiens (human)] Gene ID: 1290 (https: / / www.ncbi.nlm.nih.gov / gene / 1290). COL5A2 (negative correlation) is considered to be more effective when it is below a threshold. For example, this preferred threshold is preferably 9.62 or less, more preferably 9.42 or less, and even more preferably 9.21 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from COL5A2 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0064] <CRIP2 (Cysteine-rich protein 2)> CRIP2 cysteine-rich protein 2 [ Homo sapiens (human)] Gene ID: 1397 (https: / / www.ncbi.nlm.nih.gov / gene / 1397). CRIP2 (negative correlation) is considered to be more effective when it is below a threshold. For example, this preferred threshold is preferably 6.89 or less, more preferably 6.76 or less, and even more preferably 6.64 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from CRIP2 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0065] <HAPLN1> HAPLN1 hyaluronan and proteoglycan link protein 1 [ Homo sapiens (human)] Gene ID: 1404, (https: / / www.ncbi.nlm.nih.gov / gene / 1404) HAPLN1 (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 8.50 or less, more preferably 8.22 or less, and even more preferably 7.94 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from HAPLN1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0066] <JUP> JUP junction plakoglobin [ Homo sapiens (human)] Gene ID: 3728 (https: / / www.ncbi.nlm.nih.gov / gene / 3728). A JUP (negative correlation) below a threshold value indicates a higher level of efficacy. For example, this threshold value is preferably 2.40 or less, more preferably 2.15 or less, and even more preferably 1.89 or less; the lower the value, the better. Alternatively, a regression line derived from JUP and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case the values ​​may not necessarily match those shown.

[0067] <MMP13> MMP13 matrix metallopeptidase 13 [Homo sapiens (human)] Gene ID: 4322 (https: / / www.ncbi.nlm.nih.gov / gene / 4322). MMP13 (negative correlation) is considered to be more effective when it is below a threshold. For example, this preferred threshold is preferably 12.2 or less, more preferably 11.0 or less, and even more preferably 9.7 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from MMP13 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0068] <MMP23B> MMP23B matrix metallopeptidase 23B [ Homo sapiens (human)] Gene ID: 8510 (https: / / www.ncbi.nlm.nih.gov / gene / 8510). MMP23B (negative correlation) is considered to be more effective when it is below a threshold. For example, this preferred threshold is preferably 1.19 or less, more preferably 0.94 or less, and even more preferably 0.68 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from MMP23B and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0069] <SERPINE1 (Plasminogen Activator Inhibitor 1 (PAI-1))> SERPINE1 serpin family E member 1 [Homo sapiens (human)] Gene ID: 5054 (https: / / www.ncbi.nlm.nih.gov / gene / 5054) When SERPINE1 (negative correlation) is below a threshold, it can be determined that the efficacy is higher. This suitable threshold is preferably 8.73 or less, more preferably 8.49 or less, and even more preferably 8.24 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from SERPINE1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the exemplified values.

[0070] <THBS1 (Thrombospondin-1)> THBS1 thrombospondin 1 [Homo sapiens (human)] Gene ID: 7057 (https: / / www.ncbi.nlm.nih.gov / gene / 7057). THBS1 (negative correlation) is considered to be more effective when it is below a threshold. For example, this preferred threshold is preferably 11.8 or less, more preferably 11.5 or less, and even more preferably 11.2 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from THBS1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0071] <TINAGL1> TINAGL1 tubulointerstitial nephritis antigen like 1 [Homo sapiens (human)] Gene ID: 64129 (https: / / www.ncbi.nlm.nih.gov / gene / 64129). TINAGL1 (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 1.17 or less, more preferably 0.91 or less, and even more preferably 0.66 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from TINAGL1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0072] <Positively correlated genetic markers (Group 3)>

[0073] ADAMTS14 (ADAMTS family protein) ADAMTS14 ADAM metallopeptidase with thrombospondin type 1 motif 14 [Homo sapiens (human)] Gene ID: 140766 (https: / / www.ncbi.nlm.nih.gov / gene / 140766). When ADAMTS14 (positive correlation) is equal to or greater than a threshold, it can be determined that the efficacy is higher. For example, this preferred threshold is preferably 2.82 or greater, more preferably 3.07 or greater, and even more preferably 3.30 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from ADAMTS14 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0074] <CCT2> CCT2 chaperonin containing TCP1 subunit 2 [ Homo sapiens (human)] Gene ID: 10576 (https: / / www.ncbi.nlm.nih.gov / gene / 10576). CCT2 (positive correlation) can be determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 6.90 or greater, more preferably 6.94 or greater, and even more preferably 6.98 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from CCT2 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0075] <CYHR1> CYHR1 cysteine ​​and histidine rich 1 (ZFTRAF1 zinc finger TRAF-type containing 1 [ Homo sapiens (human)]) Gene ID: 50626, (https: / / www.ncbi.nlm.nih.gov / gene / 50626) CYHR1 (positive correlation) can be determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 3.76 or greater, more preferably 3.82 or greater, and even more preferably 3.88 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from CYHR1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0076] <MMP17 (Matrix Metallopeptidase 17)> MMP17 (positive correlation) can be determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold value is preferably 1.90 or greater, more preferably 2.05 or greater, and even more preferably 2.20 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from MMP17 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown as examples.

[0077] 1-4. Group 4 Genes The collagen-related genes in (4) above are described below, but these genes are not necessarily referred to as adhesion-related genes, and may be referred to as Group 2 genes. One or more gene markers may be selected from this group. Among these, in the case of a positive correlation, it is preferable to select and use one or more genes with a higher correlation, and / or in the case of a negative correlation, it is preferable to select and use one or more genes with a lower correlation. (4) Group 4 Genes: COL5A1, COL5A2, COL1A1, COL1A2, COL11A1.

[0078] <Negatively correlated genetic markers (Group 4)>

[0079] <COL5A1 (collagen type V alpha 1 chain)> COL5A1 collagen type V alpha 1 chain [ Homo sapiens (human)] Gene ID: 1289, (https: / / www.ncbi.nlm.nih.gov / gene / 1289) COL5A1 (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 8.37 or less, more preferably 8.09 or less, and even more preferably 7.81 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from COL5A1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0080] <COL5A2 (collagen α2(V) chain)> COL5A2 collagen type V alpha 2 chain [ Homo sapiens (human)] Gene ID: 1290 (https: / / www.ncbi.nlm.nih.gov / gene / 1290). COL5A2 (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 9.63 or less, more preferably 9.42 or less, and even more preferably 9.21 or less; the lower the value, the better. Alternatively, a regression line obtained from COL5A2 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0081] <COL1A1 (type I collagen alpha 1 chain)> COL1A1 collagen type I alpha 1 chain [ Homo sapiens (human)] Gene ID: 1277, (https: / / www.ncbi.nlm.nih.gov / gene / 1277) COL1A1 (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 12.5 or less, more preferably 12.1 or less, and even more preferably 11.8 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from COL1A1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0082] <COL1A2> COL1A2 collagen type I alpha 2 chain [Homo sapiens (human)] Gene ID: 1278 (https: / / www.ncbi.nlm.nih.gov / gene / 1278). COL1A2 (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 12.6 or less, more preferably 12.4 or less, and even more preferably 12.1 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from COL1A2 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0083] <COL11A1> COL11A1 collagen type XI alpha 1 chain [ Homo sapiens (human)] Gene ID: 1301 (https: / / www.ncbi.nlm.nih.gov / gene / 1301). COL11A1 (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 8.68 or less, more preferably 8.27 or less, and even more preferably 7.87 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from COL11A1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0084] 1-5. Group 5 Genes The (5) Cell migration-related genes are described below. These genes are not referred to as adhesion-related genes, but may also be referred to as Group 2 genes. One or more gene markers may be selected from this group. Among these, in the case of a positive correlation, it is preferable to select and use one or more genes with a higher correlation, and / or in the case of a negative correlation, it is preferable to select and use one or more genes with a lower correlation. (5) Cell migration-related genes: ARPC5, CD2AP, COL5A1, CSPG4, ITGB3, JUP, PODXL, RHOA, SDC2, SHROOM2, SNAI1, and BDKRB1, CDK5, FUT8, S1PR1, SH3KBP1, TGFBR3.

[0085] <Negatively correlated genetic markers (Group 5)>

[0086] <ARPC5> ARPC5 actin related protein 2 / 3 complex subunit 5 [Homo sapiens (human)] Gene ID: 10092 (https: / / www.ncbi.nlm.nih.gov / gene / 10092). ARPC5 (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 6.96 or less, more preferably 6.88 or less, and even more preferably 6.81 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from ARPC5 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0087] <CD2AP (protein involved in cytoskeleton regulation)> CD2AP CD2 associated protein [ Homo sapiens (human)] Gene ID: 23607 (https: / / www.ncbi.nlm.nih.gov / gene / 23607) When CD2AP (negative correlation) is below a threshold, it can be determined that the efficacy is higher. For example, this suitable threshold is preferably 4.25 or less, more preferably 4.22 or less, and even more preferably 4.18 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from CD2AP and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0088] <COL5A1> COL5A1 collagen type V alpha 1 chain [Homo sapiens (human)] Gene ID: 1289 (https: / / www.ncbi.nlm.nih.gov / gene / 1289). COL5A1 (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 8.37 or less, more preferably 8.09 or less, and even more preferably 7.81 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from COL5A1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0089] <CSPG4> CSPG4 chondroitin sulfate proteoglycan 4 [Homo sapiens (human)] Gene ID: 1464 (https: / / www.ncbi.nlm.nih.gov / gene / 1464) CSPG4 (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 5.63 or less, more preferably 5.49 or less, and even more preferably 5.35 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from CSPG4 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0090] <ITGB3 (integrin β3 subunit)> ITGB3 integrin subunit beta 3 [ Homo sapiens (human)] Gene ID: 3690 (https: / / www.ncbi.nlm.nih.gov / gene / 3690) When ITGB3 (negative correlation) is below a threshold, it can be determined that the efficacy is higher. For example, this suitable threshold is preferably 3.26 or less, more preferably 3.07 or less, and even more preferably 2.88 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from ITGB3 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0091] <JUP> JUP junction plakoglobin [ Homo sapiens (human)] Gene ID: 3728 (https: / / www.ncbi.nlm.nih.gov / gene / 3728). A JUP (negative correlation) below a threshold value indicates a higher level of efficacy. For example, this threshold value is preferably 2.40 or less, more preferably 2.15 or less, and even more preferably 1.89 or less; the lower the value, the better. Alternatively, a regression line derived from JUP and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case the values ​​may not necessarily match those shown.

[0092] <PODXL (podocalyxin-like)> PODXL podocalyxin-like [ Homo sapiens (human)] Gene ID: 5420 (https: / / www.ncbi.nlm.nih.gov / gene / 5420) When PODXL (negative correlation) is below a threshold, it can be determined that the efficacy is higher. This preferred threshold is preferably 3.44 or less, more preferably 3.07 or less, and even more preferably 2.78 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from PODXL and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the exemplified values.

[0093] <RHOA (small GTPase family)> RHOA ras homolog family member A [ Homo sapiens (human)] Gene ID: 387, (https: / / www.ncbi.nlm.nih.gov / gene / 387) When RHOA (negative correlation) is below a threshold, it can be determined that the efficacy is higher. For example, this suitable threshold is preferably 9.68 or less, more preferably 9.60 or less, and even more preferably 9.53 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from RHOA and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0094] <SDC2> SDC2 syndecan 2 [Homo sapiens (human)] Gene ID: 6383 (https: / / www.ncbi.nlm.nih.gov / gene / 6383). SDC2 (negative correlation) is considered to be more effective when it is below a threshold. For example, this preferred threshold is preferably 7.79 or less, more preferably 7.62 or less, and even more preferably 7.45 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from SDC2 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0095] <SHROOM2> SHROOM2 shroom family member 2 [ Homo sapiens (human)] Gene ID: 357 (https: / / www.ncbi.nlm.nih.gov / gene / 357) SHROOM2 (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 4.58 or less, more preferably 4.34 or less, and even more preferably 4.10 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from SHROOM2 and the ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0096] <SNAI1 (Snail family transcription factor)> SNAI1 snail family transcriptional repressor 1 [ Homo sapiens (human)] Gene ID: 6615, (https: / / www.ncbi.nlm.nih.gov / gene / 6615) SNAI1 (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 4.10 or less, more preferably 3.96 or less, and even more preferably 3.82 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from SNAI1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0097] <Positively correlated genetic markers (Group 5)>

[0098] <BDKRB1> BDKRB1 bradykinin receptor B1 [Homo sapiens (human)] Gene ID: 623 (https: / / www.ncbi.nlm.nih.gov / gene / 623) BDKRB1 (positive correlation) is determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 5.20 or greater, more preferably 5.51 or greater, and even more preferably 5.82 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from BDKRB1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0099] <CDK5 (CDK5 (cyclin-dependent kinase 5))> CDK5 cyclin-dependent kinase 5 [ Homo sapiens (human)] Gene ID: 1020, (http: / / ncbi.nlm.nih.gov / gene / 1020) CDK5 (positive correlation) can be determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 3.30 or greater, more preferably 3.35 or greater, and even more preferably 3.40 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from CDK5 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown as examples.

[0100] <FUT8 (fucosyltransferase 8)> FUT8 fucosyltransferase 8 [ Homo sapiens (human)] Gene ID: 2530 (https: / / www.ncbi.nlm.nih.gov / gene / 2530) When FUT8 (positive correlation) is equal to or greater than a threshold, it can be determined that the efficacy is higher. For example, this preferred threshold is preferably 4.80 or greater, more preferably 4.86 or greater, and even more preferably 4.92 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from FUT8 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0101] <S1PR1 (Sphingosine-1-phosphate receptor 1)> S1PR1 sphingosine-1-phosphate receptor 1 [ Homo sapiens (human)] Gene ID: 1901 (https: / / www.ncbi.nlm.nih.gov / gene / 1901) S1PR1 (positive correlation) can be determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 1.58 or greater, more preferably 1.87 or greater, and even more preferably 2.16 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from S1PR1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0102] <SH3KBP1> SH3KBP1 SH3 domain containing kinase binding protein 1 [ Homo sapiens (human)] Gene ID: 30011 (https: / / www.ncbi.nlm.nih.gov / gene / 30011). SH3KBP1 (positive correlation) can be determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 4.44 or greater, more preferably 4.55 or greater, and even more preferably 4.66 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from SH3KBP1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0103] <TGFBR3> TGFBR3 transforming growth factor beta receptor 3 [Homo sapiens (human)] Gene ID: 7049 (https: / / www.ncbi.nlm.nih.gov / gene / 7049). TGFBR3 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 2.90 or greater, more preferably 3.09 or greater, and even more preferably 3.28 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from TGFBR3 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0104] 1-6. Group 6 Genes The mitochondrial-related genes (6) are described below. These genes are not necessarily adhesion-related, but may be referred to as Group 6 genes. One or more gene markers may be selected from this group. Among these, in the case of a positive correlation, it is preferable to select and use one or more genes with a higher correlation, and / or in the case of a negative correlation, it is preferable to select and use one or more genes with a lower correlation. (6) 6th group genes: AS3MT, ATP7B, CA5B, CKB, CMC2, CTPS2, ELN, FBXO7, MARC2, ME3, NOL 3, PABPC5, PANK2, PRELID2, SEPT4, SPARC, SPTLC2, and ADCK1, ADH5, AFG3L2, AIFM1 , AKAP1, ALDH4A1, APEX1, BCS1L, C1QBP, CARS2, CHCHD3, COA4, COQ9, COX7B, DDAH2, DGAT2, DLD, DNAJA1, ECH1, ECHS1, FDXR, FIBP, FMC1, GATB, GCSH, GPX1, HSD17B10, I DH3B, LAP3, LARS2, MAPK8IP1, MDH2, METAP1D, MRPL10, MRPL24, MRPL42, MRPS12, MR PS18C, MRPS22, MRPS27, MRPS35, MTG1, MTHFD1, NDUFB9, NDUFS2, NDUFV1, OCIAD1, O XNAD1, PARS2, PDF, PHB, PHYH, POLRMT, PTCD1, PYURF, RMDN1, RMND1, SDHD, SFXN4, S LC25A11, SMIM4, TACO1, TIMM10, TIMM50, TK2, TOMM22, TOMM5, TRAP1, TUFM, UQCRC1.

[0105] <Negatively correlated genetic markers (Group 6)>

[0106] <AS3MT (an enzyme that methylates inorganic arsenic)> AS3MT arsenite methyltransferase [ Homo sapiens (human)] Gene ID: 57412, (https: / / www.ncbi.nlm.nih.gov / gene / 57412) AS3MT (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 3.38 or less, more preferably 3.16 or less, and even more preferably 2.93 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from AS3MT and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0107] <ATP7B (a protein that excretes copper from cells)> ATP7B ATPase copper transporting beta [ Homo sapiens (human)] Gene ID: 540 (https: / / www.ncbi.nlm.nih.gov / gene / 540) ATP7B (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 1.22 or less, more preferably 1.01 or less, and even more preferably 0.80 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from ATP7B and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0108] <CA5B> CA5B carbonic anhydrase 5B [ Homo sapiens (human)] Gene ID: 11238 (https: / / www.ncbi.nlm.nih.gov / gene / 11238). CA5B (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 3.11 or less, more preferably 2.97 or less, and even more preferably 2.83 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from CA5B and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0109] <CKB (creatine kinase B subunit)> CKB creatine kinase B [ Homo sapiens (human)] Gene ID: 1152 (https: / / www.ncbi.nlm.nih.gov / gene / 1152) A CKB (negative correlation) value below a threshold value indicates a higher level of efficacy. For example, this threshold value is preferably 4.37 or less, more preferably 4.02 or less, and even more preferably 3.68 or less; the lower the value, the better. Alternatively, a regression line obtained from CKB and ICSR scores may be used. Values ​​normalized by housekeeping genes may also be used, in which case the values ​​may not necessarily match those shown.

[0110] <CMC2> CMC2 C-X9-C motif containing 2 [ Homo sapiens (human)] Gene ID: 56942 (https: / / www.ncbi.nlm.nih.gov / gene / 56942). CMC2 (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 2.29 or less, more preferably 2.21 or less, and even more preferably 2.13 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from CMC2 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0111] <CTPS2> CTPS2 CTP synthase 2 [ Homo sapiens (human)] Gene ID: 56474 (https: / / www.ncbi.nlm.nih.gov / gene / 56474). CTPS2 (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 3.73 or less, more preferably 3.67 or less, and even more preferably 3.62 or less; the lower the value, the better. Alternatively, a regression line obtained from CTPS2 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0112] <ELN> ELN elastin [ Homo sapiens (human)] Gene ID: 2006, (https: / / www.ncbi.nlm.nih.gov / gene / 2006) ELN (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 8.15 or less, more preferably 7.76 or less, and even more preferably 7.37 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from ELN and ICSR scores may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0113] <FBXO7> FBXO7 F-box protein 7 [ Homo sapiens (human)] Gene ID: 25793 (https: / / www.ncbi.nlm.nih.gov / gene / 25793). FBXO7 (negative correlation) is considered to be more effective when it is below a threshold. For example, this threshold is preferably 5.33 or less, more preferably 5.31 or less, and even more preferably 5.29 or less; the lower the value, the better. Alternatively, a regression line obtained from FBXO7 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0114] <MARC2> MTARC2 mitochondrial amidoxime reducing component 2 [Homo sapiens (human)] Gene ID: 54996 (https: / / www.ncbi.nlm.nih.gov / gene / 54996). MARC2 (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 4.04 or less, more preferably 3.94 or less, and even more preferably 3.84 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from MARC2 and ICSR scores may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0115] <ME3> ME3 malic enzyme 3 [Homo sapiens (human)] Gene ID: 10873 (https: / / www.ncbi.nlm.nih.gov / gene / 10873). ME3 (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 4.27 or less, more preferably 4.14 or less, and even more preferably 4.01 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from ME3 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0116] <NOL3> NOL3 nucleolar protein 3 [Homo sapiens (human)] Gene ID: 8996 (https: / / www.ncbi.nlm.nih.gov / gene / 8996). When NOL3 (negative correlation) is below a threshold, it can be determined that the efficacy is higher. For example, this preferred threshold is preferably 4.32 or less, more preferably 4.22 or less, and even more preferably 4.12 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from NOL3 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0117] <PABPC5> PABPC5 poly(A) binding protein cytoplasmic 5 [Homo sapiens (human)] Gene ID: 140886 (https: / / www.ncbi.nlm.nih.gov / gene / 140886). PABPC5 (negative correlation) is considered to be more effective when it is below a threshold. For example, this threshold is preferably 2.42 or less, more preferably 2.16 or less, and even more preferably 1.89 or less; the lower the value, the better. Alternatively, a regression line obtained from PABPC5 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0118] <PANK2> PANK2 pantothenate kinase 2 [Homo sapiens (human)] Gene ID: 80025 (https: / / www.ncbi.nlm.nih.gov / gene / 80025). PANK2 (negative correlation) is considered to be more effective when it is below a threshold. For example, this threshold is preferably 4.46 or less, more preferably 4.42 or less, and even more preferably 4.37 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from PANK2 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0119] <PRELID2> PRELID2 PRELI domain containing 2 [ Homo sapiens (human)] Gene ID: 153768 (https: / / www.ncbi.nlm.nih.gov / gene / 153768). PRELID2 (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 1.16 or less, more preferably 1.10 or less, and even more preferably 1.03 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from PRELID2 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0120] <SEPT4> SEPTIN4 septin 4 [Homo sapiens (human)] Gene ID: 5414 (https: / / www.ncbi.nlm.nih.gov / gene / 5414). SEPT4 (negative correlation) is considered to be more effective when it is below a threshold. For example, this preferred threshold is preferably 1.20 or less, more preferably 0.96 or less, and even more preferably 0.73 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from SEPT4 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0121] <SPARC> SPARC secret protein acidic and cysteine ​​rich [ Homo sapiens (human)] Gene ID: 6678 (https: / / www.ncbi.nlm.nih.gov / gene / 6678). SPARC (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 12.5 or less, more preferably 12.2 or less, and even more preferably 11.8 or less, with lower values ​​being more preferable. Alternatively, a regression line obtained from SPARC and ICSR scores may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0122] <SPTLC2> SPTLC2 serine palmitoyltransferase long chain base subunit 2 [ Homo sapiens (human)] Gene ID: 9517 (https: / / www.ncbi.nlm.nih.gov / gene / 9517). SPTLC2 (negative correlation) can be determined to be more effective when it is below a threshold. For example, this preferred threshold is preferably 5.29 or less, more preferably 5.19 or less, and even more preferably 5.09 or less; the lower the value, the better. Alternatively, a regression line obtained from SPTLC2 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0123] <Positively correlated genetic markers (Group 6)>

[0124] <ADCK1> ADCK1 aarF domain containing kinase 1 [ Homo sapiens (human)] Gene ID: 57143 (https: / / www.ncbi.nlm.nih.gov / gene / 57143). ADCK1 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 1.03 or greater, more preferably 1.14 or greater, and even more preferably 1.26 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from ADCK1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0125] <ADH5> ADH5 alcohol dehydrogenase 5 (class III), chi polypeptide [ Homo sapiens (human)] Gene ID: 128 ( https: / / www.ncbi.nlm.nih.gov / gene / 128 ). ADH5 (positive correlation) can be determined to be more effective when it is above a threshold. For example, this preferred threshold is preferably 7.65 or higher, more preferably 7.75 or higher, and even more preferably 7.85 or higher, with higher values ​​being more preferable. Alternatively, a regression line obtained from ADH5 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0126] <AFG3L2> AFG3L2 AFG3-like matrix AAA peptidase subunit 2 [ Homo sapiens (human)] Gene ID: 10939 (https: / / www.ncbi.nlm.nih.gov / gene / 10939). AFG3L25 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. This preferred threshold is preferably 4.61 or greater, more preferably 4.69 or greater, and even more preferably 4.77 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from AFG3L2 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0127] <AIFM1> AIFM1 apoptosis-inducing factor mitochondria associated 1 [Homo sapiens (human)] Gene ID: 9131 (https: / / www.ncbi.nlm.nih.gov / gene / 9131). AIFM1 (positive correlation) is determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 4.38 or greater, more preferably 4.46 or greater, and even more preferably 4.53 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from AIFM1 and the ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0128] <AKAP1> AKAP1 A-kinase anchoring protein 1 [Homo sapiens (human)] Gene ID: 8165 (https: / / www.ncbi.nlm.nih.gov / gene / 8165). AKAP1 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 3.45 or greater, more preferably 3.54 or greater, and even more preferably 3.64 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from AKAP1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0129] <ALDH4A1> ALDH4A1 aldehyde dehydrogenase 4 family member A1 [ Homo sapiens (human)] Gene ID: 8659 (https: / / www.ncbi.nlm.nih.gov / gene / 8659). ALDH4A1 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 2.86 or greater, more preferably 2.93 or greater, and even more preferably 3.00 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from ALDH4A1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0130] <APEX1> APEX1 apurinic / apyrimidinic endodeoxyribonuclease 1 [Homo sapiens (human)] Gene ID: 328 (https: / / www.ncbi.nlm.nih.gov / gene / 328). APEX1 (positive correlation) is determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 7.73 or greater, more preferably 7.85 or greater, and even more preferably 7.98 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from APEX1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0131] <BCS1L> BCS1L BCS1 homolog, ubiquinol-cytochrome c reductase complex chaperone [ Homo sapiens (human)] Gene ID: 617, (https: / / www.ncbi.nlm.nih.gov / gene / 617) BCS1L (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 3.97 or greater, more preferably 4.04 or greater, and even more preferably 4.11 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from BCS1L and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0132] <C1QBP> C1QBP complement C1q binding protein [ Homo sapiens (human)] Gene ID: 708 (https: / / www.ncbi.nlm.nih.gov / gene / 708). C1QBP (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this threshold value is preferably 6.03 or greater, more preferably 6.10 or greater, and even more preferably 6.16 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from C1QBP and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0133] <CARS2> CARS2 cysteinyl-tRNA synthetase 2, mitochondrial [ Homo sapiens (human)] Gene ID: 79587 (https: / / www.ncbi.nlm.nih.gov / gene / 79587). CARS2 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 4.15 or greater, more preferably 4.25 or greater, and even more preferably 4.34 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from CARS2 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0134] <CHCHD3> CHCHD3 coiled-coil-helix-coiled-coil-helix domain containing 3 [ Homo sapiens (human)] Gene ID: 54927 (https: / / www.ncbi.nlm.nih.gov / gene / 54927) CHCHD3 (positive correlation) can be determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 5.23 or greater, more preferably 5.32 or greater, and even more preferably 5.41 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from CHCHD3 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0135] <COA4> CNGA4 cyclic nucleotide gated channel subunit alpha 4 [ Homo sapiens (human)] Gene ID: 1262 (https: / / www.ncbi.nlm.nih.gov / gene?Db=gene&Cmd=DetailsSearch&Term=1262). COA4 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this threshold value is preferably 5.38 or greater, more preferably 5.43 or greater, and even more preferably 5.49 or greater; higher values ​​are more preferable. Alternatively, a regression line obtained from COA4 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0136] <COQ9> COQ9 coenzyme Q9 [Homo sapiens (human)] Gene ID: 57017 (https: / / www.ncbi.nlm.nih.gov / gene / 57017). COQ9 (positive correlation) is determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 4.53 or greater, more preferably 4.61 or greater, and even more preferably 4.69 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from COQ9 and ICSR scores may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0137] <COX7B> COX7B cytochrome c oxidase subunit 7B [Homo sapiens (human)] Gene ID: 1349 (https: / / www.ncbi.nlm.nih.gov / gene / 1349). A COX7B (positive correlation) value equal to or greater than a threshold value indicates higher efficacy. For example, this threshold value is preferably 5.82 or greater, more preferably 5.87 or greater, and even more preferably 5.91 or greater; higher values ​​are preferred. Alternatively, a regression line derived from COX7B and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0138] <DDAH2> DDAH2 DDAH family member 2, ADMA-independent [ Homo sapiens (human)] Gene ID: 23564 (https: / / www.ncbi.nlm.nih.gov / gene / 23564) DDAH2 (positive correlation) can be determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 5.95 or greater, more preferably 6.03 or greater, and even more preferably 6.11 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from DDAH2 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0139] <DGAT2> DGAT2 diacylglycerol O-acyltransferase 2 [Homo sapiens (human)] Gene ID: 84649 (https: / / www.ncbi.nlm.nih.gov / gene / 84649). DGAT2 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold. For example, this preferred threshold is preferably 1.28 or greater, more preferably 1.62 or greater, and even more preferably 1.95 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from DGAT2 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0140] <DLD> DLD dihydrolipoamide dehydrogenase [ Homo sapiens (human)] Gene ID: 1738 (https: / / www.ncbi.nlm.nih.gov / gene / 1738). A DLD (positive correlation) greater than or equal to a threshold value indicates higher efficacy. For example, this threshold is preferably 5.70 or greater, more preferably 5.74 or greater, and even more preferably 5.79 or greater; higher values ​​are more preferable. Alternatively, a regression line derived from DLD and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0141] <DNAJA1> DNAJA1 DnaJ heat shock protein family (Hsp40) member A1 [Homo sapiens (human)] Gene ID: 3301, (https: / / www.ncbi.nlm.nih.gov / gene / 3301) DNAJA1 (positive correlation) can be determined to be more effective when it is above a threshold. For example, this preferred threshold is preferably 7.85 or higher, more preferably 7.92 or higher, and even more preferably 8.00 or higher, with higher values ​​being more preferable. Alternatively, a regression line obtained from DNAJA1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0142] <ECH1> ECH1 enoyl-CoA hydratase 1 [Homo sapiens (human)] Gene ID: 51798 (https: / / www.ncbi.nlm.nih.gov / gene / 51798). ECH1 (positive correlation) can be determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 6.01 or greater, more preferably 6.09 or greater, and even more preferably 6.17 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from ECH1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0143] <ECHS1> ECHS1 enoyl-CoA hydratase, short chain 1 [ Homo sapiens (human)] Gene ID: 1891 (https: / / www.ncbi.nlm.nih.gov / gene / 1891) ECHS1 (positive correlation) is judged to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 7.02 or greater, more preferably 7.04 or greater, and even more preferably 7.07 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from ECHS1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0144] <FDXR> FDXR ferredoxin reductase [ Homo sapiens (human)] Gene ID: 2232 (https: / / www.ncbi.nlm.nih.gov / gene / 2232). When FDXR (positive correlation) is equal to or greater than a threshold, it can be determined that the efficacy is higher. For example, this threshold is preferably 3.01 or greater, more preferably 3.08 or greater, and even more preferably 3.15 or greater; the higher the value, the better. Alternatively, a regression line obtained from FDXR and ICSR scores may be used. Values ​​normalized by housekeeping genes may also be used, in which case the values ​​may not necessarily match the values ​​shown.

[0145] <FIBP> FIBP FGF1 intracellular binding protein [Homo sapiens (human)] Gene ID: 9158 (https: / / www.ncbi.nlm.nih.gov / gene / 9158). FIBP (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this threshold value is preferably 5.86 or greater, more preferably 5.90 or greater, and even more preferably 5.94 or greater; the higher the value, the better. Alternatively, a regression line obtained from FIBP and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0146] <FMC1> FMC1 formation of mitochondrial complex V assembly factor 1 homolog [ Homo sapiens (human)] Gene ID: 154791 (https: / / www.ncbi.nlm.nih.gov / gene / 154791) FMC1 (positive correlation) can be determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 2.68 or greater, more preferably 2.77 or greater, and even more preferably 2.86 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from FMC1 and the ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0147] <GATB> GATB glutamyl-tRNA amidotransferase subunit B [ Homo sapiens (human)] Gene ID: 5188 (https: / / www.ncbi.nlm.nih.gov / gene / 5188). GATB (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this threshold is preferably 2.29 or greater, more preferably 2.36 or greater, and even more preferably 2.42 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from GATB and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0148] <GCSH> GCSH glycine cleavage system protein H [ Homo sapiens (human)] Gene ID: 2653, (https: / / www.ncbi.nlm.nih.gov / gene / 2653) GCSH (positive correlation) can be determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 4.11 or greater, more preferably 4.17 or greater, and even more preferably 4.24 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from GCSH and ICSR scores may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0149] <GPX1> GPX1 glutathione peroxidase 1 [Homo sapiens (human)] Gene ID: 2876 (https: / / www.ncbi.nlm.nih.gov / gene?Db=gene&Cmd=DetailsSearch&Term=2876). A positive correlation (positive correlation) of GPX1 above a threshold indicates a higher level of efficacy. For example, this threshold is preferably 8.62 or higher, more preferably 8.70 or higher, and even more preferably 8.78 or higher; higher values ​​are more preferable. Alternatively, a regression line obtained from GPX1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0150] <HSD17B10> HSD17B10 hydroxysteroid 17-beta dehydrogenase 10 [ Homo sapiens (human)] Gene ID: 3028, (https: / / www.ncbi.nlm.nih.gov / gene / 3028) HSD17B10 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 6.23 or greater, more preferably 6.33 or greater, and even more preferably 6.43 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from HSD17B10 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0151] <IDH3B> IDH3B isocitrate dehydrogenase (NAD(+)) 3 non-catalytic subunit beta [ Homo sapiens (human)] Gene ID: 3420 (https: / / www.ncbi.nlm.nih.gov / gene / 3420). IDH3B (positive correlation) can be determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 6.06 or greater, more preferably 6.11 or greater, and even more preferably 6.15 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from IDH3B and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0152] <LAP3> LAP3 leucine aminopeptidase 3 [Homo sapiens (human)] Gene ID: 51056 (https: / / www.ncbi.nlm.nih.gov / gene?Db=gene&Cmd=DetailsSearch&Term=51056). LAP3 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold. For example, this threshold is preferably 5.61 or greater, more preferably 5.68 or greater, and even more preferably 5.75 or greater; higher values ​​are more preferable. Alternatively, a regression line obtained from LAP3 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0153] <LARS2> LARS2 leucyl-tRNA synthetase 2, mitochondrial [ Homo sapiens (human)] Gene ID: 23395 (https: / / www.ncbi.nlm.nih.gov / gene / 23395). LARS2 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 3.03 or greater, more preferably 3.14 or greater, and even more preferably 3.25 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from LARS2 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0154] <MAPK8IP1> MAPK8IP1 mitogen-activated protein kinase 8 interacting protein 1 [Homo sapiens (human)] Gene ID: 9479 (https: / / www.ncbi.nlm.nih.gov / gene / 9479). MAPK8IP1 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 2.64 or greater, more preferably 2.80 or greater, and even more preferably 2.96 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from MAPK8IP1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0155] <MDH2> MDH2 malate dehydrogenase 2 [Homo sapiens (human)] Gene ID: 4191 (https: / / www.ncbi.nlm.nih.gov / gene / 4191). MDH2 (positive correlation) is determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 6.70 or greater, more preferably 6.76 or greater, and even more preferably 6.82 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from MDH2 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0156] <METAP1D> METAP1D methionyl aminopeptidase type 1D, mitochondrial [ Homo sapiens (human)] Gene ID: 254042, (https: / / www.ncbi.nlm.nih.gov / gene / 254042) METAP1D (positive correlation) can be determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 2.33 or greater, more preferably 2.43 or greater, and even more preferably 2.52 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from METAP1D and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0157] <MRPL10> MRPL10 mitochondrial ribosomal protein L10 [ Homo sapiens (human)] Gene ID: 124995 (https: / / www.ncbi.nlm.nih.gov / gene / 124995). MRPL10 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this threshold is preferably 5.42 or greater, more preferably 5.45 or greater, and even more preferably 5.49 or greater; higher values ​​are more preferable. Alternatively, a regression line obtained from MRPL10 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0158] <MRPL24> MRPL24 mitochondrial ribosomal protein L24 [Homo sapiens (human)] Gene ID: 79590 (https: / / www.ncbi.nlm.nih.gov / gene / 79590). MRPL24 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this threshold is preferably 5.95 or greater, more preferably 6.03 or greater, and even more preferably 6.10 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from MRPL24 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0159] <MRPL42> MRPL42 mitochondrial ribosomal protein L42 [Homo sapiens (human)] Gene ID: 28977 (https: / / www.ncbi.nlm.nih.gov / gene / 28977). MRPL42 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this threshold is preferably 3.12 or greater, more preferably 3.16 or greater, and even more preferably 3.20 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from MRPL42 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0160] <MRPS12> MRPS12 mitochondrial ribosomal protein S12 [ Homo sapiens (human)] Gene ID: 6183 (https: / / www.ncbi.nlm.nih.gov / gene / 6183). MRPS12 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this threshold value is preferably 5.06 or greater, more preferably 5.12 or greater, and even more preferably 5.16 or greater; higher values ​​are more preferable. Alternatively, a regression line obtained from MRPS12 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0161] <MRPS18C> MRPS18C mitochondrial ribosomal protein S18C [ Homo sapiens (human)] Gene ID: 51023 (https: / / www.ncbi.nlm.nih.gov / gene / 51023). MMRPS18C (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this threshold value is preferably 3.67 or greater, more preferably 3.75 or greater, and even more preferably 3.83 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from MRPS18C and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0162] <MRPS22> MRPS22 mitochondrial ribosomal protein S22 [Homo sapiens (human)] Gene ID: 56945 (https: / / www.ncbi.nlm.nih.gov / gene / 56945). MRPS22 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this threshold value is preferably 4.41 or greater, more preferably 4.45 or greater, and even more preferably 4.48 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from MRPS22 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0163] <MRPS27> MRPS27 mitochondrial ribosomal protein S27 [ Homo sapiens (human)] Gene ID: 23107 (https: / / www.ncbi.nlm.nih.gov / gene / 23107). MRPS27 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this threshold value is preferably 5.01 or greater, more preferably 5.06 or greater, and even more preferably 5.11 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from MRPS27 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0164] <MRPS35> MRPS35 mitochondrial ribosomal protein S35 [ Homo sapiens (human)] Gene ID: 60488 (https: / / www.ncbi.nlm.nih.gov / gene / 60488). MRPS35 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this threshold value is preferably 6.27 or greater, more preferably 6.34 or greater, and even more preferably 6.41 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from MRPS35 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0165] <MTG1> MT1G metallothionein 1G [ Homo sapiens (human)] Gene ID: 4495 (https: / / www.ncbi.nlm.nih.gov / gene / 4495). MTG1 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 3.08 or greater, more preferably 3.17 or greater, and even more preferably 3.26 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from MTG1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0166] <MTHFD1> MTHFD1 methylenetetrahydrofolate dehydrogenase, cyclohydrolase, and formyltetrahydrofolate synthetase 1 [ Homo sapiens (human)] Gene ID: 4522, (https: / / www.ncbi.nlm.nih.gov / gene / 4522) MTHFD1 (positive correlation) is determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 4.58 or greater, more preferably 4.66 or greater, and even more preferably 4.74 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from MTHFD1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0167] <NDUFB9> NDUFB9 NADH:ubiquinone oxidoreductase subunit B9 [ Homo sapiens (human)] Gene ID: 4715 (https: / / www.ncbi.nlm.nih.gov / gene / 4715). NDUFB9 (positive correlation) can be determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 6.28 or greater, more preferably 6.34 or greater, and even more preferably 6.41 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from NDUFB9 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0168] <NDUFS2> NDUFS2 NADH:ubiquinone oxidoreductase core subunit S2 [ Homo sapiens (human)] Gene ID: 4720 (https: / / www.ncbi.nlm.nih.gov / gene / 4720). NDUFS2 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 6.14 or greater, more preferably 6.17 or greater, and even more preferably 6.20 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from NDUFS2 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0169] <NDUFV1> NDUFV1 NADH:ubiquinone oxidoreductase core subunit V1 [ Homo sapiens (human)] Gene ID: 4723 (https: / / www.ncbi.nlm.nih.gov / gene / 4723). NDUFV1 (positive correlation) can be determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 5.76 or greater, more preferably 5.80 or greater, and even more preferably 5.84 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from NDUFV1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0170] <OCIAD1> OCIAD1 OCIA domain containing 1 [ Homo sapiens (human)] Gene ID: 54940 (https: / / www.ncbi.nlm.nih.gov / gene / 54940). OCIAD1 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 6.38 or greater, more preferably 6.44 or greater, and even more preferably 6.47 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from OCIAD1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0171] <OXNAD1> OXNAD1 oxidoreductase NAD binding domain containing 1 [ Homo sapiens (human)] Gene ID: 92106 (https: / / www.ncbi.nlm.nih.gov / gene / 92106) OXNAD1 (positive correlation) can be determined to be more effective when it is above a threshold. For example, this preferred threshold is preferably 1.96 or higher, more preferably 2.07 or higher, and even more preferably 2.18 or higher, with higher values ​​being more preferable. Alternatively, a regression line obtained from OXNAD1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0172] <PARS2> ARS2 prolyl-tRNA synthetase 2, mitochondrial [ Homo sapiens (human)] Gene ID: 25973 (https: / / www.ncbi.nlm.nih.gov / gene / 25973). PARS2 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 2.76 or greater, more preferably 2.87 or greater, and even more preferably 2.98 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from PARS2 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0173] <PDF> PDF peptide deformylase, mitochondrial [ Homo sapiens (human)] Gene ID: 64146, (https: / / www.ncbi.nlm.nih.gov / gene?Db=gene&Cmd=DetailsSearch&Term=64146) A PDF (positive correlation) greater than or equal to a threshold value indicates higher efficacy. For example, this threshold is preferably 4.15 or greater, more preferably 4.24 or greater, and even more preferably 4.33 or greater; higher values ​​are more preferable. Alternatively, a regression line derived from the PDF and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0174] <PHB> PHB1 prohibitin 1 [ Homo sapiens (human)] Gene ID: 5245 (https: / / www.ncbi.nlm.nih.gov / gene / 5245). The PHB gene encodes a protein called prohibitin. PHB (positive correlation) is considered more effective when it is above a threshold. For example, this threshold is preferably 6.37 or higher, more preferably 6.41 or higher, and even more preferably 6.45 or higher; higher values ​​are more preferable. Alternatively, a regression line derived from PHB and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0175] <PHYH> PHYH phytanoyl-CoA 2-hydroxylase [ Homo sapiens (human)] Gene ID: 5264 (https: / / www.ncbi.nlm.nih.gov / gene / 5264). PHYH (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this threshold value is preferably 5.17 or greater, more preferably 5.26 or greater, and even more preferably 5.36 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from PHYH and ICSR scores may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0176] <POLRMT> POLRMT RNA polymerase mitochondrial [ Homo sapiens (human)] Gene ID: 5442 (https: / / www.ncbi.nlm.nih.gov / gene / 5442). POLRMT (positive correlation) is considered to be more effective when it is equal to or greater than a threshold. For example, this threshold is preferably 4.23 or greater, more preferably 4.34 or greater, and even more preferably 4.45 or greater; higher values ​​are more preferable. Alternatively, a regression line derived from POLRMT and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0177] <PTCD1> PTCD1 pentatricopeptide repeat domain 1 [Homo sapiens (human)] Gene ID: 26024 (https: / / www.ncbi.nlm.nih.gov / gene / 26024). PTCD1 (positive correlation) can be determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 2.18 or greater, more preferably 2.24 or greater, and even more preferably 2.30 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from PTCD1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0178] <PYURF> PYURF PIGY upstream open reading frame [ Homo sapiens (human)] Gene ID: 100996939, (https: / / www.ncbi.nlm.nih.gov / gene / 100996939) PYURF (positive correlation) can be determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 7.43 or greater, more preferably 7.46 or greater, and even more preferably 7.49 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from PYURF and ICSR scores may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0179] <RMDN1> RMDN1 regulator of microtubule dynamics 1 [ Homo sapiens (human)] Gene ID: 51115 (https: / / www.ncbi.nlm.nih.gov / gene / 51115) RMDN1 (positive correlation) can be determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 4.84 or greater, more preferably 4.89 or greater, and even more preferably 4.94 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from RMDN1 and the ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0180] <RMND1> RMND1 required for meiotic nuclear division 1 homolog [Homo sapiens (human)] Gene ID: 55005 (https: / / www.ncbi.nlm.nih.gov / gene / 55005). RMND1 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold. For example, this preferred threshold is preferably 4.98 or greater, more preferably 5.06 or greater, and even more preferably 5.13 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from RMND1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0181] <SDHD> SDHD succinate dehydrogenase complex subunit D [Homo sapiens (human)] Gene ID: 6392 (https: / / www.ncbi.nlm.nih.gov / gene / 6392). SDHD (positive correlation) is considered to be more effective when it is equal to or greater than a threshold. For example, this threshold is preferably 6.92 or greater, more preferably 6.97 or greater, and even more preferably 7.02 or greater; higher values ​​are more preferable. Alternatively, a regression line obtained from SDHD and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0182] <SFXN4> SFXN4 sideroflexin 4 [Homo sapiens (human)] Gene ID: 119559 (https: / / www.ncbi.nlm.nih.gov / gene / 119559). SFXN4 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 4.59 or greater, more preferably 4.70 or greater, and even more preferably 4.81 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from SFXN4 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0183] <SLC25A11> SLC25A11 solute carrier family 25 member 11 [ Homo sapiens (human)] Gene ID: 8402 (https: / / www.ncbi.nlm.nih.gov / gene / 8402). SLC25A11 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this threshold value is preferably 5.35 or greater, more preferably 5.38 or greater, and even more preferably 5.40 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from SLC25A11 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0184] <SMIM4> UQCC5 ubiquinol-cytochrome c reductase complex assembly factor 5 [Homo sapiens (human)] Gene ID: 440957 (https: / / www.ncbi.nlm.nih.gov / gene / 440957). SMIM4 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this threshold is preferably 2.55 or greater, more preferably 2.62 or greater, and even more preferably 2.69 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from SMIM4 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0185] <TACO1> TACO1 translational activator of cytochrome c oxidase I [Homo sapiens (human)] Gene ID: 51204 (https: / / www.ncbi.nlm.nih.gov / gene / 51204). TACO1 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 4.59 or greater, more preferably 4.63 or greater, and even more preferably 4.67 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from TACO1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0186] <TIMM10> IMM10 translocase of inner mitochondrial membrane 10 [ Homo sapiens (human)] Gene ID: 26519 (https: / / www.ncbi.nlm.nih.gov / gene / 26519) TIMM10 (positive correlation) can be determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 6.03 or greater, more preferably 6.11 or greater, and even more preferably 6.19 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from TIMM10 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0187] <TIMM50 (a subunit of the TIM23 complex, a protein import apparatus of the inner mitochondrial membrane)> TIMM50 translocase of inner mitochondrial membrane 50 [ Homo sapiens (human)] Gene ID: 92609, (https: / / www.ncbi.nlm.nih.gov / gene / 92609) TIMM50 (positive correlation) is determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 3.97 or higher, more preferably 4.03 or higher, and even more preferably 4.09 or higher, with higher values ​​being more preferable. Alternatively, a regression line obtained from TIMM50 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0188] <TK2> TK2 thymidine kinase 2 [ Homo sapiens (human)] Gene ID: 7084 (https: / / www.ncbi.nlm.nih.gov / gene?Db=gene&Cmd=DetailsSearch&Term=7084). TK2 (positive correlation) is determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 4.60 or greater, more preferably 4.68 or greater, and even more preferably 4.76 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from TK2 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0189] <TOMM22> TOMM22 translocase of outer mitochondrial membrane 22 [Homo sapiens (human)] Gene ID: 56993 (https: / / www.ncbi.nlm.nih.gov / gene / 56993) TOMM22 (positive correlation) can be determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 5.98 or greater, more preferably 6.03 or greater, and even more preferably 6.09 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from TOMM22 and the ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0190] <TOMM5> TOMM5 translocase of outer mitochondrial membrane 5 [Homo sapiens (human)] Gene ID: 401505 (https: / / www.ncbi.nlm.nih.gov / gene / 401505) TOMM5 (positive correlation) can be determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 3.38 or greater, more preferably 3.44 or greater, and even more preferably 3.50 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from TOMM5 and the ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0191] <TRAP1 (TNF Receptor Associated Protein 1)> TRAP1 TNF receptor associated protein 1 [Homo sapiens (human)] Gene ID: 10131, (https: / / www.ncbi.nlm.nih.gov / gene / 10131) TRAP1 (positive correlation) can be determined to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 5.01 or greater, more preferably 5.06 or greater, and even more preferably 5.11 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from TRAP1 and the ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown as examples.

[0192] <TUFM (involved in mitochondrial protein translation)> TUFM Tu translation elongation factor, mitochondrial [ Homo sapiens (human)] Gene ID: 7284, (https: / / www.ncbi.nlm.nih.gov / gene / 7284) TUFM (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 6.96 or greater, more preferably 7.03 or greater, and even more preferably 7.10 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from TUFM and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0193] <UQCRC1 (ubiquinol-cytochrome c reductase core protein 1)> UQCRC1 ubiquinol-cytochrome c reductase core protein 1 [ Homo sapiens (human)] Gene ID: 7384, (https: / / www.ncbi.nlm.nih.gov / gene / 7384) UQCRC1 (positive correlation) is considered to be more effective when it is equal to or greater than a threshold value. For example, this preferred threshold is preferably 6.96 or greater, more preferably 7.00 or greater, and even more preferably 7.05 or greater, with higher values ​​being more preferable. Alternatively, a regression line obtained from UQCRC1 and ICSR score may be used. Values ​​normalized by housekeeping genes may also be used, in which case they may not necessarily match the values ​​shown.

[0194] The cartilage-like tissue formation property of a cell culture can be evaluated by performing or employing the analysis and determination steps described below in relation to the genetic markers. Furthermore, by performing or employing the measurement and analysis steps described below in relation to the methods, each score estimate can be estimated based on the amount of each gene (such as gene expression level) derived from the cell culture. Furthermore, various score estimates can be estimated by applying the expression levels of various genes derived from the cell culture to a model containing various association information generated by statistical analysis. Furthermore, the cartilage-like tissue formation property of a cell culture can be evaluated from one or a combination of the score estimates. The present invention also provides a marker for evaluating cartilage repair suitability. The marker may include the genetic markers described above in relation to the markers for evaluating cartilage-like tissue formation property.

[0195] In a preferred embodiment of the present invention, the expression level of each gene marker in a cell culture is measured and compared with a predetermined threshold for each gene marker to evaluate the cartilage-like tissue formation properties. Preferably, the effectiveness of the cell culture for cartilage regeneration can be predicted, estimated, or determined, or these methods can be assisted in doing so. This makes it possible to determine whether a cell culture is suitable for transplantation before transplantation, or to assist in this determination. Furthermore, the "predetermined threshold for each gene marker" may be an "effectiveness score." Based on the expression level of each gene marker in the cell culture, each score for the cell culture may be converted into an estimated value, and the estimated value may be applied to an effectiveness score (such as an ICRS score). Based on the evaluation criteria for the effectiveness score, a pass / fail judgment of the cell culture, evaluation of the cell culture, or evaluation of the starting cells may be performed, or these methods may be assisted in the evaluation.

[0196] For example, in the case of a positively correlated genetic marker, the cell culture is compared with a predetermined threshold, and if the result is equal to or greater than the threshold, it can be determined that the cell culture is highly effective or has cartilage-like tissue-forming properties; conversely, if the result is below the threshold, it can be determined that the cell culture is ineffective or does not have cartilage-like tissue-forming properties. For example, in the case of a negatively correlated genetic marker, the cell culture is compared with a predetermined threshold, and if the result is equal to or less than the threshold, it can be determined that the cell culture is highly effective or has cartilage-like tissue-forming properties; conversely, if the result is above the threshold, it can be determined that the cell culture is ineffective or does not have cartilage-like tissue-forming properties. Furthermore, in the case of a positively correlated genetic marker and / or a negatively correlated genetic marker, if it can be determined that the cell culture is highly effective or has cartilage-like tissue-forming properties, it can be determined that the pre-transplant cell culture is suitable for transplantation, or this determination can be assisted.

[0197] Here, "efficacy" means that the cell culture (preferably a cell culture or cell sheet derived from cartilage tissue, etc.) regenerates the cells (preferably cartilage) and repairs the cell defect (preferably a cartilage defect), etc. The "efficacy score" means a specific numerical value indicating the extent to which the cells (preferably cartilage) have regenerated. For example, efficacy scores for chondrocytes include the International Cartilage Repair Society (ICRS) score, O'Driscoll score, Wakitani score, and score evaluation (modified version of the ICRS grading system). The efficacy score may be a predicted numerical value based on cell data (standard data, gene expression data, etc.) obtained from publicly known literature or statistical processing, etc. Furthermore, in the present invention, the judgment or evaluation may be based on an absolute value obtained by setting a threshold value, or may be based on a relative value based on a certain standard.

[0198] When a positively correlated gene is used as a genetic marker, for example, if the expression level of a positively correlated gene in a cell culture is equal to or greater than a predetermined threshold (e.g., equal to or greater than the comparative expression level or equal to or greater than a predetermined reference value (comparative expression level + α level)) compared to the expression level of the same gene in an ineffective or less effective cell culture, the cell culture can be determined to be effective. On the other hand, if the expression level of a positively correlated gene is less than a predetermined threshold, the cell culture can be determined to be ineffective. For example, if the expression level of a positively correlated gene in a cell culture is corrected with the expression level of an internal control gene and the value is equal to or greater than a predetermined threshold, the cell culture can be determined to be effective. On the other hand, if the value is less than the predetermined threshold, the cell culture can be determined to be ineffective.

[0199] When a negatively correlated gene is used as a genetic marker, for example, if the expression level of a certain negatively correlated gene in a cell culture is below a predetermined threshold (for example, below the comparative expression level or below a set predetermined reference value (comparative expression level - α level)) compared to the expression level of the same gene in an ineffective or less effective cell culture, the cell culture can be determined to be effective. On the other hand, if the expression level of a negatively correlated gene exceeds a predetermined threshold, the cell culture can be determined to be ineffective. For example, if the expression level of a certain negatively correlated gene in a cell culture is corrected with the expression level of an internal control gene and the value is below a predetermined threshold, the cell culture can be determined to be effective. On the other hand, if the value exceeds the predetermined threshold, the cell culture can be determined to be ineffective.

[0200] The present invention also provides a kit using one or more gene markers selected from the group consisting of (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, and (6) mitochondria-related genes. The gene markers may be one or more selected from all of the genes described above. The kit of the present invention may be any of a kit for evaluating cartilage-like tissue formation characteristics, a kit used to evaluate a cell culture or the effectiveness of a cell culture, and a kit used to estimate the effectiveness score of a cell culture. It may also be a kit for use in a method such as the production method or evaluation method described below or for achieving the purpose of the method, and can be applied to the uses or purposes described herein.

[0201] As an example of an embodiment of the kit of the present invention, primers, probes, or antibodies for confirming, detecting, or measuring the gene expression or expression level of one or more of the above-mentioned genetic markers may be appropriately combined, and a solid support or kit for confirming, detecting, or measuring the markers may be provided. In this case, the primers, probes, or antibodies can be prepared by techniques well known in the fields of genetic engineering and molecular biology, and any length or size can be used. A reagent kit or a formulation kit may also be used.

[0202] The kit according to the present invention may further comprise a PCR buffer. The PCR buffer may be, for example, Tris-HCl, KCl, or (NH ４ ) ２ SO ４ , MgCl ２ Examples of suitable PCR reagents include, but are not limited to, non-ionic surfactants (e.g., Tween 20, Triton X-100), dNTPs (nucleotides necessary for DNA synthesis), and DNA polymerase (thermostable enzymes such as Taq). Pretreatment solutions such as proteinase K and surfactants may also be provided for use in PCR pretreatment. Regarding the use of this kit, an example of a method for detecting a certain genetic marker will be described, but is not limited to this. An example of PCR may include: (a) mixing a sample collected from a cell culture with a PCR buffer; (b) performing a PCR reaction using the mixture obtained in step (a); and (c) detecting the PCR product.

[0203] In addition, the present invention may involve performing expression analysis by quantitative real-time PCR using the predetermined gene markers described above. For example, RNA may be extracted from each sample, followed by synthesizing cDNA from the extracted RNA and analyzing the expression of the gene markers by quantitative real-time PCR. A gene panel may also be used for the analysis. In the present invention, a general gene expression analysis method or gene expression analysis device, such as a real-time PCR method, may be used, and examples of such methods include gene expression analysis using TaqMan Assays. In addition, detection methods include, but are not limited to, the SYBR Green method and the TaqMan probe method, and reaction formats include, but are not limited to, one-step RT-qPCR and two-step RT-qPCR.

[0204] In the present specification, the expression or expression level of a specific gene to be obtained can be determined by appropriately employing known techniques or techniques that will emerge in the future, and for example, one or more types selected from base sequences of DNA or mRNA (PCR (quantitative PCR, real-time PCR), LAMP, Northern blotting, microarray, RNA sequencing, in situ hybridization (ISH), Western blot, antibody reaction, etc.); proteins, peptides, or fragments thereof (immunostaining, Western blot, antibody reaction, aptamer, Enzyme-Linked Immunosorbent Assay, etc.) can be used.

[0205] The present invention may provide each of the nine genes or one or more genes selected from the group consisting of (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, and (6) mitochondria-related genes, or use thereof, for use in evaluating cartilage-like tissue formation properties, or may provide a method for using each of the nine or more genes as an indicator for evaluating cartilage-like tissue formation properties. The present invention may also provide a reagent containing each of the nine or more genes or an agent for detecting their expression, or a reagent combination that uses or includes the reagent, and the reagent may be for evaluating cartilage-like tissue formation properties. Furthermore, each of the nine or more genes may be used to produce the reagent, or the nine or more genes may be used in producing the reagent. The evaluation of cartilage-like tissue formation properties may also be used for other evaluations or purposes, such as predicting the effectiveness of a chondrocyte cell sheet for cartilage regeneration, or for cell cultures for cartilage-like tissue formation or cartilage-like tissue formation properties. The expression levels of the nine genes may be the gene expression levels in a cell culture (e.g., a cell sheet). Furthermore, each of the nine or more genes may be used as an indicator substance, indicator, or marker for predicting or determining the effectiveness of a chondrocyte cell sheet for cartilage regeneration. Each of the nine or more genes may be these nine genes or one or more genes selected from genes such as (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, and (6) mitochondria-related genes.

[0206] 2. cell culture

[0207] The present invention can provide a cell culture having cartilage-like tissue-forming properties, more preferably a cell culture having hyaline cartilage-like tissue-forming properties. When evaluating or assessing the cell culture, the above-mentioned (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, and (6) mitochondria-related genes, as well as all of the genes described in (1) to (6) above, can be used.

[0208] In a preferred embodiment, the expression level of at least one positively correlated gene marker selected from the group consisting of (1) to (6) may be equal to or greater than the threshold value for each gene marker, and / or the expression level of at least one negatively correlated gene marker selected from the group consisting of (1) to (6) may be equal to or less than the threshold value for each gene marker. When these gene markers are equal to or greater than a predetermined threshold, the cell culture is suitable for cartilage repair, more particularly knee cartilage repair. The preferred gene marker is at least one positively correlated gene marker selected from the group consisting of the above-mentioned Group 1 genes to Group 6 genes, and / or at least one negatively correlated gene marker selected from the group consisting of the above-mentioned Group 1 genes to Group 6 genes. These gene markers may be used alone or in combination of two or more.

[0209] Furthermore, as a cell culture having suitable cartilage-like tissue formation properties, it is preferable that the cell culture has an ICRS score estimated based on the expression level of at least one gene marker selected from the group consisting of (1) to (6) above, and the estimated ICRS score is a predetermined ICRS score (28, 29, or 30) or higher. This makes the cell culture suitable for cartilage repair, and more particularly suitable for knee cartilage repair.

[0210] Furthermore, the preferred genetic marker may be one or more genetic markers selected from the group consisting of all genetic markers described in the above-mentioned first to sixth group genes.

[0211] The genetic markers and their expression levels, as well as thresholds, ICRS score estimation methods, analysis methods, and determination methods used in the cell culture of the present invention can be appropriately adapted from the technical features and the like described in "1." above and "2." to "5." below.

[0212] The cell culture of the present invention preferably has the property of forming cartilage-like tissue, and in particular the property of forming hyaline cartilage-like tissue. The cell culture of the present invention can have the property of forming cartilage-like tissue, in particular hyaline cartilage-like tissue, when transplanted, for example, into a human body, more particularly when transplanted into the articular cartilage portion of the knee of a human body. As used herein, "cartilage-like tissue" may be a tissue having components and / or functions equivalent to or similar to those of cartilage tissue (in particular knee articular cartilage tissue), and refers, for example, to a tissue that expresses type II collagen in a manner similar to hyaline cartilage tissue.

[0213] (Cell Culture Form)

[0214] According to one embodiment of the present invention, the cell culture may be in a solid state. For example, the cell population contained in the cell culture may be organized into any shape. The shape of the solid cell culture may be, for example, a sheet, granule, fiber (thread), or mesh. According to another embodiment of the present invention, the cell culture may not be in a solid state, for example, it may be flowable. The non-solid cell culture may be, for example, in a flowable state, liquid state, or sol state.

[0215] The cell culture of the present invention is preferably in the form of a sheet, and such a sheet is also referred to as a cell sheet or a sheet-shaped cultured cell. A sheet-shaped cell culture is particularly suitable for cartilage tissue repair, particularly hyaline cartilage tissue repair. When the cell culture is in the form of a sheet, the thickness of the sheet-shaped cell culture may be, for example, 4 μm to 5 mm, particularly 4 μm to 3 mm, and more particularly 4 μm to 1 mm. The sheet-shaped cell culture may be a single sheet of cell culture, or a laminate of multiple sheets (e.g., 2 to 10 sheets, particularly 2 to 8 sheets, more particularly 2 to 5 sheets). More particularly, the thickness of the sheet-shaped cell culture may be, for example, 4 μm to 100 μm, preferably 4 μm to 70 μm, and more preferably 4 μm to 50 μm. The sheet-shaped cell culture of the present invention may be one in which cells naturally form a layer during the cell proliferation process. The sheet-shaped cell culture of the present invention does not necessarily have to be produced by artificially layering two or more sheet-shaped cell cultures that have been separately produced and culturing them continuously.

[0216] (Cell culture derived from cartilage tissue)

[0217] In one embodiment of the present invention, the cell culture may be derived from cartilage tissue. That is, the cell culture may be a culture of cells derived from cartilage tissue, and in particular, the raw cells used in the culture to obtain the cell culture may be cells derived from cartilage tissue. The cell culture is obtained by artificial culture in vitro and is therefore not a natural product. The cartilage tissue-derived cells may be, for example, a plurality of cells obtained by separating cells contained in cartilage tissue from the cartilage matrix. For example, the cartilage tissue-derived cells may be a plurality of cells recovered by treating the cartilage tissue with an enzyme to release the cells in the cartilage tissue from the cartilage matrix, and then recovering the released cells by centrifugation.

[0218] The cell culture is preferably not derived from synovial membrane. That is, the cell culture preferably does not contain cells derived from synovial membrane. The cell culture is preferably not formed from a culture of cells derived from synovial membrane, nor from a culture of synovial cells. The cell culture of the present invention may preferably be a culture of only cells derived from cartilage tissue, and in particular may be a culture of only cells derived from hyaline cartilage tissue.

[0219] In the present invention, the cartilage tissue-derived cells may be derived from the cartilage tissue of an animal with polydactyly, or may be derived from the cartilage tissue of an animal with polydactyly. The animal may be preferably a mammal, more preferably a primate, and even more preferably a human. The cartilage tissue may be harvested, for example, from tissue obtained during surgery to remove a redundant digit. The tissue may be, for example, a portion that does not appear white in an X-ray, i.e., a portion that appears black. The polydactyly may be any of the distal phalanges, middle phalanges, or proximal phalanges. The redundant digit may be any digit, for example, the thumb or little finger. If the redundant digit (limb) to be harvested is small and warty, all of the harvested subcutaneous tissue may be used. When the animal is a human, there is no limitation on the age of the human, but the human may be, for example, 5 years or younger, 3 years or younger, or 2 years or younger.

[0220] Examples of enzymes used in the enzyme treatment include collagenase, caseinase, clostripain, trypsin, hyaluronidase, elastase, pronase, and dispase. Preferably, a combination of these enzymes is used. A preferred enzyme combination is, for example, collagenase, caseinase, clostripain, and trypsin. Examples of enzyme preparations containing this combination include, but are not limited to, collagenase type I, collagenase type II, collagenase type III, collagenase type IV, and collagenase type V (all available from Fujifilm Wako Pure Chemical Industries, Ltd.). Another preferred enzyme combination is, for example, a combination of collagenase and dispase or thermolysin. Examples of enzyme preparations containing this combination include, but are not limited to, Liberase (available from Roche Diagnostics). Depending on the condition of the tissue, the enzyme treatment may be performed stepwise using multiple enzymes. For example, isolation may be performed by treating with collagenase, caseinase, clostripain, and trypsin in this order. The conditions for the enzyme treatment can be appropriately determined by those skilled in the art depending on the type of enzyme used and / or the state of the cartilage tissue. The enzyme treatment can be performed, for example, at 30 to 50°C, preferably 33 to 45°C or 35 to 40°C, for 1 to 12 hours, preferably 2 to 5 hours. If the enzyme treatment temperature is too high, problems such as cell denaturation, a decrease in viable cells, a decrease in proliferation ability, and inability to isolate cells may occur. Furthermore, if the enzyme treatment temperature is too low, sufficient enzyme activity may not be achieved, making cell isolation impossible. Highly efficient cell recovery can be achieved by adding physical stimulation during the enzyme treatment.

[0221] The enzymatic reaction can be stopped by diluting the cell suspension in which the articular cartilage has been enzymatically treated with washing. After stopping the enzymatic reaction, the cell suspension can be separated into a cell mass and a supernatant by centrifugation. For Liberase, the enzymatic reaction can be stopped by washing two or more times. The centrifugation can be performed under conditions that allow for the collection of more cells with a particle size of less than 25 μm, particularly less than 20 μm, and more than 15 μm. To collect more such cells, the centrifugation can be performed, for example, at 1000 rpm or more, 1500 rpm or more, or 2000 rpm or more, for example, for 5 minutes or more, 7 minutes or more, or 10 minutes or more.

[0222] The cartilage tissue-derived cells may be obtained by the so-called outgrowth method. The outgrowth method may include a step of finely chopping the collected cartilage tissue, and a step of seeding the finely chopped cartilage tissue fragments together with a small amount of medium onto a culture dish and culturing them. This culturing generates proliferated cells from the cartilage tissue fragments. The generated cells are recovered by enzyme treatment and centrifugation. The recovered cells can be used to produce the cell sheet of the present invention.

[0223] The cartilage tissue can be chopped into small pieces, for example, under wet conditions. This process can be performed, for example, by placing the tissue fragments and a small amount of medium in a 50 ml centrifuge tube and chopping them using Metzenbaum Scissors, SuperCut Tungsten Carbide 18 cm Long Curve (World Precision Instruments). It is preferable to obtain the smallest possible cartilage tissue fragments. The culture medium for culturing the chopped cartilage tissue fragments can be appropriately selected by those skilled in the art, but is preferably DMEM / F12 + 20% FBS + antibiotics (hereinafter also referred to as AB). After the initiation of culture, once cell adhesion to the culture dish has been confirmed, the culture medium can be replaced with, preferably, DMEM / F12 + 20% FBS + AB + ascorbic acid (hereinafter also referred to as AA). If the culture medium contains ascorbic acid from the initiation of culture, cell adhesion to the culture dish may be inhibited. The culture can be performed under general culture conditions, for example, at 37°C and 5% CO ２The culture may be performed in an incubator. The culture may be performed until subconfluence is achieved. The enzyme preparation used to recover the cells produced in the culture may include, for example, trypsin and EDTA. Centrifugation may be performed as described above.

[0224] In the present invention, the cartilage tissue-derived cells may preferably include mesenchymal stem cells. The cartilage tissue-derived cells may further include cells contained in cartilage tissue in addition to mesenchymal stem cells. That is, in the present invention, the cartilage tissue-derived cells may be a population of multiple types of cells including mesenchymal stem cells. Examples of cells other than mesenchymal stem cells include, but are not limited to, chondrocytes and chondroblasts. The cell culture of the present invention is formed from a culture of the cartilage tissue-derived cells, and is therefore more suitable for cartilage repair.

[0225] (Cell cultures derived from stem cells)

[0226] In another embodiment of the present invention, the cell culture may be derived from stem cells, which may include pluripotent stem cells, embryonic stem cells, or somatic stem cells, such as iPS cells.

[0227] In this embodiment, for example, pluripotent stem cells (particularly iPS cells) are cultured in a medium to differentiate them into chondrocytes or chondrocyte-like cells, and the chondrocytes or chondrocyte-like cells are further cultured on a surface on which a stimuli-responsive polymer is immobilized, thereby obtaining the cell culture of the present invention. For example, the cell culture of the present invention can also be obtained by seeding pluripotent stem cells (particularly iPS cells) on a substrate on which a stimuli-responsive polymer is immobilized, and differentiating them into chondrocytes or chondrocyte-like cells and culturing them.

[0228] (Uses of cell cultures)

[0229] The cell culture of the present invention may be used for repairing cartilage tissue, more preferably for repairing knee cartilage tissue, and even more preferably for repairing hyaline cartilage tissue of the knee. The cell culture of the present invention has cartilage-like tissue-forming properties, particularly hyaline cartilage-like tissue-forming properties, and is therefore suitable for such repair.

[0230] In the present invention, repair of cartilage tissue includes, but is not limited to, treating inflamed and / or damaged cartilage tissue, reinforcing cartilage tissue, filling in missing parts of cartilage tissue, and regenerating cartilage tissue. The cell culture of the present invention may also be used to prevent diseases related to cartilage tissue. For example, the cell culture of the present invention can be administered to diseased cartilage tissue or bone tissue. Examples of diseases to which the cell culture of the present invention can be applied include, but are not limited to, arthritis, arthropathy, cartilage damage, osteochondral damage, meniscus damage, and / or intervertebral disc degeneration.

[0231] The cell culture of the present invention is more particularly suitable for surgical treatment of cartilage tissue, particularly surgical therapy. Cartilage repair using the cell culture of the present invention can be performed, for example, by surgically exposing the cartilage area requiring repair and applying the cell culture to the exposed area. For example, a sheet-like cell culture of the present invention may be applied to the exposed area. The number and size of the sheet-like cell culture to be applied can be determined appropriately by those skilled in the art, taking into consideration, for example, the condition of the area to be treated or the type of disease. When applying the sheet-like cell culture of the present invention to the affected area, the area may be bonded or sutured with a biocompatible adhesive. Alternatively, the sheet-like cell culture may simply be attached to the affected area without bonding or suturing.

[0232] When the cell culture of the present invention is a sheet-shaped cell culture, the sheet-shaped cell culture may have a substrate on all sides. Preferably, the substrate may contain fibronectin. When cells are cultured on a substrate, a substrate is typically produced between the cells and the substrate, and no substrate is produced on the side opposite the substrate, i.e., the portion not in contact with the substrate. The sheet-shaped cell culture of the present invention may have a substrate produced not only on the side in contact with the substrate, but also on the side not in contact with the substrate. By having a substrate on all sides of the sheet-shaped cell culture of the present invention, the sheet-shaped cell culture may be more suitable for cartilage repair.

[0233] In the sheet-shaped cell culture of the present invention, the basement membrane-like protein formed between the cells and the porous membrane during culture is preferably one that has not been destroyed by enzymes such as proteolytic enzymes, e.g., dispase and trypsin. That is, the sheet-shaped cell culture of the present invention may have a basement membrane-like protein between the cells and the porous membrane. In particular, the sheet-shaped cell culture of the present invention may have a basement membrane-like protein between the cells and the porous membrane on one side of the sheet-shaped cell culture or on both sides of the sheet-shaped cell culture. When the sheet-shaped cell culture has the basement membrane-like protein, the sheet-shaped cell culture may exhibit better cartilage repair ability.

[0234] The sheet-shaped cell culture of the present invention preferably has a cell density of 100×10 ５ ~100 x 10 ８ pieces / cm ３ , more preferably 100×10 6 ~100 x 10 7 pieces / cm ３ , more preferably 100×10 ６ ~500 x 10 6 pieces / cm ３ , and even more preferably 200×10 6 ~300 x 10 6 pieces / cm ３ It is possible.

[0235] The cell culture of the present invention preferably does not contain artificial scaffold components. That is, the cell culture of the present invention may consist only of cells in the culture and components produced by the cells (as well as medium components attached to the cell sheet). The cell culture of the present invention may preferably be transplanted into a patient in a state free of artificial scaffold components.

[0236] The cell culture of the present invention can be produced by the production method of the present invention described below in "3." Therefore, with regard to the preparation or production of the cell culture of the present invention, and the production method of the cell culture of the present invention, "3." can be referred to below, and these explanations can be appropriately adopted as technical features, etc.

[0237] 3. Method for producing cell cultures

[0238] The present invention also provides a method for producing a cell culture having cartilage-like tissue-forming properties. The production method includes a culturing step of culturing cells to obtain a cell culture. The production method may further include an analysis step of analyzing the expression level of at least one of the gene markers described above, and a determination step based on the expression level obtained in the analysis step. The determination step is preferably a determination step of determining whether the cell culture has cartilage-like tissue-forming properties based on the expression level obtained in the analysis step. Each step is described below.

[0239] 3-1. Culture process

[0240] The present invention preferably includes a culturing step of culturing cells to obtain a cell culture. In the culturing step, the above-mentioned (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, (6) mitochondria-related genes, and all of the genes described in (1) to (6) above can be used. In a preferred embodiment, the culturing step may be performed such that the expression level of at least one positively correlated gene marker selected from the group consisting of (1) to (6) is equal to or greater than a threshold value for each gene marker, and / or the expression level of at least one negatively correlated gene marker selected from the group consisting of (1) to (6) is equal to or less than a threshold value for each gene marker.

[0241] In addition, in a preferred embodiment, the culturing step may be performed using an ICRS score estimated based on the expression level of at least one gene marker selected from the group consisting of (1) to (6) above, so that the estimated ICRS score is a predetermined value or more (e.g., 28 or more, 29 or more, or 30 or more).

[0242] The cell culture obtained by the culturing step has excellent cartilage-like tissue-forming properties, particularly hyaline cartilage-like tissue-forming properties. When the cell culture is transplanted into a human body, for example, particularly into the knee cartilage portion of a human body, it can form cartilage-like tissue, particularly hyaline cartilage-like tissue. That is, the cell culture can exert a cartilage repair effect, particularly a hyaline cartilage repair effect. Furthermore, the cell culture exerts functions similar to those of cartilage tissue.

[0243] The cells cultured in the culturing step may be the cartilage tissue-derived cells described in "1." and "2." above, or may be stem cell-derived cells. For example, the starting cells used for culturing may be the cartilage tissue-derived cells, which may be cells obtained by culturing cells in cartilage tissue in DMEM / F12 containing FBS for at least two days. In a preferred embodiment of the present invention, the starting cells used in the culturing to obtain the cell culture may be prepared by a starting cell preparation method including: a first culture step in which cells in cartilage tissue are cultured in a medium to become confluent; a dissociation step in which the cell populations that have become confluent in the first culture step are dissociated from each other; and a second culture step in which the cell populations dissociated in the dissociation step are further cultured in the same fresh medium as the first culture step. The starting cells prepared by this starting cell preparation method are suitable for producing a tissue culture suitable for cartilage repair.

[0244] In the culturing step, cells may be cultured in a medium containing a substrate having a surface on which a stimuli-responsive polymer is immobilized. For example, cells may be cultured on the surface of a porous membrane having a surface on which a stimuli-responsive polymer is immobilized. By culturing cells in a medium containing a substrate (particularly a porous membrane) having a surface on which a stimuli-responsive polymer is immobilized, a cell culture having cartilage-like tissue formation properties can be obtained, and the cell culture can also be detached from the substrate without damage. The stimuli-responsive polymer may be, for example, a temperature-responsive polymer, a pH-responsive polymer, or a photoresponsive polymer. More specifically, it may be a polymer whose properties (e.g., hydration strength) change in response to a temperature stimulus (e.g., a temperature change), a pH stimulus (e.g., a pH change), or a light stimulus (e.g., light irradiation), respectively. The change in properties may be, for example, a change in properties that facilitates detachment of the culture from the substrate. For example, a cell culture is formed by seeding cells on a surface on which a stimuli-responsive polymer is immobilized and culturing the cells in a culture medium at a temperature range where the hydration strength of the polymer is weak. After the cell culture is formed, the temperature of the culture medium is changed to a temperature at which the hydration force of the polymer is strong, thereby facilitating detachment of the cell culture from the surface. For example, the temperature range in which the culture is performed (i.e., the temperature range in which the hydration force is weak) may be, for example, 33°C to 40°C. The temperature for detachment (i.e., the temperature range in which the hydration force is strong) may be lower than the temperature range, for example, 31°C or lower.

[0245] In one embodiment of the present technology, the stimuli-responsive polymer may be a temperature-responsive polymer. When the cells are cultured in a medium containing a substrate having a surface on which a temperature-responsive polymer is immobilized, for example, after culture, the temperature of the medium may be increased to above the upper critical dissolution temperature or below the lower critical dissolution temperature of the temperature-responsive polymer, thereby changing the surface from hydrophobic to hydrophilic, thereby facilitating peeling between the culture and the porous membrane. Note that the culture in the production method of the present invention may be, for example, two-dimensional culture (also called plate culture) or three-dimensional culture (e.g., suspension culture or pellet culture).

[0246] When the porous membrane is used in the culturing step, the cells may be in contact with the culture medium above the porous membrane and in contact with the culture medium below the porous membrane via the pores of the porous membrane. By culturing under such conditions, cells more suitable for cartilage repair can be obtained.

[0247] When the cell culture is detached from the substrate after the culturing step, treatment with proteolytic enzymes such as dispase and trypsin is not required. By utilizing the properties of the temperature-responsive polymer and changing the temperature of the medium, the cell culture can be detached from the substrate. Therefore, the cell culture produced by the production method of the present invention has the advantage that it can be detached from the substrate without being damaged by the enzymes.

[0248] Treatment with proteases results in the degradation of desmosome structures between cells and basement membrane-like proteins between cells and the substrate, which can result in individual cells in the cell culture. On the other hand, the cell culture obtained by the production method of the present invention can be detached from the substrate by changing the temperature of the medium without treatment with proteases. As a result, the desmosome structures are maintained and defects in the cell culture can be reduced. Furthermore, when the cell culture obtained by the production method of the present invention is detached from the substrate by changing the temperature of the medium, the basement membrane-like proteins are not destroyed by the enzymes. Therefore, better adhesion to the affected tissue can be achieved during transplantation, allowing for more efficient treatment.

[0249] Dispase, a protease, is known to be able to detach a cell sheet while maintaining 10 to 60% of the desmosome structure, but it destroys most of the basement membrane-like proteins, resulting in weak cell cultures. The cell cultures produced by the production method of the present invention can be detached from the substrate with 80% or more of both the desmosome structure and the basement membrane-like proteins remaining.

[0250] The upper or lower critical solution temperature of the temperature-responsive polymer used in the present invention may be preferably 0° C. to 80° C., more preferably 20° C. to 50° C., and even more preferably 25 to 45° C. If the upper or lower critical solution temperature is too high, cells may die. If the upper or lower critical solution temperature is too high, the cell growth rate may decrease or the cells may die.

[0251] In the production method of the present invention, the temperature-responsive polymer may be either a homopolymer or a copolymer. The polymer may be, for example, a homopolymer of a (meth)acrylamide compound, an N-(or N,N-di)alkyl-substituted (meth)acrylamide derivative, or a vinyl ether derivative, or may be a copolymer of these monomers. The (meth)acrylamide compound may be, for example, acrylamide or methacrylamide. One or more selected from these may be used. The N-alkyl substituted (meth)acrylamide derivative may be, for example, N-ethylacrylamide (lower critical solution temperature of homopolymer: 72°C), N-n-propylacrylamide (same: 21°C), N-n-propylmethacrylamide (same: 27°C), N-isopropylacrylamide (same: 32°C), N-isopropylmethacrylamide (same: 43°C), N-cyclopropylacrylamide (same: 45°C), N-cyclopropylmethacrylamide (same: 60°C), N-ethoxyethylacrylamide (same: about 35°C), N-ethoxyethylmethacrylamide (same: about 45°C), N-tetrahydrofurfurylacrylamide (same: about 28°C), or N-tetrahydrofurfurylmethacrylamide (same: about 35°C). The N,N-dialkyl-substituted (meth)acrylamide derivative may be, for example, N,N-dimethyl(meth)acrylamide, N,N-ethylmethylacrylamide (lower critical solution temperature of homopolymer: 56°C), or N,N-diethylacrylamide (lower critical solution temperature of homopolymer: 32°C). The vinyl ether derivative may be, for example, methyl vinyl ether (lower critical solution temperature of homopolymer: 35°C).

[0252] In the present invention, the temperature-responsive polymer may be a copolymer with a monomer other than the above-mentioned monomer, a polymer obtained by graft polymerization or copolymerization of polymers, or a mixture of polymers or copolymers. Crosslinking may also be performed to the extent that the inherent properties of the polymer are not impaired. The above-mentioned polymers may be appropriately selected to select a temperature-responsive polymer having a critical solution temperature more suitable for the culture or detachment of the present invention, to adjust the interaction between the porous membrane and the culture, or to adjust the hydrophilicity or hydrophobicity of the surface of the porous membrane. In a preferred embodiment, the temperature-responsive polymer is poly(N-isopropylacrylamide).

[0253] In the production method of the present invention, the amount of the temperature-responsive polymer immobilized on the surface of the membrane is preferably 0.3 to 5.0 μg / cm ２ and more preferably 0.3 to 4.8 μg / cm ２ Furthermore, when the membrane is a porous membrane, particularly when the membrane is a porous membrane and the culture is performed using a cell culture insert, the amount of the temperature-responsive polymer immobilized on the membrane surface is preferably 0.3 to 1.5 μg / cm ２ In addition, when the membrane is not a porous membrane, the amount of the temperature-responsive polymer immobilized on the surface of the membrane is preferably 1 to 2 μg / cm ２ When the immobilized amount of the temperature-responsive polymer is within this range, the culture can be more efficient. When the immobilized amount is outside this range, the cell culture may not be formed or may not be produced efficiently. Furthermore, when the immobilized amount is within this range, the cell culture may be more easily detached from the membrane.

[0254] In the production method of the present invention, preferably, the substrate is a porous membrane, and in the culturing step, the cells are in contact with the culture medium above the porous membrane and with the culture medium below the porous membrane via the pores of the porous membrane. By culturing in this state, the culture can be carried out more efficiently.

[0255] In the production method of the present invention, the material of the membrane, particularly the porous membrane, may be, for example, polycarbonate, polyester, polyethylene terephthalate (PET), polystyrene, or polytetrafluoroethylene. Of these, PET is particularly preferred. By using a PET porous membrane, the cell culture of the present invention can be produced more efficiently.

[0256] A method for immobilizing a temperature-responsive polymer on the surface of a porous membrane can be performed, for example, by the method described in Patent Document 3 (JP-A-2-211865). That is, the immobilization can be performed by bonding the porous membrane and the temperature-responsive polymer through a chemical reaction or by bonding through physical interaction. These methods may be used in combination. In the method for bonding through a chemical reaction, for example, electron beam (EB) irradiation, gamma-ray irradiation, ultraviolet irradiation, visible light irradiation, LED irradiation, plasma treatment, or corona treatment can be performed. Alternatively, the temperature-responsive polymer can be immobilized on the porous membrane by commonly used organic reactions such as radical reactions, anion radical reactions, and cation radical reactions. Furthermore, a block copolymer having a structure in which a water-insoluble polymer segment and a temperature-responsive polymer segment are bonded can be coated on the surface of the substrate as the temperature-responsive polymer component. Immobilization can also be performed by physical adsorption or hydrophobicity. In the method for bonding through physical interaction, the temperature-responsive polymer or a mixture of the polymer and any medium can be applied to the porous membrane.

[0257] The medium used for culturing in the production method of the present invention may be a medium that can be used for cell culture, particularly mammalian cell culture, such as DMEM / F12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12). The medium may contain supplementary factors. Examples of supplementary factors include cell growth factors, hormones, binding proteins, cell adhesion factors, lipids, and other components. One or more selected from these may be used.

[0258] Examples of the cell growth factors include TGF-β, b-FGF, IGF, EGF (Epidermal Growth Factor), BMP (Bonemorphogenetic protein), Fibroblast growth factor receptor 3 (FGFR-3), Frizzled-related protein (FRZB), CDMP-1, and Growth Differentiation Factor (GFR). Factor 5 (GDF-5), G-CSF (Granulocyte Colony Stimulating Factor), LIF (Leukemia Inhibitory Factor), interleukin, PDGF (Platelet-Derived Growth Factor), NGF (Nerve Growth Factor), TGF (Transforming Growth Factor) family such as activin A, Wnt family, in particular Wnt-3a (Wingless-type MMTV integration site family, member 3A), and the like, but are not limited to these. The TGF family includes TGF-β1, TGF-β2, and TGF-β3, and one or more selected from these may be used.

[0259] Examples of the hormone include, but are not limited to, insulin, transferrin, dexamethasone, estradiol, prolactin, glucagon, thyroxine, growth hormone, FSH (Follicle Stimulating Hormone), LH (Leutenizing Hormone), glucocortinoid, and prostaglandin, and one or more selected from these may be used.

[0260] Examples of the cell adhesion factor include, but are not limited to, collagen, collagen-like peptides, fibronectin, laminin, and vitronectin. Examples of collagen-like peptides include recombinant peptides in which an RGD sequence-containing region in collagen is linked. Examples of such recombinant peptides include cellnest (Fujifilm Corporation). One or more selected from these may be used.

[0261] The lipids include, but are not limited to, phospholipids and unsaturated fatty acids, and one or more selected from these may be used.

[0262] Examples of other components that can be added to the culture medium include, but are not limited to, ascorbic acid, serum, insulin transferrin selenite (ITS), transferrin, sodium selenite, pyruvate, proline, albumin, lipoproteins, and certoplasmin. Serum can include, but is not limited to, fetal bovine serum (FBS) and human serum. One or more selected from these may be used. In the production method of the present invention, the culture medium is preferably a culture medium containing FBS, and may particularly be DMEM / F12 containing FBS. For more efficient culture, the FBS content is preferably 1 to 30% by volume, preferably 10 to 30% by volume, more preferably 12 to 28% by volume, and even more preferably 15 to 25% by volume, based on the total volume of the culture medium.

[0263] Furthermore, in the production method of the present invention, the medium may be a serum-free medium. Since supplemental factors such as ITS can replace the function of serum, by including such supplemental factors in the medium, culture in the production method of the present invention can be carried out in a serum-free medium. Furthermore, by using a serum-free medium, the risks of using biological raw material resources can be avoided when transplanting into humans.

[0264] Preferably, the medium used in the present invention contains ascorbic acid at a concentration of, for example, 0.01 to 1 mg / mL, preferably 0.05 to 0.5 mg / mL, and more preferably 0.07 to 0.3 mg / mL relative to the medium volume, for better cell growth. Ascorbic acid can promote the production of articular cartilage-specific matrices from cultured cells and / or make the phenotype expression of cell cultures more suitable for cartilage repair. In other words, ascorbic acid can contribute to the regeneration of damaged areas of articular cartilage with hyaline cartilage. If the ascorbic acid concentration is too high, it can hinder the adhesion of cultured cells to the porous membrane. If the ascorbic acid concentration is too low, the effect may not be achieved.

[0265] In a preferred embodiment, the cell culture of the present invention is obtained by culturing cells in a medium, preferably by culturing cells in a medium containing FBS and / or ascorbic acid, and may be, for example, obtained by culturing cells in DMEM / F12 containing FBS and / or ascorbic acid.

[0266] In the production method of the present invention, the period for culturing cells on a membrane may be appropriately selected depending on the state of the culture, such as the state of phenotype expression, and may be, for example, 10 to 20 days, preferably 11 to 18 days, and more preferably 12 to 16 days. Depending on the culture period, the cell culture may become more suitable for cartilage repair.

[0267] The culture can be performed in a state in which the cell population is in contact with the culture medium above the porous membrane and in contact with the culture medium below the porous membrane via the pores of the porous membrane. By culturing in this state, adhesion proteins can be produced not only on the surface of the cell sheet adhered to the porous membrane, but also on the surface opposite the adhered surface, that is, the surface in contact with the upper culture medium. The cell sheet of the present invention can be made more suitable for cartilage repair by having a substrate on both sides of the cell sheet. For culturing in this state, a culture vessel having, for example, a cell insert (also called a cell culture insert) or a similar structure can be used. For example, a culture vessel having a cell insert with a stimuli-responsive polymer (e.g., a temperature-responsive polymer) immobilized on the porous membrane portion can be used in the manufacturing method of the present invention. The cell population can be cultured on the surface of the porous membrane on which the stimuli-responsive polymer (e.g., a temperature-responsive polymer) is immobilized. The stimuli-responsive polymer can be immobilized by methods known to those skilled in the art, for example, by the method described in Patent Document 3 (JP Patent Publication No. 2-211865). In addition, commercially available cell inserts on which stimuli-responsive polymers are immobilized may be used, such as Thermo Scientific™ Nunc™ CC Insert #140660 and BD Falcon Cell Culture Inserts #353090, #353490, 353102, #353091, #353092, and #353093.

[0268] In the production method of the present invention, the culture vessel may be appropriately selected by those skilled in the art, and examples thereof include, but are not limited to, dishes, multi-well plates, flasks, and vessels of similar shapes.

[0269] The characteristics of the cell culture obtained by the production method of the present invention are as described above in "2.". The cells cultured in the culture step and the method for preparing the same are as described above in "2."

[0270] 3-2. Analysis process

[0271] The production method of the present invention preferably includes an analysis step of analyzing gene markers contained in the cell culture. The analysis step may be an analysis step of analyzing gene expression in the cell culture (e.g., sheet-like cultured cells, etc.). The analysis step may be a measurement step. In the analysis step, the above-mentioned (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, (6) mitochondria-related genes, and all of the above-mentioned genes (1) to (6) can be used.

[0272] Preferably, in the analysis, the expression level of at least one genetic marker selected from the group consisting of (1) to (6) is analyzed. The expression levels of one or more genetic markers selected from all of (1) to (6) may be analyzed. The analysis may be measurement, and the measurement is not particularly limited, but is preferably performed by a PCR method or the like.

[0273] For example, the expression level of at least one positively correlated genetic marker selected from the group consisting of (1) to (6) above, and / or the expression level of at least one negatively correlated genetic marker selected from the group consisting of (1) to (6) above, is measured.

[0274] In one embodiment of the present invention, the analyzing step measures at least one gene selected from (1) to (6). These markers described in (1) to (6), such as the nine cell surface markers in (1), are particularly suitable for evaluating cartilage-like tissue formation characteristics, particularly hyaline cartilage-like tissue formation characteristics.

[0275] 3-3. Judgment process

[0276] Preferably, the production method of the present invention may include a determination step of determining whether the cell culture has cartilage-like tissue-forming properties (particularly whether it has hyaline cartilage-like tissue-forming properties) based on the expression levels of gene markers obtained in the analysis step. The expression levels of the gene markers may be the expression levels of each of the genes described above. In the determination step, it may be determined whether the cell culture is suitable for repairing cartilage tissue, particularly repairing hyaline cartilage tissue, and more particularly repairing knee hyaline cartilage tissue, based on the expression levels of the gene markers obtained in the analysis step. In the determination step, the above-mentioned (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, (6) mitochondria-related genes, and all of the genes described in (1) to (6) above can be used.

[0277] In the determination step, it is preferably determined whether the expression level of at least one positively correlated genetic marker selected from the group consisting of (1) to (6) is equal to or greater than a threshold value for each genetic marker, and / or whether the expression level of at least one negatively correlated genetic marker selected from the group consisting of (1) to (6) is equal to or less than a threshold value for each genetic marker.

[0278] In a preferred embodiment, the determination step is preferably performed using an ICRS score estimated based on the expression level of at least one gene marker selected from the group consisting of (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, and (6) mitochondria-related genes. Gene markers selected from all of the gene markers listed in (1) to (6) may also be used without classifying them into the above categories (1) to (6).

[0279] In a more preferred embodiment, in the determination step, an ICRS score estimated based on the expression level of at least one gene marker selected from the group consisting of (1) to (6) is used, and it is determined whether the sheet-shaped cell culture has cartilage-like tissue formation properties when the estimated ICRS score is a predetermined value or more.

[0280] In a more preferred embodiment, in the determination step, an ICRS score estimated based on the expression level of at least one gene marker selected from the group consisting of (1) to (6) is used, and a regression equation that models the relationship between the amount of gene marker and the ICRS score is applied to determine whether the sheet-shaped cell culture has cartilage-like tissue formation properties when the estimated ICRS score is a predetermined value or more.

[0281] The cell culture determined to be suitable for cartilage repair in the determination step may be used for transplantation into a human. Furthermore, the determination step may determine that the cell culture is suitable for transplantation into a human knee. Furthermore, the step may be a step for assisting evaluation, etc.

[0282] The criteria used in the determination step may be publicly known criteria (preferably efficacy scores) and / or pre-generated criteria. The publicly known criteria may be criteria established by academic societies or academic papers. The pre-generated criteria may be criteria obtained by statistical processing.

[0283] In the determination step, it is preferable to use a determination criterion to which the amount of the genetic marker is applied. The determination criterion is not particularly limited, but may include a threshold (cutoff value), a multi-stage evaluation criterion, a hierarchical evaluation criterion, etc., and one or more of these may be used. The determination criterion may also be an evaluation criterion. Furthermore, the determination or determination criterion used in the present invention may be determination, discrimination, appraisal, or any of these criteria or indexes, and may employ a scoring system or grading.

[0284] Generally, in multi-stage evaluation and hierarchical evaluation, the evaluation target is divided into multiple levels or criteria and judged. For example, multi-stage evaluation can be a method of dividing the evaluation target into multiple stages (levels) (e.g., A to C levels, 1 to 5 levels, etc.) and judging them. For example, hierarchical evaluation can be a method of organizing evaluation criteria into a hierarchical structure and evaluating different elements at each level, such as a hierarchy of certain states or a classification system of certain states, or an evaluation model using a classification table, etc.

[0285] The amount of each gene marker used in the present invention correlates with a known efficacy score for chondrocytes (preferably an ICRS score), and therefore, by using an evaluation model including this correlation (e.g., an estimation formula, etc.), the amount of each gene marker can be converted into a known judgment criterion (e.g., an efficacy score, etc.). This makes it possible to estimate or predict an estimated value of a known efficacy score based on the analyzed amount of each gene marker. The known efficacy score may be an efficacy score (preferably an ICRS score) proposed by a chondrocyte-related academic society or the like. The correlation may be determined by obtaining a statistical treatment (e.g., a regression formula, a conversion formula, a threshold (cut-off value), etc.) using statistical processing or statistical analysis based on statistical variables, features, or feature amounts including at least each gene marker, and using the statistical treatment or an evaluation model including the statistical treatment.

[0286] Examples of the evaluation model include a threshold (cutoff value)-based model, a scoring model, a statistical model, a time series analysis model, a Bayesian inference model, a machine learning model (random forest, support vector machine), a deep learning model, etc. One or more of these may be selected.

[0287] Here, the statistically processed product used in the present invention is preferably a model generated by statistical analysis and including various association information (formula, inflection points, classification table, protocol, coefficients (factors), conversion table, etc.), and the formula may be, for example, an estimated formula derived by regression analysis (e.g., linear function: y = ax + b, where a is the slope and b is the y-intercept; quadratic function, etc.). The evaluation model may also indicate a cutoff value. The regression analysis may be either simple regression analysis when there is one independent variable or multiple regression analysis when there are two or more independent variables, but simple regression analysis is preferred when determining a regression line formula.

[0288] As described above, another aspect of the present invention provides a method for estimating an efficacy score, in which the amount of each gene marker derived from a cell culture is applied to a model including various related information generated by statistical analysis corresponding to each gene marker to obtain an estimated efficacy score (preferably an ICRS score, more preferably a score evaluation (modified version of the ICRS grading system)). Another aspect of the present invention provides a method for estimating an efficacy score, in which the amount of each gene marker derived from a cell culture is applied to an estimation formula derived by regression analysis corresponding to each gene marker to obtain an estimated efficacy score of the cell culture. Since an estimated efficacy score can be derived from the amount of each gene marker derived from a cell culture, this estimated value can be applied to a judgment criterion or evaluation criterion (preferably an efficacy score (ICRS score, etc.)), and the cell culture can be evaluated based on the application results. It can also be used to evaluate whether a starting cell can be used as a starting material for an effective cell culture.

[0289] Here, with regard to the "a:slope" of the estimated equation derived by the regression analysis generated by the statistical processing used in the present invention, a suitable numerical range for "a:slope" can be set appropriately based on "a:slope", for example, preferably within the range of ±("a:slope" × 0.2), more preferably within the range of ±("a:slope" × 0.1), even more preferably within the range of ±("a:slope" × 0.05), and particularly preferably remaining as "a:slope". Furthermore, with regard to the "b:y intercept", a suitable numerical range for "b:y intercept" can be set appropriately based on "b:y intercept", for example, preferably within the range of ±("b:y intercept" × 0.2), more preferably within the range of ±("b:y intercept" × 0.1), even more preferably within the range of ±("b:y intercept" × 0.05), and particularly preferably remaining as "b:y intercept".

[0290] Furthermore, for the "threshold value (cut-off value)" used in the present invention, which is generated by statistical processing, the width of a suitable numerical range may be appropriately set based on the generated "threshold value (cut-off value)", and for example, is preferably within the range of ±("threshold value" × 0.2), more preferably within the range of ±("threshold value" × 0.1), even more preferably within the range of ±("threshold value" × 0.05), and particularly preferably remains the "threshold value".

[0291] 3-4. Other Steps The present invention preferably includes a providing step of providing the results of assessment or evaluation of the cell culture based on the assessment criteria or an evaluation model including the assessment criteria, and it is desirable that the results be provided to users, subjects, and related parties. Furthermore, the results of assessment of each gene marker and the results of assessment or evaluation of the cell culture may be provided, and the provision may be, for example, in the form of images, videos, diagrams, text, printed matter, audio, data, etc.

[0292] The present invention may include a treatment or administration step of transplanting, administering, or administering a cell culture that meets the standards obtained by the above-mentioned production method to a target animal at an application site or an affected area. The cell culture may also be a transplant material.

[0293] 4. Methods for evaluating cell cultures

[0294] The present invention also provides a method for evaluating a cell culture. The evaluation method may include a step of determining whether the cell culture has cartilage-like tissue-forming properties (particularly, whether the cell culture has hyaline cartilage-like tissue-forming properties) based on the expression levels of gene markers contained in the cell culture. Furthermore, in the determination step, it may be determined whether the cell culture is suitable for repairing cartilage tissue, particularly repairing hyaline cartilage tissue, and more particularly repairing knee hyaline cartilage tissue, based on the expression levels of gene markers obtained in the analysis step described in "2.", "3.", etc. above. The determination step may be the same as the determination step described in "2.", "3.", etc. above. For example, the determination may be made based on whether the expression levels of specific gene markers in a positive or negative correlation, such as those listed in "2.", "3.", etc. above, satisfy a predetermined threshold condition. The determination may be made based on an ICRS score estimation method. The evaluation method may further include an analysis step of performing analysis by PCR. The determination in the determination step may be made based on the analysis results in the analysis step. The analysis step may be the same as the analysis step described above in "2.", "3.", etc.

[0295] The expression levels and thresholds of specific gene markers in the positive or negative correlation described above are effective indicators for determining whether a cell culture has the ability to form cartilage-like tissue (particularly whether it has the ability to form hyaline cartilage-like tissue). Therefore, the evaluation method of the present invention enables appropriate evaluation of cartilage repair suitability, particularly hyaline cartilage repair suitability. Furthermore, the evaluation method of the present invention determines whether a cell culture is suitable for cartilage repair based on the expression levels of specific gene markers in the positive or negative correlation, making it possible to determine cartilage repair suitability without inducing and culturing the cell culture to form cartilage-like tissue. Furthermore, the evaluation method of the present invention can similarly determine cartilage repair suitability using an ICRS score estimated based on the amount of specific gene markers. Therefore, the evaluation method of the present invention can reduce the time required for determining cartilage repair suitability. Furthermore, since the evaluation method of the present invention can be performed in a short time, it can be incorporated as one of the steps in the production of a cell culture for cartilage repair, thereby more reliably producing a cell culture with cartilage repair suitability. Furthermore, the evaluation method of the present invention also enables cartilage repair suitability to be evaluated more inexpensively than when actually inducing and culturing a cell culture to form cartilage-like tissue and then assessing cartilage repair suitability.

[0296] In the methods (e.g., evaluation methods, production methods, etc.) according to the present invention, the culturing step, measuring step, determination step, evaluation step, etc. may be performed by a computer or by a determination device, evaluation device, etc. Furthermore, the present invention may also be a method, provision system, or provision device that provides information or data such as the results of each step, for example, the determination results or evaluation results, to a person (a user, a manufacturer, etc.) via wired or wireless communication.

[0297] Various data used in the present invention may also be stored in a memory unit or the like. The various data may then be appropriately acquired from the memory unit or the like, and the acquired data may be appropriately used in each step, such as the culturing step, the analysis step, and the evaluation step. Examples of the various data used in the present invention include, but are not limited to, data relating to the steps or flow of a method such as a production method, an evaluation method, or an evaluation method, and data relating to the analysis and evaluation of each gene. Examples of the data relating to the analysis and evaluation of each gene include, but are not limited to, threshold data relating to thresholds; statistical processing data relating to statistical processing such as correlation analysis and the results thereof (e.g., methods for estimating ICRS score values ​​and regression equations); measurement data relating to the measurement results of test substances, gene expression, etc.; and cell culture data relating to the efficacy, characteristics, evaluation, etc. of cell cultures. One or more types selected from these may be used.

[0298] The method of the present invention can also be realized by a control device or control unit including a CPU in an apparatus or system (e.g., a computer, PLC, server, cloud service, etc.) used for evaluation, etc. In addition, the method of the present invention can be stored as a program in hardware resources including a recording medium (non-volatile memory (e.g., USB memory), SSD, HDD, CD, DVD, Blu-ray Disc, etc.) and realized by a control unit. It is also possible to provide a control unit including a system for executing the culture method, measurement method, determination or evaluation method, etc. of the present invention, or an apparatus including such a system. The control unit may be a processing unit that processes data, determination, etc., and the processing unit may control each unit.

[0299] An apparatus or system for carrying out a method such as an evaluation method or a manufacturing method according to the present invention can include an input unit such as a keyboard, a communication unit such as a network, an output unit such as a display, a storage unit such as an HDD, a measurement unit, etc. The apparatus or system preferably includes an input unit, an output unit, and a storage unit, and more preferably includes a communication unit and / or a measurement unit.

[0300] Furthermore, the system relating to the present invention for culturing, evaluation, determination, production, etc. can be implemented by utilizing a program and hardware. One embodiment (not shown) of a computer 1 according to one embodiment of the present invention is not limited thereto, but may include at least a CPU as components of the computer 1, and may further include one or two components selected from RAM, a memory unit, an output unit, an input unit, a communication unit, a ROM, and a measurement unit, etc., of which RAM, a memory unit, an output unit, and an input unit are preferred, and at least one of a communication unit, a measurement unit, a ROM, etc. are also preferred. It is preferable that the respective components are connected, for example, by a bus as a data transmission path.

[0301] The present invention can provide a culture device for cell cultures, and the culture device may be a culture control device. The present invention can also provide a cell culture evaluation device. The device may be a system. In a preferred embodiment, a cell culture device can be provided, including a processing unit that applies the amounts of each gene marker in the cell culture to a criterion to determine or evaluate the cell culture. The processing unit is configured to appropriately execute the above-described method or each step. The processing unit may be configured to transmit and receive various information data, or to control or process the transmission and reception of information data in each unit. The processing unit is preferably configured to control the culture unit, analysis unit or measurement unit, memory unit, communication unit, input unit, output unit, etc. The processing unit is preferably configured to apply the analysis data obtained in the above-described analysis step to a model generated by statistical processing to estimate a known efficacy score. The processing unit may be configured to apply the analysis data obtained in the above-described analysis step to a previously generated criterion to determine the pass / fail of the cell culture, or to apply the above-described known efficacy score estimate to the known efficacy score to determine the pass / fail of the cell culture.

[0302] A simplified example of the processing procedure of specialized AI used as a trained model in the present invention can be, but is not limited to, "(1) training data → (2) algorithm → (3) trained model" and "(4) input data → (3) trained model → (5) result." In broad terms, specialized AI is a mechanism in which a result is obtained by applying any input data to a trained model constructed by incorporating training data (teacher data) into an algorithm that functions as a learning program.

[0303] In the present invention, when a pre-generated judgment criterion is obtained, it may be obtained by machine learning information data of the subject. In the present invention, the subjects in the training data (teacher data) may include new participants. The processing unit acquires various data of the subject (such as cell culture, the genes and gene amounts, and known efficacy scores) as training data. As the training data, one or more gene amounts selected from the various genes in the cultured cell product are acquired. The average value of the acquired data may be set as the judgment criterion. A correlation between the judgment criterion and known efficacy scores may be determined. Furthermore, standardized acquired data may be set as the judgment criterion. Standardization is also referred to as normalization or standardization. It can be calculated by the following formula: standard value (x) = (individual value (x) - average value of x group) / standard deviation (S). The storage unit can store various data acquired from multiple subjects as data in advance.

[0304] Next, the processing unit can incorporate the learning data read from the memory unit into a pre-set algorithm, thereby constructing, for example, an estimation formula and a judgment criterion based on this (trained model), a threshold value, an ICRS score value estimation method, etc. and a judgment criterion based on this (trained model).

[0305] The above-described algorithm can function as, for example, a machine learning algorithm. The type of machine learning algorithm is not particularly limited, and may be, for example, an algorithm using a neural network such as a recurrent neural network (RNN), a convolutional neural network (CNN), or a multilayer perceptron (MLP), or may be any algorithm.

[0306] Next, the processing unit inputs input data from the operator ((4) input data (input layer)) into the constructed evaluation model (trained model), thereby generating data related to clinical diagnosis predictions ((5) result (output layer)) to be output from the display. The trained model may be a trained model generated by, for example, deep learning. For example, the trained model may be a multilayer neural network, such as a deep neural network (DNN), or more specifically, a convolutional neural network (CNN). A multilayer neural network may be used as the trained model, and the multilayer neural network may have an input layer to which a subject or a target person inputs measurements, age, etc., an output layer that outputs the clinical diagnosis result of the subject or a target person, and at least one intermediate layer provided between the input layer and the output layer.

[0307] The processing unit can execute and realize various methods, such as a method for generating a trained model, a method for determining whether a cell culture is pass or fail, or a method for evaluating a cell culture using a trained model, a method for manufacturing a cell culture, or a method for providing these.

[0308] 5. Other aspects of the present invention

[0309] The present technology may also employ the following configurations. [1] A marker for evaluating cartilage-like tissue formation characteristics, comprising one or more gene markers selected from the group consisting of (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, and (6) mitochondria-related genes. Preferably, (1) to (6) are the markers or gene markers described in the first to sixth groups above. The markers may be one or more markers, genes, or gene markers selected from the group consisting of all the markers, all the genes, or all the gene markers described in (1) to (6), without being grouped as such. [2] A marker for evaluating cartilage-like tissue formation characteristics, comprising one or more gene markers selected from the group consisting of positively correlated genes in (1) to (6) and / or one or more gene markers selected from the group consisting of negatively correlated genes in (1) to (6). [3] The marker according to [1] or [2], wherein the marker is used to predict the effectiveness of a cartilage cell sheet for cartilage regeneration. [4] A method for evaluating a cell culture, a method for producing a cell culture having cartilage-like tissue formation properties, and a cell culture obtained by the evaluation method or production method, using one or more of the gene markers according to any one of [1] to [3]. [5] A gene marker for use in or for evaluating cartilage-like tissue formation properties, wherein the gene marker is one or more gene markers selected from the group of markers or gene marker groups described in each of the groups (1) to (6). [6] Use of the sequence (nucleotide sequence) of the following gene or the sequence (amino acid sequence) of the protein produced from the gene in the production of a marker for evaluating cartilage-like tissue formation properties, wherein the gene is one or more genes selected from the group consisting of (1) to (6).

[0310] [7] A cell culture having cartilage-like tissue-forming properties as described in (7a) or (7b) below. (7a) A cell culture having cartilage-like tissue-forming properties, in which the expression level of at least one positively correlated gene marker selected from the group consisting of (1) to (6) is equal to or greater than the threshold value for each gene marker, and / or the expression level of at least one negatively correlated gene marker selected from the group consisting of (1) to (6) is equal to or less than the threshold value for each gene marker. (7b) A cell culture having cartilage-like tissue-forming properties, in which the estimated ICRS score value is equal to or greater than a predetermined value (e.g., 28 or more, 29 or more, or 30 or more) using an ICRS score value estimated based on the expression level of at least one gene marker selected from the group consisting of (1) to (6). [8] The cell culture according to [7] above, wherein the cell culture is in the form of a sheet. [9] The cell culture according to [7] or [8] above, wherein the cell culture is derived from cartilage tissue.

[10] The cell culture according to any one of [7] to [9], wherein the cell culture is not derived from synovial membrane.

[11] The cell culture according to any one of [7] to

[10] , wherein the cell culture is derived from stem cells.

[12] The cell culture according to any one of [7] to

[11] , wherein the cell culture is derived from stem cells, and the stem cells include pluripotent stem cells, embryonic stem cells, or somatic stem cells.

[13] The cell culture according to any one of [7] to

[12] , wherein the cell culture is used for repairing cartilage tissue.

[14] The cell culture according to any one of [7] to

[13] , wherein the cell culture is used for repairing knee cartilage tissue.

[15] The cell culture according to any one of [7] to

[14] , wherein the cell culture has hyaline cartilage-like tissue-forming properties.

[16] The cell culture according to any one of [7] to

[15] above, wherein the cell culture is obtained by culturing cells in a medium containing a substrate having a surface on which a stimuli-responsive polymer is immobilized.

[17] The cell culture according to any one of [7] to

[16] above, wherein the cell culture is obtained by culturing cells on a surface of a porous membrane having a surface on which a stimuli-responsive polymer is immobilized.

[18] The cell culture according to any one of [7] to

[17] above, wherein the cell culture is obtained by culturing cells in a medium.

[19] The cell culture according to any one of [7] to

[18] above, wherein the starting cells used in the culture to obtain the cell culture are cartilage tissue-derived cells, and the cartilage tissue-derived cells are cells obtained by culturing cells in cartilage tissue in DMEM / F12 containing FBS for at least two days.

[20] The cell culture according to any one of [7] to

[19] above, wherein the starting cells used in the culture to obtain the cell culture are prepared by a starting cell preparation method comprising: a first culture step of culturing cells in cartilage tissue in a medium to make them confluent; a dissociation step of dissociating cell populations that have become confluent in the first culture step from each other; and a second culture step of further culturing the cell populations dissociated in the dissociation step in the same fresh medium as the medium.

[0311]

[21] A method for evaluating a cell culture according to (21a) or (21b) below:

[0312] (21a) A method for evaluating a cell culture, comprising a determining step of determining whether the expression level of at least one positively correlated genetic marker selected from the group consisting of (1) to (6) is equal to or greater than a threshold value for each genetic marker, and / or whether the expression level of at least one negatively correlated genetic marker selected from the group consisting of (1) to (6) is equal to or less than a threshold value for each genetic marker. A preferred determining step in (21a) is determining that the cell culture has cartilage-like tissue formation properties when the expression level of the positively correlated genetic marker is equal to or greater than the threshold value and / or when the expression level of the negatively correlated genetic marker is equal to or less than the threshold value. A preferred determining step in (21a) may convert the "equal to or greater than a threshold value" and the "equal to or less than a threshold value" to "equal to or greater than a predetermined value of the estimated ICRS score value" in (21b).

[0313] (21b) A method for evaluating a cell culture, comprising a determination step of using an ICRS score estimated based on the expression level of at least one gene marker selected from the group consisting of (1) to (6). A preferred determination step uses an ICRS score estimated based on the expression level of at least one gene marker selected from the group consisting of (1) to (6), and determines that the sheet-shaped cell culture has cartilage-like tissue-forming properties when the estimated ICRS score is equal to or greater than a predetermined value. A more preferred determination step applies the expression levels of the selected predetermined gene markers to a regression equation that models the relationship between the gene marker levels and the ICRS score, and determines that the sheet-shaped cell culture has cartilage-like tissue-forming properties when the estimated ICRS score is equal to or greater than a predetermined value. The method of

[21] may suitably employ any of the details of any of [7] to

[19] , such as the cell culture, sheet, origin, medium, substrate, porous membrane, and process. The method may also be a method for assisting evaluation, a method for providing evaluation, or a device for performing the method.

[22] The method according to

[21] above may suitably employ any of the contents of [7] to

[19] above, such as a cell culture, a sheet-like form, a source, a medium, a substrate, a porous membrane, and a process. The method may also be a method for assisting evaluation, a method for providing evaluation, or a device for performing the method.

[23] The method includes a culturing step of culturing cells to obtain a cell culture, and the culturing step may select either (23a) or (23b) below. (23a) A method for producing a cell culture having cartilage-like tissue-forming properties, wherein the culturing is performed such that the expression level of at least one positively correlated gene marker selected from the group consisting of (1) to (6) is equal to or greater than a threshold value for each gene marker, and / or the expression level of at least one negatively correlated gene marker selected from the group consisting of (1) to (6) is equal to or less than a threshold value for each gene marker.(23b) A method for producing a cell culture having cartilage-like tissue-forming properties, in which the cell culture is cultured so that the ICRS score estimated based on the expression level of at least one gene marker selected from the group consisting of (1) to (6) is equal to or greater than a predetermined value. The production method may suitably employ any of the contents of any of (7) to (19), such as the cell culture, sheet, origin, medium, substrate, porous membrane, and process. The method may also be a method for assisting evaluation, providing evaluation, or a device for carrying out the method.

[24] The method for producing a cell culture according to (23) is preferably (24a) or (24b) below. (23a) The method for producing a cell culture according to

[23] , further comprising: a step of analyzing genetic markers; and a determination step of determining that the sheet-shaped cell culture has cartilage-like tissue-forming properties when the expression level of the positively correlated genetic marker obtained based on the analysis step is equal to or greater than the threshold and / or when the expression level of the negatively correlated genetic marker is equal to or less than the threshold. (23a) The method for producing a cell culture according to

[23] , further comprising: a step of analyzing genetic markers; and a determination step of determining that the sheet-shaped cell culture has cartilage-like tissue-forming properties using an ICRS score estimated based on the expression level of at least one genetic marker selected from the group consisting of (1) to (6) obtained based on the analysis step. The production method may suitably employ any of the contents of [7] to

[19] , such as the cell culture, sheet-shaped, origin, medium, substrate, porous membrane, and process. The method may also be a method for assisting evaluation, providing evaluation, or a device for performing the method.

[0314]

[25] A method for estimating the effectiveness score of a cell culture based on the amount of one gene marker described in any of [1] to [6] above or the amount of at least one gene marker selected from the group consisting of (1) to (6).

[26] A method for estimating the effectiveness score of a cell culture by applying the amount of one gene marker described in any of [1] to [6] above or the amount of at least one gene marker selected from the group consisting of (1) to (6) to information relating the gene marker to the effectiveness score (preferably a protocol, a classification table, an estimation formula, a regression formula, etc.).

[27] A method for evaluating a cell culture based on the estimated value of the effectiveness score of the cell culture obtained by the method of

[25] or

[26] above.

[0315]

[28] A kit for evaluating cartilage-like tissue formation characteristics, using any one of the gene markers described in [1] or [2] above.

[29] A kit for use in the methods of

[21] to

[28] above, using any one of the gene markers described in [1] or [2] above.

[30] The kit according to

[28] or

[29] above, in which evaluation is performed by real-time PCR or a gene panel.

[0316] In this specification, a "doing" such as "culturing" may be expressed as a "process" or a "step," a "process" may be expressed as a "doing" or a step, and a "step" may be expressed as a "doing" or a "process." Furthermore, in this specification, a "process" such as a "process" may be expressed as an "apparatus or unit configured to perform the process, or a circuit configured to perform the process or "doing" (a "doing" or "to do")," a "device" may be expressed as a "mechanism or unit," and a "unit" may be expressed as a "unit or device for providing in a mechanism, apparatus, system, or the like, or a circuit configured to perform "doing" (a "doing" or "to do")." Any of a kit, a set, and a combination may be used.

[0317] Hereinafter, the present technology will be described in further detail based on examples, etc. Note that the examples, etc. described below are examples of typical examples, etc. of the present technology, and the scope of the present technology will not be construed as being narrow.

[0318] <Test Example 1: Method for extracting efficacy factors>

[0319] [Preparation of P1 and Pn stocks] Cartilage tissue obtained from the extra fingers (toes) of polydactyly patients was digested with Liberase. The resulting chondrocytes were cultured until they reached subconfluence or confluence, and then stored in STEM-CELLBANKER. （R） The cells were suspended in GMP grade water and frozen (P1 stock). Subsequently, a portion of the frozen cell stock was thawed and cultured until the cells reached subconfluence and confluence. (R) The process of suspending the cells in GMP-grade solution and freezing and storing them was repeated multiple times (Pn stock). Only the P2 stock was used to identify the 10 efficacy factors (plus the P1 stock). The P2, P3, P5, and P7 stocks from eight lots of three donors (Table 2) were used to narrow down the list to nine.

[0320] [Test Example 1-1: Preparation of chondrocyte cell sheets used in the first pharmacological efficacy test] Frozen cell stocks from 8 lots of 5 donors (Table 1) were thawed and cultured until they reached subconfluence to confluence. (R) Cells were seeded onto the inserts and cultured for 14 days to produce chondrocyte sheets. For example, a P1 stock, which was prepared by seeding a frozen cell stock at (passage number of the cell sheet) - 1, can be thawed to produce a P2 sheet.

[0321]

[0322] [Chondron cell sheet transplantation into nude rats] The efficacy of chondrocyte cell sheets was evaluated in a xenotopic transplantation test using a nude rat cartilage injury model. Nude rats (F344 / NJcl-rnu / rnu) were anesthetized with 2-4% isoflurane inhalation, and anesthesia was maintained during transplantation by adjusting the isoflurane concentration appropriately to 0.5-3%. The rats were placed in a supine position. The medial side of the right hind knee joint was incised, the soft tissue was cut and dissected, and the patella was dislocated laterally to dislocate it. A circular marking was made on the trochlea 5 mm proximal to the distal end of the medial femoral condyle using a 2 mm biopsy punch (Kai Industries) (where the line 5 mm from the distal end meets the distal end of the circle). The cartilage tissue at the marked location was scraped off in a circular pattern approximately 1 mm deep using a manual drill (precision screwdriver type), and the resulting depression was washed with sterile saline. One-half cell sheet was transplanted into each rat's depression. The patellar dislocation was reduced and sutured to reconstitute the joint capsule. The muscle layer was sutured, and then the skin was sutured with buried sutures. 28 days after transplantation, cartilage regeneration in the injured area was evaluated using a score (modified version of the ICRS grading system).

[0323] [Sample collection and specimen preparation] Twenty-eight days after administration, rats were euthanized by exsanguination under 2% isoflurane inhalation anesthesia, and the right hind knee joint was collected and preserved by immersion in 10% neutral buffered formalin. Paraffin sections were prepared from the collected formalin-fixed specimens of the right hind knee joint, and then stained with hematoxylin and eosin and double stained with Safranin O and fast green (decalcified with 10% EDTA solution).

[0324] [Identification of efficacy factors] We identified new efficacy predictive marker candidates using chondrocyte sheets subjected to xenogeneic orthotopic transplantation tests in a nude rat cartilage injury model. We then narrowed down efficacy factors by comparing these results with previous results. Specifically, we obtained 21,121 gene expression data from comprehensive gene expression data obtained using RNA-Seq for pre-implantation chondrocyte sheets (Table 1) from different donors and passage numbers. Meanwhile, we obtained 7,596 protein expression data from comprehensive secreted protein data obtained using SOMA-Scan (https: / / somalogic.com / ) for analysis of chondrocyte sheet culture supernatant. Next, we attempted to identify efficacy marker candidates by comparing cartilage regeneration in the nude rat cartilage injury model with the aforementioned comprehensive gene and protein data. As a result, several functional molecules were identified. From these, we focused on adhesion-related molecules and identified 10 molecules (ELANE, LAP3, TENM4, and PTHLH (all positively correlated), and THBS3, IL27RA, NTN4, COL1A1, CHRD, and VCAM1 (all negatively correlated)) that were highly correlated with clinical scores after transplantation in a nude rat cartilage injury model using both RNA-Seq and SOMA-Scan data. These molecules were extracted based on correlation coefficients of 0.5 or higher between ICRS score (Hgtot) and RNA-Sequence, and 0.5 or higher between ICRS score (Hgtot) and SOMA-Scan. Furthermore, these adhesion-related molecules were extracted from adhesion-related GO (Gene Ontology) terms.

[0325] [Test Example 1-2: Preparation of chondrocyte cell sheets used in the second pharmacological efficacy test and confirmation of reproducibility] New chondrocyte cell sheets (Table 2) from different donors and passage numbers were prepared, and cartilage regeneration in a nude rat cartilage injury model and comprehensive expression data were obtained using RNA-Seq. Correlation analysis was performed between the gene expression levels of each chondrocyte cell sheet and the ICRS score for the 10 identified types, and reproducibility was observed for nine types, except for ELANE (Figure 2 and Table 3). The regression line was calculated using the linear approximation formula in Excel.

[0326]

[0327]

[0328] Figure 1 shows the histological evaluation of repair tissues based on ICRS scores. These were also subjected to microscopic observation for histological and immunohistochemical analyses. Figure 2 shows a scatter plot between ICRS scores and the genes selected in Test Example 1-1. Of the 10 genes, nine, excluding ELANE, showed a similar correlation (Pearson correlation coefficient) in Test Example 1-2. ELANE showed an inverse correlation between Test Example 1-1 and Test Example 1-2.

[0329] Example 2 Gene groups for keywords (GO terms) related to gene function of angiogenesis, ECM (extracellular matrix), collagen, cell migration, and mitochondria were extracted using comprehensive expression data obtained by RNA-Seq in each of Test Examples 1-1 and 1-2. Correlation analysis was used to narrow down the search to genes with a correlation coefficient of 0.5 or higher in each of Test Examples 1-1 and 1-2.

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337] <GeneScore calculation> RNA-seq expression data was normalized by the average expression level for each gene. Genes with a positive correlation with the ICRS score were multiplied by a plus sign, and genes with a negative correlation were multiplied by a minus sign. The normalized gene expression levels were then added together to calculate the GeneScore.

Claims

1. A marker for evaluating cartilage-like tissue formation characteristics, comprising one or more gene markers selected from the group consisting of (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, and (6) mitochondria-related genes. (1) Adhesion-related genes: LAP3, TENM4, PTHLH, THBS3, IL27RA, NTN4, COL1A1, CHRD, VCAM1. (2) Angiogenesis-related genes: PDGFA, VEGFC, SERPINE1, CSPG4, RASIP1, WARS, AAMP, FGF18, S1PR1. (3) ECM-related genes: BGN, CLEC3B, COL11A1, COL1A1, COL1A2, COL5A1, COL5A2, CRIP2, HAPLN1, JUP, MMP13, MMP23B, SERPINE1, THBS1, TINAGL1, ADAMTS14, CCT2, CYHR1, MMP17. (4) Collagen-related genes: COL5A1, COL5A2, COL1A1, COL1A2, COL11A1. (5) Cell migration-related genes: ARPC5, CD2AP, COL5A1, CSPG4, ITGB3, JUP, PODXL, RHOA, SDC2, SHROOM2, SNAI1, BDKRB1, CDK5, FUT8, S1PR1, SH3KBP1, TGFBR3.(6) Mitochondria-related genes: AS3MT, ATP7B, CA5B, CKB, CMC2, CTPPS2, ELN, FBXO7, MARC2, ME3, NOL3, PABPC5, PANK2, PRELID2, SEPT4, SPARC, SPTLC2, ADCK1, ADH5, AFG3L2, AIFM1, AKAP1, ALDH4A1, APEX1, BCS1L, C1QBP, CARS2, CHCHD3, COA4, COQ9, COX7B, DDAH2, DGAT2, DLD, DNAJA1, ECH1, ECHS1, FDXR, FIBP, FMC1, GATB, GCSH, GPX1, HSD17B10, IDH3B, LAP3, LARS2, MAPK8IP1, MDH2, METAP1D, MRPL10, MRPL24, MRPL42, MRPS12, MRPS18C, MRPS22, MRPS27, MRPS35, MTG1, MTHFD1, NDUFB9, NDUFS2, NDUFV1, OCIAD1, OX NAD1, PARS2, PDF, PHB, PHYH, POLRMT, PTCD1, PYURF, RMDN1, RMND1, SDHD, SFXN4, SLC25A11, SMIM4, TACO1, TIMM10, TIMM50, TK2, TOMM22, TOMM5, TRAP1, TUFM, UQCRC1.

2. The marker according to claim 1, which is used to predict the effectiveness of a chondrocyte sheet for cartilage regeneration.

3. A method for evaluating a cell culture, comprising a determining step of determining whether the expression level of at least one positively correlated gene marker selected from the group consisting of (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, and (6) mitochondria-related genes is equal to or greater than the threshold value for each gene marker, and / or whether the expression level of at least one negatively correlated gene marker selected from the group consisting of (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, and (6) mitochondria-related genes is equal to or less than the threshold value for each gene marker. (1) Adhesion-related genes: (positive correlation) LAP3, TENM4, PTHLH, and (negative correlation) THBS3, IL27RA, NTN4, COL1A1, CHRD, VCAM1. (2) Angiogenesis-related genes: (negative correlation) PDGFA, VEGFC, SERPINE1, CSPG4, and (positive correlation) RASIP1, WARS, AAMP, FGF18, S1PR1. (3) ECM-related genes: (negative correlation) BGN, CLEC3B, COL11A1, COL1A1, COL1A2, COL5A1, COL5A2, CRIP2, HAPLN1, JUP, MMP13, MMP23B, SERPINE1, THBS1, TINAGL1, and (positive correlation) ADAMTS14, CCT2, CYHR1, MMP17. (4) Collagen-related genes: (negative correlation) COL5A1, COL5A2, COL1A1, COL1A2, COL11A1. (5) Cell migration-related genes: (negative correlation) ARPC5, CD2AP, COL5A1, CSPG4, ITGB3, JUP, PODXL, RHOA, SDC2, SHROOM2, SNAI1, and (positive correlation) BDKRB1, CDK5, FUT8, S1PR1, SH3KBP1, TGFBR3.(6) Mitochondria-related genes: (negative correlation) AS3MT, ATP7B, CA5B, CKB, CMC2, CTPS2, ELN, FBXO7, MARC2, ME3, NOL3, PABPC5, PANK2, PRELID2, SEPT4, SPARC, SPTLC2, and (positive correlation) ADCK1, ADH5, AFG3L2, AIFM1, AKAP1, ALDH4A1, APEX1, BCS1L, C1QBP, CARS2, CHCHD3, COA4, COQ9, COX7B, DDAH2, DGAT2, DLD, DNAJA1, ECH1, ECHS1, FDXR, FIBP, FMC1, GATB, GCSH, GPX1, HSD 17B10, IDH3B, LAP3, LARS2, MAPK8IP1, MDH2, METAP1D, MRPL10, MRPL24, MRPL42, MRPS 12, MRPS18C, MRPS22, MRPS27, MRPS35, MTG1, MTHFD1, NDUFB9, NDUFS2, NDUFV1, OCIAD 1, OXNAD1, PARS2, PDF, PHB, PHYH, POLRMT, PTCD1, PYURF, RMDN1, RMND1, SDHD, SFXN4, SLC25A11, SMIM4, TACO1, TIMM10, TIMM50, TK2, TOMM22, TOMM5, TRAP1, TUFM, UQCRC1.

4. The cell culture evaluation method described in claim 3, wherein the determination step determines that the sheet-shaped cell culture has cartilage-like tissue formation properties when the expression level of the positively correlated gene marker is equal to or greater than the threshold value and / or when the expression level of the negatively correlated gene marker is equal to or less than the threshold value.

5. A method for evaluating a cell culture, comprising a step of using an ICRS score estimated based on the expression level of at least one gene marker selected from the group consisting of (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, and (6) mitochondria-related genes, and determining that the sheet-shaped cell culture has cartilage-like tissue formation properties when the estimated ICRS score is a predetermined value or higher. (1) Adhesion-related genes: (positive correlation) LAP3, TENM4, PTHLH, and (negative correlation) THBS3, IL27RA, NTN4, COL1A1, CHRD, VCAM1. (2) Angiogenesis-related genes: (negative correlation) PDGFA, VEGFC, SERPINE1, CSPG4, and (positive correlation) RASIP1, WARS, AAMP, FGF18, S1PR1. (3) ECM-related genes: (negative correlation) BGN, CLEC3B, COL11A1, COL1A1, COL1A2, COL5A1, COL5A2, CRIP2, HAPLN1, JUP, MMP13, MMP23B, SERPINE1, THBS1, TINAGL1, and (positive correlation) ADAMTS14, CCT2, CYHR1, MMP17. (4) Collagen-related genes: (negative correlation) COL5A1, COL5A2, COL1A1, COL1A2, COL11A1. (5) Cell migration-related genes: (negative correlation) ARPC5, CD2AP, COL5A1, CSPG4, ITGB3, JUP, PODXL, RHOA, SDC2, SHROOM2, SNAI1, and (positive correlation) BDKRB1, CDK5, FUT8, S1PR1, SH3KBP1, TGFBR3.(6) Mitochondria-related genes: (negative correlation) AS3MT, ATP7B, CA5B, CKB, CMC2, CTPS2, ELN, FBXO7, MARC2, ME3, NOL3, PABPC5, PANK2, PRELID2, SEPT4, SPARC, SPTLC2, and (positive correlation) ADCK1, ADH5, AFG3L2, AIFM1, AKAP1, ALDH4A1, APEX1, BCS1L, C1QBP, CARS2, CHCHD3, COA4, COQ9, COX7B, DDAH2, DGAT2, DLD, DNAJA1, ECH1, ECHS1, FDXR, FIBP, FMC1, GATB, GCSH, GPX1, HSD 17B10, IDH3B, LAP3, LARS2, MAPK8IP1, MDH2, METAP1D, MRPL10, MRPL24, MRPL42, MRPS 12, MRPS18C, MRPS22, MRPS27, MRPS35, MTG1, MTHFD1, NDUFB9, NDUFS2, NDUFV1, OCIAD 1, OXNAD1, PARS2, PDF, PHB, PHYH, POLRMT, PTCD1, PYURF, RMDN1, RMND1, SDHD, SFXN4, SLC25A11, SMIM4, TACO1, TIMM10, TIMM50, TK2, TOMM22, TOMM5, TRAP1, TUFM, UQCRC1.

6. The method for evaluating a cell culture described in claim 5, wherein the expression levels of the selected predetermined gene markers are applied to a regression equation that models the relationship between the amount of the selected gene markers and ICRS score values, and the sheet-shaped cell culture is determined to have cartilage-like tissue formation properties when the estimated ICRS score value is a predetermined value or higher.

7. A cell culture having cartilage-like tissue-forming properties, wherein the expression level of at least one gene marker selected from the group consisting of (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, and (6) mitochondria-related genes is equal to or greater than the threshold value for each gene marker, and / or the expression level of at least one negatively correlated gene marker selected from the group consisting of (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, and (6) mitochondria-related genes is equal to or less than the threshold value for each gene marker. (1) Adhesion-related genes: (positive correlation) LAP3, TENM4, PTHLH, and (negative correlation) THBS3, IL27RA, NTN4, COL1A1, CHRD, VCAM1. (2) Angiogenesis-related genes: (negative correlation) PDGFA, VEGFC, SERPINE1, CSPG4, and (positive correlation) RASIP1, WARS, AAMP, FGF18, S1PR1. (3) ECM-related genes: (negative correlation) BGN, CLEC3B, COL11A1, COL1A1, COL1A2, COL5A1, COL5A2, CRIP2, HAPLN1, JUP, MMP13, MMP23B, SERPINE1, THBS1, TINAGL1, and (positive correlation) ADAMTS14, CCT2, CYHR1, MMP17. (4) Collagen-related genes: (negative correlation) COL5A1, COL5A2, COL1A1, COL1A2, COL11A1. (5) Cell migration-related genes: (negative correlation) ARPC5, CD2AP, COL5A1, CSPG4, ITGB3, JUP, PODXL, RHOA, SDC2, SHROOM2, SNAI1, and (positive correlation) BDKRB1, CDK5, FUT8, S1PR1, SH3KBP1, TGFBR3.(6) Mitochondria-related genes: (negative correlation) AS3MT, ATP7B, CA5B, CKB, CMC2, CTPS2, ELN, FBXO7, MARC2, ME3, NOL3, PABPC5, PANK2, PRELID2, SEPT4, SPARC, SPTLC2, and (positive correlation) ADCK1, ADH5, AFG3L2, AIFM1, AKAP1, ALDH4A1, APEX1, BCS1L, C1QBP, CARS2, CHCHD3, COA4, COQ9, COX7B, DDAH2, DGAT2, DLD, DNAJA1, ECH1, ECHS1, FDXR, FIBP, FMC1, GATB, GCSH, GPX1, HSD 17B10, IDH3B, LAP3, LARS2, MAPK8IP1, MDH2, METAP1D, MRPL10, MRPL24, MRPL42, MRPS 12, MRPS18C, MRPS22, MRPS27, MRPS35, MTG1, MTHFD1, NDUFB9, NDUFS2, NDUFV1, OCIAD 1, OXNAD1, PARS2, PDF, PHB, PHYH, POLRMT, PTCD1, PYURF, RMDN1, RMND1, SDHD, SFXN4, SLC25A11, SMIM4, TACO1, TIMM10, TIMM50, TK2, TOMM22, TOMM5, TRAP1, TUFM, UQCRC1.

8. A cell culture having cartilage-like tissue-forming properties, wherein the ICRS score estimated based on the expression level of at least one gene marker selected from the group consisting of: (1) adhesion-related genes, (2) angiogenesis-related genes, (3) ECM-related genes, (4) collagen-related genes, (5) cell migration-related genes, and (6) mitochondria-related genes, is a predetermined ICRS score or higher. (1) Adhesion-related genes: LAP3, TENM4, PTHLH, and THBS3, IL27RA, NTN4, COL1A1, CHRD, and VCAM1. (2) Angiogenesis-related genes: PDGFA, VEGFC, SERPINE1, CSPG4, RASIP1, WARS, AAMP, FGF18, and S1PR1. (3) ECM-related genes: BGN, CLEC3B, COL11A1, COL1A1, COL1A2, COL5A1, COL5A2, CRIP2, HAPLN1, JUP, MMP13, MMP23B, SERPINE1, THBS1, TINAGL1, ADAMTS14, CCT2, CYHR1, and MMP17. (4) Collagen-related genes: COL5A1, COL5A2, COL1A1, COL1A2, and COL11A1. (5) Cell migration-related genes: ARPC5, CD2AP, COL5A1, CSPG4, ITGB3, JUP, PODXL, RHOA, SDC2, SHROOM2, SNAI1, and BDKRB1, CDK5, FUT8, S1PR1, SH3KBP1, and TGFBR3.(6) Mitochondrial-related genes: AS3MT, ATP7B, CA5B, CKB, CMC2, CTPS2, ELN, FBXO7, MARC2, ME3, NOL3, PABPC5, PANK2, PRELID2, SEPT4, SPARC, SPTLC2, and ADCK1, ADH5, AFG3L2, AIFM1, AKAP1, ALDH4A1, APEX1, BCS1L, C1QBP, CARS2, CHCHD3, COA4, COQ9, COX7B, DDAH2, DGAT2, DLD, DNAJA1, ECH1, ECHS1, FDXR, FIBP, FMC1, GATB, GCSH, GPX1, HSD17B10, IDH3B, LAP3, LARS2, MAPK8IP1, MDH2, METAP1D, MRPL10, MRPL24, MRPL42, MRPS12, M RPS18C, MRPS22, MRPS27, MRPS35, MTG1, MTHFD1, NDUFB9, NDUFS2, NDUFV1, OCIAD1, O XNAD1, PARS2, PDF, PHB, PHYH, POLRMT, PTCD1, PYURF, RMDN1, RMND1, SDHD, SFXN4, S LC25A11, SMIM4, TACO1, TIMM10, TIMM50, TK2, TOMM22, TOMM5, TRAP1, TUFM, UQCRC1.

9. The cell culture according to claim 7 or 8, wherein the cell culture is in the form of a sheet.

10. The cell culture of claim 7 or 8, wherein the cell culture is derived from cartilage tissue.

11. The cell culture of claim 7 or 8, wherein the cell culture is derived from stem cells.

12. The cell culture according to claim 7 or 8, which is used for repairing cartilage tissue.

13. A kit for evaluating cartilage-like tissue formation characteristics, using one or more gene markers according to claim 1 or 2.

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