Marker gene search method of chondrocyte sheet and marker gene search device of chondrocyte sheet, evaluation method of chondrocyte sheet and evaluation device of chondrocyte sheet, chondrocyte sheet, manufacturing method of chondrocyte sheet, and cartilage regeneration method using chondrocyte sheet

Abstract

To provide a marker gene search method for searching for a marker gene related to the posttransplant efficacy of a chondrocyte sheet used for cartilage regeneration from a pretransplant chondrocyte sheet, and the like.SOLUTION: The present invention relates to a marker gene search method of a chondrocyte sheet comprising: a measurement step of measuring the expression level of a gene in a pretransplant chondrocyte sheet; an analysis step of evaluating the gene expression and secretory protein of a posttransplant chondrocyte sheet using a transplantation model to analyze a gene expression profile characteristic of the chondrocyte sheet that has showed posttransplant efficacy from the measurement results obtained in the measurement step; and a search step for searching for a gene showing a positive correlation and / or a gene showing a negative correlation with respect to the posttransplant efficacy, based on the gene expression profile analyzed in the analysis step.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to 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 producing a cartilage cell sheet, and a method for regenerating cartilage using a cartilage cell sheet. [Background technology]

[0002] It is well known that articular cartilage does not easily regenerate. Natural repair of cartilage after damage due to trauma or osteoarthritis (OA) cannot be expected. In fact, OA is designated as a degenerative cartilage disease, and it is estimated that approximately half of people over the age of 40 are potential patients (Non-Patent Document 1). OA accounts for the majority of cartilage damage requiring treatment, but no definitive treatment for OA has been established.

[0003] Chondrocyte sheets can be produced without using animal-derived collagen or other materials by using a temperature-responsive culture device such as a thermoresponsive polymer graft culture dish to allow cells to attach, proliferate, and form into a sheet (Non-Patent Documents 2 and 3). These sheets can be harvested without enzymatic digestion by lowering the temperature. This technique allows cartilage sheets to be transplanted into cartilage lesions while retaining the extracellular matrix and adhesion molecules produced by the chondrocytes themselves (Non-Patent Document 4). Because chondrocyte sheets can be attached to the transplant site without suturing, the chondrocytes remain at the transplant site. Therefore, cartilage sheet transplantation is a promising approach for the treatment of articular cartilage lesions. Regeneration of articular cartilage using hyaline cartilage, which is important for joint function, has been demonstrated using partial-thickness and osteochondral defect models in rats, rabbits, and pigs (Non-Patent Documents 4-7).

[0004] Patients aged 20 to 60 years with knee articular cartilage damage due to trauma or OA degeneration have been treated with cartilage sheets made from the patient's own cells. Hyaline cartilage was used to repair the articular cartilage, and treatment improved the clinical condition score of the joint (Non-Patent Document 8). These results suggest that cell sheet transplantation may be a definitive treatment for OA-related articular cartilage lesions.

[0005] Based on many years of experience with allogeneic transplantation, it is well known that cartilage tissue has immune tolerance. Allogeneic cartilage fragments are commercially available and are widely transplanted in the United States (Non-Patent Document 9). Therefore, the use of allogeneic cartilage sheets is considered a viable treatment. In considering the application of allogeneic chondrocyte sheets to the treatment of joint diseases, the application of chondrocytes obtained from surgical specimens of patients with juvenile polydactyly has been investigated. To date, chondrocyte sheets (PD (polydactyly patient-derived) sheets: also known as PD cartilage sheets) have been reported to have properties similar to autologous chondrocytes, such as the expression of mesenchymal cell surface markers and the production of some chondrogenic anabolic factors (Non-Patent Document 10).

[0006] Since most polydactyly surgeries are performed during infancy, it is possible to obtain highly proliferative chondrocytes. If the number of chondrocytes obtained from a single donor is insufficient to produce many sheets, the chondrocytes can be cultured and expanded, and then cryopreserved as material for future PD sheet production.

[0007] It has been reported that the efficacy of human adult knee chondrocyte sheets for osteochondral defects can be directly evaluated using a xenotransplantation model using rabbits administered immunosuppressants (Non-Patent Document 11). [Prior art documents] [Patent documents]

[0008] [Non-Patent Document 1] Yoshimura, N.; Muraki, S.; Oka, H.; Mabuchi, A.; En-Yo, Y.; Yoshida, M.; Saika, A.; Yoshida, H.; Suzuki, T.; Yamamoto, S.; et al. Prevalence of knee osteoarthritis, lumbar spondylosis, and osteoporosis in Japanese men and women: the research on osteoarthritis / osteoporosis against disability study. J. Bone Miner. Metab. 2009, 27, 620-628. [Non-Patent Document 2] Okano, T.; Yamada, N.; Sakai, H.; Sakurai, Y. A novel recovery system for cultured cells using plasma-treated polystyrene dishes grafted with poly(N-isopropylacrylamide). J. Biomed. Mater. Res. 1993, 27, 1243-1251. [Non-Patent Document 3] Okano, T.; Yamada, N.; Okuhara, M.; Sakai, H.; Sakurai, Y. Mechanism of cell detachment from temperature-modulated, hydrophilic-hydrophobic polymer surfaces. Biomaterials 1995, 16, 297-303. [Non-Patent Document 4] Kaneshiro, N.; Sato, M.; Ishihara, M.; Mithani, G.; Sakai, H.; Mochida, J. Bioengineered chondrocyte sheets may be potentially useful for the treatment of partial thickness defects of articular cartilage. Biochem. Biophys. Res. Commun. Rev. 2006, 349, 723–731.

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[0009] However, cartilage tissue obtained from polydactyly surgery varies greatly depending on the donor, with respect to size, maturation stage of cartilage development, proportion of perichondrium, cartilage depth, and hypertrophic zone. Furthermore, chondrocytes are known to be prone to dedifferentiation when cultured in a dish, and it is expected that the characteristics of PD sheets will be affected by variations in culture period and number of passages. For these reasons, it is necessary to clarify the impact of the diversity associated with allogeneic cell sheets on the efficacy of cell sheet transplantation.

[0010] An object of the present invention is to provide a method and device for searching for marker genes of chondrocyte cells, which are used to search for marker genes associated with the post-transplant effectiveness of chondrocyte cells used for cartilage regeneration from chondrocyte cells before transplantation. Another object of the present invention is to provide a method and device for evaluating chondrocyte cells, which are used to predict the post-transplant effectiveness of chondrocyte cells used for cartilage regeneration from chondrocyte cells before transplantation. Another object of the present invention is to provide a chondrocyte cell sheet that is highly effective after transplantation for use in cartilage regeneration, and a method for producing the same. Yet another object of the present invention is to provide a cartilage regeneration method using a chondrocyte cell sheet that is highly effective after transplantation for use in cartilage regeneration. [Means for solving the problem]

[0011] The present inventors used the above xenograft model with immunosuppressant-treated rabbits to evaluate the efficacy of PD sheets for osteochondral defects, and also evaluated gene expression and secreted proteins in the transplanted PD sheets. Transcriptome and proteome analyses revealed gene expression profiles and secreted factors characteristic of effective PD sheets. Furthermore, the present inventors estimated the biological functions of the molecules that contribute to the efficacy of PD sheets, leading to the completion of this invention.

[0012] The present invention provides a method for searching for marker genes in cartilage cell sheets, for searching for marker genes associated with the post-transplant effectiveness of cartilage cell sheets used for cartilage regeneration from cartilage cell sheets before transplantation, the method comprising: a sheet preparation step of preparing multiple lots of cartilage cell sheets before transplantation; a protein measurement step of measuring the secretion levels of multiple proteins for each of the multiple lots of cartilage cell sheets before transplantation; a score calculation step of transplanting each of the multiple lots of cartilage cell sheets into an osteochondral defect model organism and calculating a score of cartilage regeneration effectiveness for each cartilage cell sheet; and a gene identification step of identifying positively correlated genes that show a positive correlation with the score and / or negatively correlated genes that show a negative correlation with the score, based on the correlation between the secretion levels of proteins measured in the protein measurement step and the score calculated in the score calculation step.

[0013] In this case, it is preferable that the method further comprises a gene measurement step of measuring the expression levels of multiple genes for each of the multiple lots of cartilage cell sheets before implantation, and that the gene identification step identifies positively correlated genes that show a positive correlation with the score and / or negatively correlated genes that show a negative correlation with the score based on the correlation between the expression levels of the multiple genes measured in the gene measurement step and the score calculated in the score calculation step.

[0014] The present invention also provides a cartilage cell sheet marker gene screening device for screening marker genes associated with the post-transplant effectiveness of a cartilage cell sheet used for cartilage regeneration from a cartilage cell sheet before transplantation, the device comprising: a sheet preparation means for preparing multiple lots of cartilage cell sheets before transplantation; a protein measurement means for measuring the secretion levels of multiple proteins for each of the multiple lots of cartilage cell sheets before transplantation; a score calculation means for transplanting each of the multiple lots of cartilage cell sheets into an osteochondral defect model organism and calculating a score of the effectiveness in cartilage regeneration for each cartilage cell sheet; and a gene identification means for identifying positively correlated genes that show a positive correlation with the score and / or negatively correlated genes that show a negative correlation with the score, based on the correlation between the secretion levels of proteins measured by the protein measurement means and the score calculated by the score calculation means.

[0015] In this case, it is preferable that the system further comprises a gene measurement means for measuring the expression levels of multiple genes for each of the multiple lots of cartilage cell sheets before implantation, and that the gene identification means identifies positively correlated genes that show a positive correlation with the score and / or negatively correlated genes that show a negative correlation with the score based on the correlation between the expression levels of the multiple genes measured by the gene measurement means and the score calculated by the score calculation means.

[0016] The present invention also provides a method for evaluating a chondrocyte sheet to predict the efficacy of a chondrocyte sheet used for cartilage regeneration after transplantation from the chondrocyte sheet before transplantation, the method comprising: determining the expression levels of positively correlated genes selected from the group consisting of ESM1, GREM1, SERPINA3, DKK1, MIA, NTN4, FABP3, PDGFA, COLEC12, CTSS, FTL, FTH1, GRN, CYCS, IL6R, THBS2, PRKCD, SLPI, IL12B, IL23A, MRC2, DKK4, UFC1, and SST, and / or RARRES2, APOE, PG, and the like, in the chondrocyte sheet before transplantation. The method for evaluating a cartilage cell sheet comprises: a measuring step of measuring the expression levels of negatively correlated genes selected from the group consisting of F, NACA, CXCL6, SMPDL3A, DKKL1, PRL, TEC, CCL11, IL1B, IFNGR1, CXCL12, MAP3K7, TAB1, TNFRSF11B, TIMP1, EEF1B2, EPHB4, KDR, ANGPTL4, STK16, and TNFSF12; and an estimation step of estimating the effectiveness of the cartilage cell sheet after transplantation based on the expression levels of the positively correlated genes and / or the negatively correlated genes measured in the measuring step.

[0017] In this case, the positively correlated gene is preferably selected from the group consisting of ESM1, GREM1, SERPINA3, DKK1, MIA, NTN4, FABP3, and PDGFA.

[0018] Furthermore, it is preferable that the estimation step estimates that the effectiveness of the pre-implantation chondrocyte sheet is high when the expression level of the positively correlated gene is equal to or higher than a predetermined expression level.

[0019] In this case, the negatively correlated gene is preferably selected from the group consisting of RARRES2, APOE, and PGF.

[0020] Furthermore, it is preferable that the estimation step estimates that the effectiveness of the chondrocyte sheet before implantation is high when the expression level of the negatively correlated gene is equal to or lower than a predetermined expression level.

[0021] In the above case, the measurement step preferably measures the expression level of the correlated gene by quantifying RNA transcribed from the correlated gene and / or quantifying the protein encoded by the correlated gene.

[0022] The present invention also provides a chondrocyte cell sheet evaluation device for predicting the post-transplant efficacy of a chondrocyte cell sheet used for cartilage regeneration from the chondrocyte cell sheet before transplantation, which predicts the post-transplant efficacy of a chondrocyte cell sheet used for cartilage regeneration from the chondrocyte cell sheet before transplantation, and is characterized in that the device is capable of predicting the post-transplant efficacy of a chondrocyte cell sheet used for cartilage regeneration from the chondrocyte cell sheet before transplantation by measuring the expression levels of positively correlated genes selected from the group consisting of ESM1, GREM1, SERPINA3, DKK1, MIA, NTN4, FABP3, PDGFA, COLEC12, CTSS, FTL, FTH1, GRN, CYCS, IL6R, THBS2, PRKCD, SLPI, IL12B, IL23A, MRC2, DKK4, UFC1, and SST, and / or RARRES2, APOE, P The present invention provides an apparatus for evaluating cartilage cell sheets, comprising: a measuring means for measuring the expression level of negatively correlated genes selected from the group consisting of GF, NACA, CXCL6, SMPDL3A, DKKL1, PRL, TEC, CCL11, IL1B, IFNGR1, CXCL12, MAP3K7, TAB1, TNFRSF11B, TIMP1, EEF1B2, EPHB4, KDR, ANGPTL4, STK16, and TNFSF12; and an estimation means for estimating the effectiveness of the cartilage cell sheet after transplantation based on the expression levels of the positively correlated genes and / or negatively correlated genes measured by the measuring means.

[0023] In this case, the positively correlated gene is preferably selected from the group consisting of ESM1, GREM1, SERPINA3, DKK1, MIA, NTN4, FABP3, and PDGFA.

[0024] In this case, it is preferable that the estimation means estimates that the effectiveness of the pre-implantation cartilage cell sheet is high when the expression level of the positively correlated gene is equal to or higher than a predetermined expression level.

[0025] In this case, the negatively correlated gene is preferably selected from the group consisting of RARRES2, APOE, and PGF.

[0026] In this case, it is preferable that the estimation means estimates that the effectiveness of the pre-implantation chondrocyte sheet is high when the expression level of the negatively correlated gene is equal to or lower than a predetermined expression level.

[0027] In the above case, it is preferable that the measurement means measures the expression level of the correlated gene by quantifying RNA transcribed from the correlated gene and / or by quantifying the protein encoded by the correlated gene.

[0028] The present invention also relates to a chondrocyte cell sheet used for cartilage regeneration, wherein the expression level of a positively correlated gene selected from the group consisting of ESM1, GREM1, SERPINA3, DKK1, MIA, NTN4, FABP3, PDGFA, COLEC12, CTSS, FTL, FTH1, GRN, CYCS, IL6R, THBS2, PRKCD, SLPI, IL12B, IL23A, MRC2, DKK4, UFC1, and SST in the chondrocyte cell sheet before implantation is equal to or higher than a predetermined expression level. and / or the expression level of a negatively correlated gene selected from the group consisting of RARRES2, APOE, PGF, NACA, CXCL6, SMPDL3A, DKKL1, PRL, TEC, CCL11, IL1B, IFNGR1, CXCL12, MAP3K7, TAB1, TNFRSF11B, TIMP1, EEF1B2, EPHB4, KDR, ANGPTL4, STK16, and TNFSF12 is below a predetermined expression level.

[0029] Furthermore, the present invention relates to a method for producing a chondrocyte sheet used for cartilage regeneration, the method comprising the steps of: (a) producing a chondrocyte sheet using a chondrocyte culture medium containing 1000 cells / ml of 10 ... and / or RARRES2, APOE, PGF, NACA, CXCL6, SMPDL3A, DKKL1, PRL, TEC, CCL11, IL1B, IFNGR1, CXCL12, MAP3K7, TAB1, TNFRSF11B, TIMP1, EEF1B2, EPHB4, KDR, ANGPTL4, STK16, and TNFSF12, is cultured so that the expression level of a negatively correlated gene selected from the group consisting of RARRES2, APOE, PGF, NACA, CXCL6, SMPDL3A, DKKL1, PRL, TEC, CCL11, IL1B, IFNGR1, CXCL12, MAP3K7, TAB1, TNFRSF11B, TIMP1, EEF1B2, EPHB4, KDR, ANGPTL4, STK16, and TNFSF12 is below a predetermined expression level.

[0030] Furthermore, the present invention relates to a method for regenerating cartilage using a chondrocyte cell sheet, wherein the expression level of a positively correlated gene selected from the group consisting of ESM1, GREM1, SERPINA3, DKK1, MIA, NTN4, FABP3, PDGFA, COLEC12, CTSS, FTL, FTH1, GRN, CYCS, IL6R, THBS2, PRKCD, SLPI, IL12B, IL23A, MRC2, DKK4, UFC1 and SST is equal to or higher than a predetermined expression level, and / or the expression level of a positively correlated gene selected from the group consisting of RARRES2, A A cartilage regeneration method characterized by comprising the steps of: preparing a chondrocyte sheet in which the expression level of a negatively correlated gene selected from the group consisting of POE, PGF, NACA, CXCL6, SMPDL3A, DKKL1, PRL, TEC, CCL11, IL1B, IFNGR1, CXCL12, MAP3K7, TAB1, TNFRSF11B, TIMP1, EEF1B2, EPHB4, KDR, ANGPTL4, STK16, and TNFSF12 is below a predetermined expression level; and transplanting the chondrocyte sheet into a patient. [Effects of the Invention]

[0031] According to the present invention, it is possible to provide a method and device for searching for marker genes of cartilage cell sheets, which are used to search for marker genes associated with the post-transplant effectiveness of a cartilage cell sheet used for cartilage regeneration from a cartilage cell sheet before transplantation.Furthermore, according to the present invention, it is possible to provide a method and device for evaluating cartilage cell sheets, which are used to predict the post-transplant effectiveness of a cartilage cell sheet used for cartilage regeneration from a cartilage cell sheet before transplantation.Furthermore, according to the present invention, it is possible to provide a cartilage cell sheet that is highly effective after transplantation for use in cartilage regeneration, and a method for producing the same.Furthermore, according to the present invention, it is possible to provide a cartilage regeneration method using a cartilage cell sheet that is highly effective after transplantation for use in cartilage regeneration. [Brief explanation of the drawings]

[0032] [Figure 1] FIG. 1 is a schematic diagram showing an overview of a device for searching for marker genes of cartilage cell sheets and a device for evaluating cartilage cell sheets. [Figure 2] Comparison of 12 types of polydactyly patient-derived cartilage sheets (PD sheets) for their efficacy in hyaline cartilage regeneration in a rabbit xenograft model. (a) Study overview. (b) Representative images of rabbit knee cartilage defects treated with or without cell sheet transplantation using histological staining with Safranin O and immunohistochemical staining for COL2, COL1, and vimentin. A: No sheet transplantation; B: Poor cartilage repair; C: Good cartilage repair. Scale bar = 1 mm. (c) ICRS score of PD sheets. Each circle represents the average ICRS score of rabbits within a group. D represents the group without PD sheet transplantation. Numbers indicate each lot of PD sheets. *p<0.05. (d) Distribution of PD sheet characteristics. Cell sheets were dispersed by enzymatic digestion, and the cell number and viability of the cell suspension were measured. [Figure 3] Heatmap and hierarchical clustering of gene expression associated with efficacy. PD sheets showed different gene expression profiles depending on in vivo efficacy. Red indicates high expression, green indicates low expression. [Figure 4] Meta-enrichment analysis of drug efficacy-related genes. (a) Heat map of the top 20 enriched clusters based on the positively and negatively correlated gene list. Colors indicate statistical significance, with gray indicating no significance. (b) Visualization of the enrichment network. Cluster annotations are displayed in different colors. Node size indicates the number of genes included in the ontology term or pathway. (c) The statistical significance color scale was applied to the same cluster network. [Figure 5] Potential efficacy-related markers of PD sheets. The scatter plot shows a weak correlation between gene expression levels in the PD sheets and ICRS scores (n=12). The horizontal axis shows the gene expression level at the delta Ct value, which is the Ct value of the target gene subtracted from the Ct value of ACTB as the reference gene. [Figure 6] Analysis of proteins secreted by PD sheets. (a) Meta-enrichment analysis of selected proteins. (b) Venn diagram of genes selected from microarray and SOMA scan assays. (c) List of genes showing positive correlation with both mRNA expression (GE) and protein secretion (SOMA). (d) Scatter plot showing a weak correlation between protein concentration determined by ELISA for PD sheet supernatants and ICRS score. DETAILED DESCRIPTION OF THE INVENTION

[0033] Below, the method for searching for marker genes in cartilage cell sheets and the marker gene searching method of the present invention are described. Also below, the method for evaluating cartilage cell sheets, the device for evaluating cartilage cell sheets, cartilage cell sheets, methods for producing cartilage cell sheets, and methods for cartilage regeneration using cartilage cell sheets are described. Note that, although the following embodiments and examples use PD sheets (sheets derived from patients with polydactyly), which are a type of cartilage cell sheet, the cartilage cell sheets are not limited to PD sheets.

[0034] 1. Method for searching for marker genes for cartilage cell sheets The present invention provides a method for screening for marker genes in cartilage cell sheets, for screening marker genes associated with the post-transplant effectiveness of cartilage cell sheets used for cartilage regeneration from cartilage cell sheets before transplantation. The method for screening for marker genes in cartilage cell sheets of the present invention comprises a sheet preparation step of preparing multiple lots of cartilage cell sheets before transplantation, a protein measurement step of measuring the secretion levels of multiple proteins for each of the multiple lots of cartilage cell sheets before transplantation, a score calculation step of transplanting each of the multiple lots of cartilage cell sheets into an organism model of osteochondral defect and calculating a score of cartilage regeneration effectiveness for each cartilage cell sheet, and a gene identification step of identifying positively correlated genes and / or negatively correlated genes that show a positive correlation with the score, based on the correlation between the secretion levels of proteins measured in the protein measurement step and the score calculated in the score calculation step.

[0035] 1.1.Sheet preparation process In the sheet preparation step, multiple lots of cartilage cell sheets are prepared before transplantation. PF sheets, an example of a cartilage cell sheet, can be obtained by culturing cartilage tissue obtained from a polydactyly patient and forming it into a sheet. In this embodiment, multiple lots of cartilage cell sheets are obtained using PD sheets derived from multiple polydactyly patients.

[0036] 1.2.Protein measurement process In the protein measurement step, the amount of protein secreted from the chondrocyte sheet before transplantation is measured. In this embodiment, the proteins secreted by culturing the chondrocyte sheet and contained in the supernatant of the culture medium are quantified by a multiple adaptamer assay. An example of a multiple adaptamer assay is an assay using the SOMAscan assay platform.

[0037] 1.3. Gene measurement process In the gene measurement process, the expression levels of genes in the chondrocyte sheet before implantation are measured. Specifically, total RNA from the chondrocyte sheet is purified, amplified, and quantified. After labeling the RNA with cyanine 3 or similar, it is reverse transcribed into double-stranded cDNA to generate fluorescent cRNA, which is then hybridized using a microarray, allowing the genes expressed in the chondrocyte sheet to be identified and quantified.

[0038] 1.4.Score calculation process In the score calculation step, each of the multiple lots of chondrocyte sheets is transplanted into an organism modeling an osteochondral defect. Rabbits and other animals can be used as the osteochondral defect model. Two to six weeks after chondrocyte sheet transplantation, the transplanted area is harvested from the organism modeling an osteochondral defect, and the regenerated cartilage is histologically scored. Here, "efficacy" refers to the regeneration of cartilage by the chondrocyte sheet and repair of the cartilage defect, and "efficacy score" refers to a specific numerical value indicating the extent to which the cartilage has regenerated. Examples of efficacy scores include the International Cartilage Repair Society (ICRS) score, O'Driscoll score, and Wakitani score. For example, in the Examples described below, the ICRS score of the untreated group after four weeks was approximately 19 to 25; if the ICRS score is higher than this by a predetermined standard value (e.g., 10), it can be determined that the treatment is effective.

[0039] 1.5. Gene identification process In the gene identification step, the correlation between the amount of protein secretion (expression level) measured in the protein measurement step and the score calculated in the score calculation step is analyzed. Specifically, the Pearson correlation coefficient is calculated between the amount of protein secretion and the score, and genes with a predetermined value (e.g., 0.4) or more are identified as genes showing a positive correlation, and genes with a value less than the predetermined value (e.g., 0.4) are identified as genes showing a negative correlation.

[0040] In this gene identification step, positively correlated genes that exhibit a positive correlation with the score and / or negatively correlated genes that exhibit a negative correlation with the score may be identified based on the correlation between the expression levels of multiple genes measured in the gene measurement step and the score calculated in the score calculation step. In this case, the Pearson correlation coefficient is calculated between the gene expression levels and the score, and genes with a predetermined value (e.g., 0.4) or more are identified as genes exhibiting a positive correlation, and genes with a value less than the predetermined value (e.g., 0.4) are identified as genes exhibiting a negative correlation.

[0041] Furthermore, in the gene identification step, genes that are common to both the genes that show a correlation between the amount of protein secreted and the score and the genes that show a correlation between the gene expression level and the score may be identified as genes that show a positive correlation and / or genes that show a negative correlation. In this way, by identifying correlated genes based on both the amount of protein secreted in the chondrocyte cell sheet and the expression levels of genes expressed in the chondrocyte cell sheet, effective genes can be identified more reliably.

[0042] 2. Evaluation method of cartilage cell sheets The method for evaluating cartilage cell sheets of the present invention is a method for evaluating cartilage cell sheets for predicting the post-transplant effectiveness of a cartilage cell sheet used for cartilage regeneration from the cartilage cell sheet before transplantation, and comprises: (1) a measurement step of measuring the expression levels of positively correlated genes and / or negatively correlated genes; and (2) an estimation step of estimating the post-transplant effectiveness of the cartilage cell sheet based on the expression levels of positively correlated genes and / or negatively correlated genes measured in the measurement step.

[0043] Here, the positively correlated genes were ESM1 (endothelial cell-specific molecule 1), GREM1 (gremlin 1, DAN family BMP antagonist), SERPINA3 (serpin family A member 3), DKK1 (Dick-Kopf WNT signaling pathway inhibitor 1), MIA (melanoma inhibitory activity), NTN4 (netrin 4), FABP3 (fatty acid binding protein 3), PDGFA (platelet-derived growth factor A subunit), COLEC12 (collectin subfamily member 12), CTSS (cathepsin S), FTL (ferritin light chain), and FTH1 (ferritin heavy chain 1). The gene is selected from the group consisting of GRN (granulin precursor), CYCS (cytochrome c, somatic), IL6R (interleukin 6 receptor), THBS2 (thrombospondin 2), PRKCD (protein kinase C delta), SLPI (secretory leukocyte peptidase inhibitor), IL12B (interleukin 12B), IL23A (interleukin 23 subunit alpha), MRC2 (mannose receptor type C 2), DKK4 (Dick-Kopf WNT signaling pathway inhibitor 4), UFC1 (ubiquitin-modifying enzyme-conjugating enzyme 1), and SST (somatostatin).

[0044] Among these, the positively correlated genes are preferably genes selected from the group consisting of ESM1, GREM1, SERPINA3, DKK1, MIA, NTN4, FABP3, and PDGFA. As shown in the Examples below, these genes show a positive correlation between the efficacy score and both proteins secreted from chondrocyte cells and genes expressed in chondrocyte cells.

[0045] Here, the negatively correlated genes were RARRES2 (retinoic acid receptor responder 2), APOE (apolipoprotein E), PGF (placental growth factor), NACA (nascent polypeptide-associated complex alpha subunit), CXCL6 (C-X-C motif chemokine ligand 6), SMPDL3A (sphingomyelin phosphodiesterase acid-like 3A), DKKL1 (Dick-Kopf-like acrosome protein 1), PRL (prolactin), TEC (Tech protein tyrosine kinase), CCL11 (C-C motif chemokine ligand 11), IL1B (interleukin-1 beta), IFNGR1 (interferon gamma receptor 1), CXCL12 ( The gene is selected from the group consisting of C-X-C motif chemokine ligand 12), MAP3K7 (mitogen-activated protein kinase kinase kinase 7), TAB1 (TGF-β-activated kinase 1 (MAP3K7) binding protein 1), TNFRSF11B (TNF receptor superfamily member 11b), TIMP1 (TIMP metallopeptidase inhibitor 1), EEF1B2 (nucleic acid translation elongation factor 1 beta 2), EPHB4 (EPH receptor B4), KDR (kinase insert domain receptor), ANGPTL4 (angiopoietin-like 4), STK16 (serine / threonine kinase 16), and TNFSF12 (TNF superfamily member 12).

[0046] Of these, the positively correlated genes are preferably genes selected from the group consisting of RARRES2 (retinoic acid receptor responder 2), APOE (apolipoprotein E), and PGF (placental growth factor). As shown in the Examples below, these genes show a negative correlation between efficacy scores for both proteins secreted from chondrocyte sheets and genes expressed in chondrocyte sheets.

[0047] 3.1.Measurement process The measurement step is a step of measuring the expression level of a gene expressed in a chondrocyte cell sheet before transplantation. As explained above in "1.2. Protein measurement step," the gene expression level can be quantified from the amount of protein secreted by the gene in the chondrocyte cell sheet before transplantation. Furthermore, as explained above in "1.3. Gene measurement step," the gene expression level can be quantified from the amount of RNA transcribed from the gene (DNA) in the chondrocyte cell sheet before transplantation.

[0048] 3.2. Estimation process In the estimation step, the effectiveness of the chondrocyte cell sheet after transplantation is estimated based on the gene expression levels measured in the measurement step. Here, the expression levels of genes judged to be effective vary depending on the type of gene, and are therefore determined relatively by comparing effective chondrocyte cell sheets with ineffective or low-effective chondrocyte cell sheets. Since the ICRS score of the untreated group after 4 weeks, as explained in "1. Method for searching for marker genes for chondrocyte cell sheets" above, is approximately 19 to 25 (average 22), chondrocyte cell sheets with an ICRS score 10 points higher than this, i.e., an ICRS score of less than 32, can be determined to be ineffective or low-effective chondrocyte cell sheets.

[0049] Positively correlated genes will be explained below. When the expression level of a positively correlated gene in a chondrocyte cell sheet is equal to or greater than the expression level of a gene that increases the ICRS score by a predetermined standard value (e.g., 10) compared to the expression level of a gene in an ineffective or low-effective chondrocyte cell sheet, the chondrocyte cell sheet can be determined to be effective. Conversely, when the expression level of a positively correlated gene is less than the expression level of this predetermined standard value, the chondrocyte cell sheet can be determined to be ineffective.

[0050] This predetermined reference value is preferably 10 under the conditions of the Examples described below, but can be set appropriately depending on the evaluation conditions of the cartilage cell sheet, and may be, for example, 20, 15, 5, 4, 3, 2, etc., other than 10. This reference value may be the same or different for the gene expressed in the cartilage cell sheet and the amount of protein secreted from the cartilage cell sheet. An example in which the predetermined reference value is 10 will be described below.

[0051] For representative positively correlated genes, the table below lists the gene expression levels associated with a 10-fold increase in ICRS score. For example, in the case of ITGA10, a 19.1-fold increase in expression level results in a 10-fold increase in ICRS score, indicating post-transplant efficacy. In the case of ACAN, a 28.3-fold increase in expression level results in a 10-fold increase in ICRS score. Similarly, for individual genes, the increase is 6.0-fold for NGEF, 3.4-fold for C15orf52, and 3.9-fold for HOXA3. The expression level ratio associated with a 10-fold increase in ICRS score was calculated by performing a regression analysis in the correlation analysis between gene expression values ​​and ICRS and applying the least squares method to derive the relationship between the expression level ratio and the ICRS score from the slope obtained. The same applies to the expression level ratios of secreted proteins, described below. The regression line formula for the results of the regression analysis is shown in the table below. Note that the gene expression level is calculated as a doubling of the amount for every 1-fold increase in acquired data (signal intensity). The absolute value of the expression level can be calculated from this regression line and the ICRS score, so it is possible to determine that the effectiveness of a cartilage cell sheet is high, for example, when the expression level is such that the ICRS score is 32 or higher.

[0052] [Table 1]

[0053] Furthermore, for representative positively correlated genes, the table below lists the expression levels (secretion levels) of secreted proteins that increase the ICRS score by 10. As a ratio of the secretion level of proteins secreted from chondrocyte sheets, a 6.5-fold increase in ESM1 increases the ICRS score by 10. Similarly, a 3.4-fold increase in GREM1 increases the ICRS score by 10. Similarly, SERPINA3 increases by 9.8-fold, DKK1 by 9.1-fold, MIA by 14.0-fold, NTN4 by 7.0-fold, PDGFA by 3.1-fold, and FABP3 by 135-fold.

[0054] [Table 2]

[0055] Next, representative negatively correlated genes will be described. When the expression level of a negatively correlated gene in a chondrocyte cell sheet is compared with the expression level of a gene in an ineffective or low-effective chondrocyte cell sheet and is equal to or lower than the expression level of a gene that reduces the ICRS score by a predetermined reference value (e.g., 10), the chondrocyte cell sheet can be determined to be effective. Conversely, when the expression level of a negatively correlated gene is equal to or higher than the expression level of the gene that corresponds to this predetermined reference value, the chondrocyte cell sheet can be determined to be ineffective.

[0056] This predetermined reference value is preferably 10 under the conditions of the Examples described below, but can be set appropriately depending on the evaluation conditions of the cartilage cell sheet, and may be, for example, 20, 15, 5, 4, 3, 2, etc., other than 10. This reference value may be the same or different for the gene expressed in the cartilage cell sheet and the amount of protein secreted from the cartilage cell sheet. An example in which the predetermined reference value is 10 will be described below.

[0057] For example, the table below lists the expression levels of representative negatively correlated genes that result in a 10-point decrease in ICRS score. In the case of BRD9, a 3.6-fold increase in gene expression results in a 10-point decrease in the ICRS score, which indicates post-transplant efficacy. In the case of TUSC1, a 6.5-fold increase in expression results in a 10-point decrease in the ICRS score, which indicates post-transplant efficacy. Similarly, SPRY1 is 6.2-fold, C1orf229 is 5.1-fold, and HAGHL is 7.0-fold.

[0058] [Table 3]

[0059] Additionally, for negatively correlated genes, the table below shows the expression levels (secretion amounts) of secreted proteins that result in a 10-point decrease in the ICRS score. As a ratio of the amount of protein secreted from the chondrocyte sheet, a 13.1-fold increase in RARRES2 results in a 10-point decrease in the ICRS score. Similarly, the increase is 4.8-fold for APOE and 18.6-fold for PGF.

[0060] [Table 4]

[0061] 3. Cartilage cell sheet marker gene detection device and cartilage cell sheet evaluation device The marker gene screening device and cartilage cell sheet evaluation device of the present invention are devices for carrying out the above-mentioned marker gene screening method and cartilage cell sheet evaluation method, respectively. FIG. 1 is a schematic diagram showing an overview of the marker gene screening device and cartilage cell sheet evaluation device. First, the above-mentioned cartilage cell sheet is prepared (sheet preparation means). The cartilage cell sheet is then analyzed using a PCR panel or the like to measure gene expression levels (gene measurement means: measurement means). The cartilage cell sheet is then subjected to protein expression analysis to measure protein secretion levels (protein measurement means: measurement means). The analyzed data is stored in an analysis and digitization execution database. Note that the analysis and digitization execution database may also store data analyzed using public databases.

[0062] Meanwhile, the system is equipped with an integrated information analysis system (score calculation means) that transplants a cartilage cell sheet into this cartilage defect model organism, analyzes the cartilage regeneration status, and calculates a score for the effectiveness of cartilage regeneration, as well as a cell information database that stores this information. The cell information data stored in the cell information database is analyzed by the integrated information analysis system along with data from the analysis and quantification execution database. As with the above-mentioned "gene identification process," this integrated information analysis system uses a program algorithm (gene identification means) to analyze the correlation between protein secretion amount (expression level) and effectiveness score, identifying genes that show a correlation with effectiveness. Genes can also be identified by analyzing the correlation between gene expression level and effectiveness score. Furthermore, it is possible to identify genes that are common to both protein secretion amount and gene expression level.

[0063] Furthermore, the integrated information analysis system (estimation means) can predict the effectiveness of markers based on the expression levels of positively correlated genes and negatively correlated genes. That is, chondrocyte cells sheets whose expression levels of positively correlated genes stored in the analysis and quantification execution database are equal to or greater than a predetermined value are evaluated as effective, and chondrocyte cells sheets whose expression levels of negatively correlated genes are equal to or greater than a predetermined value are evaluated as ineffective. Alternatively, chondrocyte cells sheets whose expression levels of positively correlated genes are less than a predetermined value may be evaluated as ineffective, and chondrocyte cells sheets whose expression levels of negatively correlated genes are less than a predetermined value may be evaluated as effective. The evaluation results can be output as a cell function and quality evaluation report.

[0064] 4. Cartilage cell sheet and its manufacturing method Next, the cartilage cell sheet of the present invention and its production method will be described. In the cartilage cell sheet of the present invention, the expression level of positively correlated genes in the cartilage cell sheet before transplantation is equal to or higher than a predetermined expression level, and / or the expression level of negatively correlated genes is equal to or lower than a predetermined expression level. Here, the positively correlated genes and negatively correlated genes are the genes listed above in "2. Method for evaluating cartilage cell sheets."

[0065] Furthermore, the method for producing a cartilage cell sheet of the present invention is a method for culturing a cultured cartilage sheet so that the expression levels of positively correlated genes in the cartilage cell sheet before transplantation are equal to or higher than a predetermined expression level and / or so that the expression levels of negatively correlated genes are equal to or lower than a predetermined expression level. Here, the positively correlated genes and negatively correlated genes are the genes listed in "2. Method for evaluating cartilage cell sheets" above.

[0066] The method for producing a cartilage cell sheet involves first isolating cartilage tissue from the multiple fingers of a patient with juvenile polydactyly by digesting it in a prepared medium to isolate chondrocytes, which are then placed on a culture dish and cultured. Examples of prepared medium include DMEM / F12 (Dulbecco's modified Eagle's medium-nutrient mixture F-12). The cultured chondrocytes are then seeded on culture inserts and cultured to produce a cartilage culture sheet.

[0067] In the present invention, the above culture is carried out under conditions such that the expression level of positively correlated genes in the chondrocyte cell sheet before transplantation is equal to or higher than a predetermined expression level. Such conditions may include culturing with the addition of a factor (e.g., a compound) that promotes the expression of positively correlated genes. Furthermore, in the present invention, the above culture is carried out under conditions such that the expression level of negatively correlated genes in the chondrocyte cell sheet before transplantation is equal to or lower than a predetermined expression level. Such conditions may include culturing with the addition of a factor (e.g., a compound) that reduces the expression of negatively correlated genes.

[0068] 5. Cartilage regeneration method using cartilage cell sheets The present invention provides a method comprising the steps of preparing a chondrocyte cell sheet in which the expression levels of positively correlated genes are equal to or higher than a predetermined expression level and / or the expression levels of negatively correlated genes are equal to or lower than a predetermined expression level before transplantation, and transplanting the chondrocyte cell sheet into a patient. Here, the positively correlated genes and negatively correlated genes are the genes listed in "2. Method for evaluating chondrocyte cell sheets" above.

[0069] In the cartilage regeneration method of the present invention, a chondrocyte sheet that is highly effective after transplantation is transplanted into a patient, and therefore, cartilage regeneration efficiency is high and the prognosis after transplantation of the chondrocyte sheet is good. [Example]

[0070] The present invention will be described in detail below with reference to examples, but the object of the present invention is not limited to these examples. In the following examples, "%" is based on mass (mass percent) unless otherwise specified.

[0071] 1. Materials and Methods 1.1. Ethics statement Experiments were conducted under the approval and guidance of the Tokai University School of Medicine Clinical Research Review Board. Informed consent was obtained from the donor's guardian in all cases. Some surgical specimens were irreversibly deidentified. All experiments involving human cells and tissues were conducted in accordance with the tenets of the Declaration of Helsinki. Animal experiments were conducted with the approval of the Tokai University Institutional Animal Experimentation Committee, and animal handling and care were carried out in accordance with the "Facility Regulations for Animal Experiments" and the "Basic Guidelines for the Proper Conduct of Animal Experiments, etc. at Academic Research Institutions," both of which are administered by the Ministry of Education, Culture, Sports, Science and Technology.

[0072] 1.2. Preparation of PD sheets 1.2.1. Preparation of chondrocytes Chondrocytes were isolated according to the preparation procedure described above (Non-Patent Document 10). Briefly, cartilage tissue obtained from juvenile polydactyly patients (11 patients, ages 8-23 months; 5 girls, 4 boys, and 2 unidentified donors) was digested with 5 mg / ml CLS1 (collagenase type I) (Worthington Biochemical Corp., Lakewood, NJ, USA) in a conditioned medium (DMEM / F12 (Dulbecco's modified Eagle's medium-nutrient mixture F-12), Gibco, Waltham, MA, USA) supplemented with 20% fetal bovine serum and 1% antibiotic-antimycotic solution (Gibco, Waltham, MA, USA) at 37°C for 2-3 hours with agitation in a humidified atmosphere of 5% CO2 and 95% air. The isolated chondrocytes were washed with the conditioned medium and seeded onto culture dishes. After the cells attached to the culture dish and began to proliferate, the medium was replaced with a conditioned medium containing 100 μg / mL ascorbic acid (Nissin Pharmaceuticals, Yamagata, Japan). In two cases, the tissue was cut into 1 mm pieces. 3 The cells were minced to the following sizes, placed on a culture dish in the prepared medium, and cultured until the cells were incomplete. Chondrocytes were collected using TrypLE Express (Thermo Fisher Scientific, Tokyo, Japan) and stored in a STEM-CELLBANKER TM (Zenoac, Fukushima, Japan) or TC-Protector Cell Freezing Medium (KAC, Kyoto, Japan). Some donors were passaged up to five times before cryopreservation.

[0073] 1.2.2. Preparation of PD sheet Twelve batches of cryopreserved chondrocytes were thawed, passaged once in culture medium, and cultured at 1 × 10 cells per 100 ml in a temperature-responsive culture insert (CellSeed Co., Ltd., Tokyo, Japan). 4 cells / cm 2 The cells were seeded at 1000 kJ / well and cultured for two weeks. The resulting PD sheets were divided into four groups and used for efficacy evaluation, characterization, gene expression analysis, and protein secretion analysis.

[0074] 1.2.3.Characteristics evaluation of PD sheets To determine cell number, PD sheets were digested with TrypLE Express (Thermo Fisher Scientific) for 30 minutes at 37°C and then incubated with 0.25 mg / mL Collagenase-P (Roche, Basel, Switzerland) for 30 minutes at 37°C. Dispersed cells were washed with culture medium, and cell number and viability were determined by trypan blue dye exclusion assay. To determine the thickness of the PD sheets, each PD sheet was embedded in Tissue-Tek OCT compound (Sakura Fine Tech, Tokyo, Japan) and frozen at -80°C. 10 μm-thick cross sections of the PD sheets were cut, mounted on glass slides, air-dried, and fixed in 4% paraformaldehyde in 0.01 M phosphate buffer for 30 minutes at room temperature. The sections were stained with hematoxylin and eosin (H&E) according to standard protocols. Microscopic images were taken, and the distance between pairs of dots was measured using a BZ-8000 microscope (Keyence, Osaka, Japan).

[0075] 1.3.Evaluation of the effectiveness of PD sheets for osteochondral defects 1.3.1. Immunosuppressed rabbit osteochondral defect model The efficacy of PD sheets in rabbits was evaluated as described in Non-Patent Document 11. A total of 69 female Japanese white rabbits (average body weight = 3.0 kg; Tokyo Experimental Animal Science Co., Ltd.) were used. Before surgery, rabbits were randomly assigned based on body weight to either a defect-only group or a PD sheet implantation group. For immunosuppression, tacrolimus (1.6 mg / kg / day) was administered intramuscularly daily for 10 days starting 2 days before implantation, and then every other day for up to 4 weeks after surgery. Before surgery and implantation, rabbits were anesthetized with 2 L / min of nitrous oxide, 1 L / min of oxygen, and 2.5–3.0% isoflurane (Pfizer, New York, NY, USA). Using a 5 mm biopsy punch (Kai Kogyo, Gifu Prefecture, Japan) as a marking guide, an osteochondral defect (diameter = 5 mm, depth = 3 mm) was created in the patellar groove of the femur using a 5 mm drill. Slight bleeding from the subchondral bone was confirmed, and the defect was washed with saline (Nipro, Osaka, Japan) to prevent thermal damage.

[0076] 1.3.2. Transplantation of PD sheets For PD sheet transplantation, the culture plate containing the PD sheet was kept at 25°C for 30 minutes to allow for detachment. One PD sheet per knee was transplanted into the osteochondral defect using a polyvinylidene difluoride membrane. After repair of the patella, the quadriceps muscle and tendon were sutured to prevent dislocation.

[0077] 1.3.3. Histological scoring of regenerated cartilage Four weeks after PD sheet implantation, rabbits were euthanized by intravenous administration of 50 mg / ml pentobarbital (Tokyo Chemical Industry Co., Ltd., Tokyo, Japan). The operated femurs were harvested and fixed in 20% formalin (Wako Pure Chemical Industries, Ltd.) for 3–5 days. The specimens were decalcified in 10% EDTA (ethylenediaminetetraacetic acid) (Wako Pure Chemical Industries, Ltd.) for 3–4 weeks and embedded in paraffin. Serial sections (3 μm) were cut parallel to the long axis of the femur near the center of the defect. For histological examination, sections were stained with hematoxylin and eosin (H&E), safranin O, and fast green according to standard protocols. Stained sections were randomized and independently scored by two trained orthopedic surgeons using a modified version of the O'Driscoll score and the International Cartilage Repair Society (ICRS) score [44,45].

[0078] 1.3.4. Immunostaining For immunostaining of type I collagen (COL1) and type II collagen (COL2), deparaffinized 3-μm sections were treated with 0.4% pepsin (Agilent Technologies, Santa Clara, CA, USA) for 30 minutes at 37°C to retrieve antigens. Next, sections were incubated with 0.4% pepsin (DAKO, Glostrup, Denmark) for 30 minutes at 37°C, washed with distilled water, and then incubated with 0.3% hydrogen peroxide / methanol solution for 30 minutes at room temperature (RT) to block endogenous peroxidase activity. Next, sections were washed with phosphate-buffered saline (PBS), blocked with 2.5% normal goat serum (NGS) for 10 minutes at room temperature (RT), and incubated with either human COL1 or human COL2 mouse monoclonal antibody (Kyowa Pharmaceutical Co., Ltd., Toyama, Japan) diluted 1:100 in 1% bovine serum albumin (Sigma-Aldrich) in PBS for 3 hours at room temperature (RT). Finally, the stained sections were washed with PBS, then incubated with ImmPRESS reagent anti-mouse Ig (Vector Laboratories, Burlingame, CA, USA) at RT, immersed in Tris-HCl buffer (pH 7.6) containing 0.02% diaminobenzidine and 0.005% hydrogen peroxide for 2–8 min, and counterstained with H&E.

[0079] For human vimentin detection, deparaffinized sections were treated with 10 mM sodium citrate buffer (pH 6.0) in a microwave oven at 98°C for 10 minutes for antigen retrieval. The sections were then incubated in PBS containing 5% NGS for blocking, followed by overnight incubation at 4°C with Alexa Fluor 647-conjugated rabbit monoclonal antibody (Cell Signaling Technology, Danvers, MA, USA) diluted 1:100 with 1% bovine serum albumin in PBS to detect human vimentin. The sections were washed with distilled water, then mounted and cured with 4',6-diamino-2-phenylindole (Vector Laboratories) according to the manufacturer's instructions. All microscopic images were obtained using a BZ-9000 Generation II fluorescence microscope (Keyence Corporation).

[0080] 1.4.Gene expression analysis of PD sheets RNA isolation PD sheets were disrupted in TRIzol Reagent (Life Technologies) using a SHAKE Master Neo (Bio Medical Science, Japan), and total RNA was further purified using the Qiagen RNeasy Mini Kit (QIAGEN, Valencia, CA, USA) according to the manufacturer's instructions. RNA quantity and quality were determined using a NanoDropOne spectrophotometer (Thermo Fisher Scientific) and an Agilent Bioanalyzer (Agilent Technologies) as recommended.

[0081] 1.4.2.cRNA Amplification and Labeling Total RNA was amplified and labeled with cyanine 3 (Cy3) using the Agilent Low Input Quick Amp Labeling Kit, one-color (Agilent Technologies) according to the manufacturer's instructions. Briefly, total RNA was reverse transcribed into double-stranded cDNA using a poly(dT)-T7 promoter primer. Primers, template RNA, and quality control transcripts of known concentration and quality were first denatured at 65°C for 10 minutes and then incubated at 40°C for 2 hours with 5X First-Strand Buffer, 0.1 M dithiothreitol, 10 mM deoxyribonucleotide triphosphate mix, and AffinityScript RNase Block Mix. After inactivating the AffinityScript enzyme at 70°C for 15 minutes, the cDNA product was used as a template for in vitro transcription to generate fluorescent cRNA. The cDNA product was mixed with a transcription master mix in the presence of T7 RNA polymerase and Cy3-labeled CTP and incubated at 40°C for 2 hours. The labeled cRNA was purified using QIAGEN RNeasy mini spin columns and eluted in 30 μL of nucleose-free water. After amplification and labeling, the amount of cRNA and the degree of cyanine incorporation were determined using a Nanodrop ND-1000 spectrophotometer and an Agilent Bioanalyzer.

[0082] 1.4.3. Sample Hybridization For each hybridization, 0.60 μg of Cy3-labeled cRNA was fragmented using an Agilent SurePrint G3 Human GE v3 8x60K microarray (design ID: 072363) and hybridized for 17 hours at 65°C. After washing, the microarray was scanned using an Agilent DNA microarray scanner.

[0083] 1.4.4. Identification of efficacy-correlated genes The intensity of each scanned feature was quantified using Agilent's feature extraction software, version 11.5.1.1.1. Only features flagged as error-free ("Detected" flag) were used, while features flagged as not positive, not significant, not uniform, not above background, saturated, or population outliers ("Not Detected" and "Compromised" flags) were excluded. Quantile normalization was performed using Agilent GeneSpring software, version 14.9.1. After quantile normalization of the raw signal data, probes were filtered based on existing gene symbol annotations and the mean signal intensity across all samples (with a coveted log2 intensity of 5 or greater). Marker genes were extracted by calculating the Pearson correlation coefficient between the expression level of each probe and its ICRS score, and probes with an ICRS score greater than or equal to 0.4 or less were selected. Ultimately, 443 genes were identified using these criteria, with high variance and a log2 intensity of 0.5 or greater across all samples. Hierarchical clustering analysis was performed using Agilent GeneSpring software version 14.9.1 (similarity measure: Euclidean, linkage rule: average).

[0084] 1.4.5. Enrichment Analysis Genes associated with efficacy were analyzed using the online tool Metascape (http: / / metascape.or. / )

[12] . Functional enrichment was performed on the default settings. Multiple input gene lists, including positively and negatively correlated genes, were analyzed.

[0085] 1.4.6. RT-PCR (Reverse Transcriptase Polymerase Chain Reaction) Total RNA from the PD sheets was converted to cDNA using the QuantiTect Reverse Transcription Kit (Qiagen). The cDNA was preamplified using the TaqMan PreAmp Master Mix Kit (Applied Biosystems). TaqMan Gene Expression Assays (Applied Biosystems) containing fluorescent probes and forward and reverse primers (see table below) were diluted with Tris and EDTA (TE) buffer (1x) according to the manufacturer's instructions to obtain a pooled assay mix with a final concentration of 0.2x. 25 μL of TaqMan PreAmp Master Mix (2x), 12.5 μL of pooled assay mix (0.2x), 1 μL of cDNA sample, and nucleotase-free water to adjust the total to 50 μL. The reaction was performed with this 50 μL mixture. Preamplification PCR was performed using a GeneAmp PCR System 9700 (Applied Biosystems) at 95°C for 10 minutes, followed by 14 15-s cycles at 95°C and 60°C for 4 minutes. The preamplification cDNA product was diluted 1:20 in TE buffer (1x) for use as a template for real-time RT-PCR analysis. TaqMan real-time PCR was performed using a 7500 Real-Time PCR System (Applied Biosystems). The PCR reaction mixture consisted of 10 μL of TaqMan Gene Expression Master Mix (2x), 1 μL of TaqMan Gene Expression Assay (20x), 5 μL of preamplification cDNA, and nucleotase-free water to adjust the volume to 20 μL. The 7500 Real-Time PCR System was run for amplification at 50°C for 2 minutes, 95°C for 10 minutes, followed by 40 15-s cycles at 95°C and 60°C for 1 minute. Cycle threshold (Ct) values ​​were determined by 7500 Real-Time PCR System Software v.2.0.6 (Thermo Fisher Scientific).The relative expression value (ΔCt value) of each gene was derived relative to the Ct of the internal control ACTB.

[0086] [Table 5]

[0087] 1.5.Analysis of proteins secreted by PD sheets 1.5.1. Preparation of PD sheet supernatant Each PD sheet was cultured for 72 hours in 3 mL of DMEM / F12 (Gibco) supplemented with 1% fetal bovine serum and 1× antibiotic-antimycotic solution (Gibco). The supernatant was collected, centrifuged at 13,000 g for 10 minutes to remove debris, divided into aliquots, and stored at −80°C until use.

[0088] 1.5.2.Proteomics analysis using SOMAscan Supernatants were analyzed using the SOMAscan assay platform (SomaLogic), a multiplexed aptamer-based assay that detects 1,129 proteins using slow-off-rate modified aptamers (SOMAmers)

[13] . This assay uses chemically modified nucleotides to convert protein signals into nucleotide signals that can be quantified using relative fluorescence on the microarray. Rank-based quantile normalization was applied to the raw signal data. Probes were then filtered based on signal intensity (coveted log2 intensity ≥ 6) to obtain the average intensity across all samples. For marker protein extraction, the Pearson correlation coefficient was calculated between the protein expression level of each probe and its individual ICRS score, and those greater than or less than 0.4 were selected. Finally, 112 genes with high variance were identified using these criteria, and those with high variance were confirmed to have a log2 intensity of ≥ 0.5 across all samples.

[0089] 1.5.3. Enzyme-linked immunosorbent assay The supernatant was thawed, and the protein concentration was determined using MIA (melanoma inhibitory activity) (Roche) and DKK1 (Dickkopf WNT signaling pathway inhibitor 1) (R&D Systems, Minneapolis, MN) enzyme-linked immunosorbent assay (ELISA) kits.

[0090] 1.6.Statistical analysis Numerical results were summarized as means and standard deviations. Analysis of ICRS scores was performed using analysis of variance, with Dunnett's multiple comparisons used for post-hoc analysis.

[0091] 2.Results 2.1.Effect of PD sheet on hyaline cartilage regeneration The overall experimental design of this study is shown in Figure 2(a). To evaluate the effect of PD sheet variability on efficacy and transcriptome and proteome profiles, PD sheets were created using 12 lots of PD chondrocytes obtained from 11 donors. These lots included different passage numbers, culture expansion under different conditions, and cryopreservation media (see the table below). Figure 2(b) shows representative macroscopic images of defect areas in the transplantation group and defect areas with good and poor histological repair. Figures 2(c) and (d) show the evaluation results of PD sheets for osteochondral defects in the rabbit orthotopic xenograft model used.

[0092] [Table 6]

[0093] PD sheets promoted the regeneration of hyaline cartilage in the defect area, but the effect varied, with some lots being ineffective. The ICRS score after 4 weeks in the untreated group was 22.13 ± 3.09 (n = 5), while the ICRS scores in the transplanted groups varied from 18.1 to 35.2 (see Figure 2(c)). Figure 2(d) shows the distribution of PD sheet characteristics regarding thickness, cell number, and cell viability contained in the PD sheets. The PD sheets formed a piled cell structure, with thicknesses ranging from 7.7 to 18.3 μm (average 11.52 to 3 ± 3.23 μm). The cell number per PD sheet ranged from 1.7 to 4.2 × 10 6 pieces (average 2.65±0.67×10 6 The number of patients varied within a range of 100 (individuals).

[0094] 2.2. Gene expression profile related to the efficacy of PD sheets Gene expression of 12 lots of PD sheets, the efficacy of which was examined using an orthotopic xenograft model, was analyzed using microarrays. Genes were extracted based on the correlation between array signal and ICRS score, yielding 205 genes with a positive correlation with ICRS score and 238 genes with a negative correlation. The 20 genes with the highest positive correlation are shown in the table below (top 20 positively correlated genes), and the 20 genes with the highest negative correlation are shown in the table below (top 20 negatively correlated genes). Figure 3 shows the results of cluster analysis of the 443 extracted genes using a gene expression heat map. These genes can be considered efficacy-related genes. Of the five lots of PD sheets with the lowest ICRS scores, four were classified into the same cluster, suggesting that the expression levels of these gene sets may be useful for identifying effective PD sheets.

[0095] [Table 7]

[0096] [Table 8]

[0097] 2.3. Function prediction based on efficacy-related genes To clarify the biological functions of these efficacy-related genes, we performed enrichment analysis of these genes using Metascape

[12] . The results suggested that positively correlated genes are involved in skeletal development pathways, cartilage development, and mesenchymal stem cell differentiation (Figure 4). In addition, some genes were involved in angiogenesis and extracellular matrix organization.

[0098] The table below shows the efficacy-related genes described and annotated from the perspective of enriched gene ontology (GO: Gene Ontology). Furthermore, some genes related to extracellular matrix organization (GO:0030198) were also related to cartilage development regulation (GO:0061035). We selected five GO-annotated genes for reverse transcription-polymerase chain reaction (RT-PCR) assays. Expression of aggrecan (ACAN), integrin alpha 10 (ITGA10), growth differentiation factor 5 (GDF5), SRY (sex-determining region Y)-BOX transcription factor 9 (SOX9), and SRY-BOX transcription factor 5 (SOX5) was verified by RT-PCR and analyzed for correlation with ICRS scores. Results showed a correlation between CΔ and ICRS scores for actin beta (ACTB) for ACAN, ITGA10, GDF5, SOX9, and SOX5, but not for SOX5 (Figure 5).

[0099] [Table 9]

[0100] 2.4. Analysis of secretory factors related to the effectiveness of PD sheets Humoral factors continuously supplied by the PD sheet are thought to contribute to the promotion of hyaline cartilage regeneration. Therefore, we used the SOMAscan platform to analyze the correlation between the relative amounts of proteins secreted from the PD sheet and the ICRS score [13,14]. Among the protein factors analyzable by SOMAscan, 49 proteins were identified as positively correlated with efficacy scores, and 63 proteins were identified as negatively correlated (see Tables (Top 20 Secreted Proteins with Positive Correlation) and (Top 20 Secreted Proteins with Negative Correlation) below). Figure 6(a) shows the enrichment analysis results for efficacy-correlated factors. Factors showing negative correlations were concentrated in the GO: apoptosis signaling pathway. Factors showing positive correlations were concentrated in connective tissue development, the parent term of skeletal development, and vascular development, the parent term of angiogenesis. These pathways were concentrated in the same way as in the gene expression analysis. Of the proteins showing expression of genes related to efficacy, eight proteins correlated with efficacy were identified in the supernatant. Eight proteins were identified in the supernatant: ESM1 (endothelial cell-specific molecule 1), GREM1 (gremlin 1), SERPINA3 (serpin family A member 3), DKK1, MIA, NTN4 (netrin 4), FABP3 (fatty acid binding protein 3), and PDGFA (platelet-derived growth factor A subunit) (Figure 6(b) and (c)). Three proteins showed a negative correlation: RARRES2 (retinoic acid receptor responder 2), APOE (apolipoprotein E), and PGF (placental growth factor).

[0101] Among these, we focused on DKK1

[15] , which is expected to have an OA prevention effect, and MIA [16-18], a cartilage differentiation marker and anabolic factor. Quantitation of these factors using enzyme-linked immunosorbent assay (ELISA) revealed that the protein amounts varied between PD sheets, with MIA ranging from 0 to 38.4 ng / mL and DKK1 ranging from 2.6 to 26.6 ng / mL. Furthermore, there was a weak correlation with ICRS score (Figure 6(d)).

[0102] [Table 10]

[0103] [Table 11]

[0104] 3. Discussion In this study, we confirmed that the use of PD sheets in an orthotopic xenotransplantation model was as effective as that using adult knee cartilage sheets. When creating adult human chondrocyte sheets, chondrocyte proliferation in a 2D culture environment leads to decreased Col2A1 expression, suggesting that the chondrocyte sheets dedifferentiate. However, animal models

[11] and human clinical studies [8] have shown that PD sheets redifferentiate into hyaline cartilage in transplanted joints. We have observed the formation of hyaline cartilage-like tissue in the defect site derived from human cells using PD sheets, suggesting that PD sheets can also redifferentiate into hyaline cartilage in vivo. Using an in vivo imaging system to track orthotopic transplantation of chondrocyte sheets in rats, the results showed that the luciferase signal of allogeneic chondrocytes in the joint largely disappeared within several months

[19] , suggesting that in the long term, allograft-derived tissue is replaced by recipient-derived tissue. Our results suggest that, at least in the short term, PD sheets may act as chondrogenic progenitors.

[0105] Cartilage sheets attached to cartilage lesions have been shown to act as a barrier against catalytic factors while preventing the loss of cartilage matrix [4, 20]. Furthermore, anabolic factors produced by cartilage sheets have been suggested to act as paracrine factors to promote cartilage regeneration [10, 21, 22]. Our study demonstrates that both the expression of genes related to extracellular matrix, skeletal formation, ossification, and angiogenesis, as well as the secretion of protein factors, contribute to the effectiveness of PD sheets. We previously reported that PD sheets secrete MIA as an anabolic factor

[10] , and this study confirmed this. Furthermore, we have identified other candidate anabolic factors (GREM1, DKK1, and PDGF) that contribute to the effectiveness of PD sheets.

[0106] GREM1 and DKK1 are known to be involved in the regulation of Wnt signaling, and impaired Wnt signaling in articular cartilage is thought to cause impaired cartilage homeostasis [23-26]. It has been reported that GREM1 inhibits hypertrophic chondrocyte differentiation and increases the cartilage surface and quiescent chondrocyte layer

[27] . It has also been reported that DKK1 transgenic mice are protected from OA

[15] . PDGF-AA has been identified as a chondrotrophic factor that promotes cartilage proteoglycan production and chondrocyte proliferation [28,29], and a decrease in PDGF-AA promotes cartilage degeneration

[30] .

[0107] Our results suggest that PD sheets promote hyaline cartilage regeneration by producing these factors. Because PD sheets are living tissues, they may be able to adjust their factor production depending on the microenvironment—the superficial, deep, or subchondral cartilage layer—during the process of redifferentiation into hyaline cartilage. The production of these factors may be useful markers for predicting the efficacy of PD sheets when selecting donors and improving culture methods for cell sheet production.

[0108] Furthermore, our study demonstrated that the production of NTN4 and ESM1, which have not previously been reported to have specific roles in cartilage tissue, correlated with the effectiveness of PD sheets. NTN4 is a netrin family factor that has been reported to promote osteoblast differentiation

[31] and modify SMAD signaling in chondrocytes

[32] , suggesting that NTN4 may have some effect on chondrocytes themselves and subchondral bone. NTN4 has also been reported to suppress angiogenesis [33,34]. ESM1 is a dermatan sulfate proteoglycan whose expression is enhanced by VEGF [35,36]. ESM1 has been reported to be more highly expressed in the deep cartilage layer than in other parts of cartilage

[37] , suggesting a biological role related to chondrocyte differentiation. Rocha et al. reported that ESM1 increases the bioavailability of VEGF-165 by competitively binding to fibronectin

[38] . Because PD sheets express abundant fibronectin, ESM1 secreted from PD sheets is speculated to bind to and saturate fibronectin in the PD sheets themselves or in recipient tissues, potentially preventing VEGF binding and the invasion of new blood vessels.

[0109] Articular cartilage angiogenesis is thought to be a cause of cartilage degeneration and osteophyte formation. We previously reported that administration of anti-VEGF antibodies inhibited osteophyte formation in a rabbit model of anterior cruciate ligament resection [39,40]. These results suggest that inhibition of angiogenesis is a promising approach for preventing OA. NTN4 and ESM1 produced by PD sheets may contribute to hyaline cartilage regeneration by inhibiting angiogenesis and subsequent mineralization during cartilage defect repair.

[0110] SERPINA3 is a member of the serine protease family and has been reported to be a gene whose expression level changes during mesenchymal stem cell (MSC) differentiation into chondrocytes

[41] . FABP3 is a fatty acid-binding protein required for the accumulation of intracellular lipid droplets and has been reported to enhance MSC survival [42,43]. However, because FABP3 is normally localized in the Golgi apparatus, increased levels in the supernatant may be related to cell death in FABP3-expressing cells. The contribution of these factors to the effects is currently unknown and requires further analysis.

[0111] One limitation of this study is that the results were obtained from a xenotransplantation model using immunosuppressants. Therefore, there may be differences in the processes of entrapment and rejection between xenotransplantation and allotransplantation that should be considered when applying this to humans. The engraftment of the PD sheet is considered to be very important from the perspective of efficacy, and in xenotransplantation, there may be factors that promote engraftment rather than promoting cartilage regeneration.

[0112] In regenerative medicine using allogeneic cells, the properties of which may vary depending on the donor, stabilizing the properties of tissue-engineered cells is an unavoidable challenge for clinical application. To achieve this, it is essential to clarify the biological properties that contribute to in vivo efficacy. This will enable us to verify donor cells and culture conditions based on the identified biological properties.

[0113] In this study, we identified biological pathways and related genes that may contribute to hyaline cartilage regeneration by PD sheet implantation. These components may be used as markers to predict the in vivo efficacy of PD sheets. To use these markers to predict efficacy, further correlations with efficacy using independent data and thresholds for markers must be determined. Ultimately, how these factors contribute to the efficacy of PD sheets must be confirmed in human clinical trials before they can be used as quality markers.

[0114] 4. Conclusion In this study, we identified efficacy-related genes that may contribute to hyaline cartilage regeneration by PD sheet implantation. These identified features may serve as markers predicting the in vivo efficacy of PD sheet use. Furthermore, further elucidation of the mechanisms by which these factors promote cartilage regeneration may provide a new approach to OA treatment.

[0115] Cartilage sheet transplantation is a novel and promising approach for treating patients with cartilage defects associated with osteoarthritis. Hyaline cartilage regeneration using autologous cartilage sheets has already been demonstrated in clinical studies. In this study, we investigated the efficacy of porous chondrocyte sheets (PD sheets) as an alternative allograft model to standard chondrocyte sheets using a standard chondrocyte sheet allograft model. Furthermore, we analyzed the expression of genes and secreted proteins in PD sheets using microarrays and DNA aptamer arrays. The efficacy of PD sheets in treating cartilage defects was evaluated using a histological score, and genes and proteins contained in signals that correlated with efficacy were identified. Enrichment analysis of genes and proteins correlated with efficacy revealed that they were related to the extracellular matrix, skeletal development, and angiogenesis. Eight genes (ESM1, GREM1, SERPINA3, DKK1, MIA, NTN4, FABP3, PDGFA) showed a positive correlation with the efficacy of PD sheets, while three genes (RARRES2, APOE, PGF) showed a negative correlation in both transcriptome and proteome analyses. Of these, MIA and DKK1, which are involved in the skeletal development pathway, showed a weak correlation between secretion levels and efficacy. These results suggest that DKK1 and MIA are useful for predicting the efficacy of PD sheets and may contribute to the regeneration of hyaluronic acid cartilage via PD sheets.

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Claims

1. A method for searching for marker genes of chondrocyte sheets, for searching for marker genes associated with the post-transplant effectiveness of chondrocyte sheets used for cartilage regeneration from chondrocyte sheets before transplantation, comprising: a sheet preparation step of preparing multiple lots of cartilage cell sheets before transplantation; a protein measurement step of measuring the secretion amounts of a plurality of proteins for each of the plurality of lots of chondrocyte sheets before implantation; a score calculation step of transplanting each of the multiple lots of chondrocyte sheets into an organism model of osteochondral defect and calculating a score of the effectiveness of each chondrocyte sheet in cartilage regeneration; a gene identification step of identifying positively correlated genes and / or negatively correlated genes based on the correlation between the amount of protein secretion measured in the protein measurement step and the score calculated in the score calculation step; A method for searching for marker genes in cartilage cell sheets, comprising:

2. The method further comprises a gene measurement step of measuring the expression levels of a plurality of genes for each of the plurality of lots of chondrocyte sheets before implantation, The method for searching for marker genes for cartilage cell sheets, as described in claim 1, characterized in that the gene identification process identifies positively correlated genes that show a positive correlation with the score and / or negatively correlated genes that show a negative correlation with the score based on the correlation between the expression levels of multiple genes measured in the gene measurement process and the score calculated in the score calculation process.

3. A device for searching for marker genes of cartilage cell sheets, for searching for marker genes related to the post-transplant effectiveness of cartilage cell sheets used for cartilage regeneration from cartilage cell sheets before transplantation, comprising: a sheet preparation means for preparing multiple lots of cartilage cell sheets before transplantation; a protein measurement means for measuring the secretion amounts of a plurality of proteins for each of the plurality of lots of chondrocyte sheets before implantation; a score calculation means for transplanting each of the plurality of lots of chondrocyte sheets into an organism model of osteochondral defect and calculating a score of the effectiveness of each chondrocyte sheet in terms of cartilage regeneration; a gene identification means for identifying positively correlated genes and / or negatively correlated genes based on the correlation between the amount of secreted protein measured by the protein measurement means and the score calculated by the score calculation means; A screening device for marker genes of cartilage cell sheets, comprising:

4. The method further comprises a gene measurement means for measuring the expression levels of a plurality of genes for each of the plurality of lots of chondrocyte sheets before implantation, The cartilage cell sheet marker gene search device described in claim 3, characterized in that the gene identification means identifies positively correlated genes that show a positive correlation with the score and / or negatively correlated genes that show a negative correlation with the score based on the correlation between the expression levels of multiple genes measured by the gene measurement means and the score calculated by the score calculation means.

5. A method for evaluating a chondrocyte sheet for predicting the post-transplant efficacy of a chondrocyte sheet used for cartilage regeneration from the chondrocyte sheet before transplantation, comprising: the expression level of a positively correlated gene selected from the group consisting of ESM1, GREM1, SERPINA3, DKK1, MIA, NTN4, FABP3, PDGFA, COLEC12, CTSS, FTL, FTH1, GRN, CYCS, IL6R, THBS2, PRKCD, SLPI, IL12B, IL23A, MRC2, DKK4, UFC1, and SST in the chondrocyte sheet before transplantation, and / or measuring the expression level of negatively correlated genes selected from the group consisting of RARRES2, APOE, PGF, NACA, CXCL6, SMPDL3A, DKKL1, PRL, TEC, CCL11, IL1B, IFNGR1, CXCL12, MAP3K7, TAB1, TNFRSF11B, TIMP1, EEF1B2, EPHB4, KDR, ANGPTL4, STK16, and TNFSF12; A method for evaluating a cartilage cell sheet, characterized by comprising an estimation step of estimating the effectiveness of the cartilage cell sheet after transplantation based on the expression level of the positively correlated gene and / or the expression level of the negatively correlated gene measured in the measurement step.

6. The method for evaluating a cartilage cell sheet according to claim 5, wherein the positively correlated gene is selected from the group consisting of ESM1, GREM1, SERPINA3, DKK1, MIA, NTN4, FABP3, and PDGFA.

7. The method for evaluating a cartilage cell sheet according to claim 5, characterized in that the estimation step estimates that the effectiveness of the cartilage cell sheet before transplantation is high when the expression level of the positively correlated gene is above a predetermined expression level.

8. The method for evaluating a cartilage cell sheet according to claim 5, wherein the negatively correlated gene is selected from the group consisting of RARRES2, APOE, and PGF.

9. The method for evaluating a cartilage cell sheet according to claim 5, characterized in that the estimation step estimates that the effectiveness of the cartilage cell sheet before transplantation is high when the expression level of the negatively correlated gene is below a predetermined expression level.

10. The method for evaluating a cartilage cell sheet described in claim 5, characterized in that the measurement process measures the expression level of the correlated gene by quantifying RNA transcribed from the correlated gene and / or quantifying the protein encoded by the correlated gene.

11. A cartilage cell sheet evaluation device for predicting the post-transplant efficacy of a cartilage cell sheet used for cartilage regeneration from the cartilage cell sheet before transplantation, comprising: the expression level of a positively correlated gene selected from the group consisting of ESM1, GREM1, SERPINA3, DKK1, MIA, NTN4, FABP3, PDGFA, COLEC12, CTSS, FTL, FTH1, GRN, CYCS, IL6R, THBS2, PRKCD, SLPI, IL12B, IL23A, MRC2, DKK4, UFC1, and SST in the chondrocyte sheet before transplantation, and / or a measuring means for measuring the expression level of a negatively correlated gene selected from the group consisting of RARRES2, APOE, PGF, NACA, CXCL6, SMPDL3A, DKKL1, PRL, TEC, CCL11, IL1B, IFNGR1, CXCL12, MAP3K7, TAB1, TNFRSF11B, TIMP1, EEF1B2, EPHB4, KDR, ANGPTL4, STK16, and TNFSF12; A cartilage cell sheet evaluation device characterized by comprising an estimation means for estimating the effectiveness of the cartilage cell sheet after transplantation based on the expression level of the positively correlated gene and / or the expression level of the negatively correlated gene measured by the measurement means.

12. The cartilage cell sheet evaluation device according to claim 11, wherein the positively correlated genes are selected from the group consisting of ESM1, GREM1, SERPINA3, DKK1, MIA, NTN4, FABP3, and PDGFA.

13. The cartilage cell sheet evaluation device described in claim 11, characterized in that the estimation means estimates that the effectiveness of the cartilage cell sheet before transplantation is high when the expression level of the positively correlated gene is above a predetermined expression level.

14. The cartilage cell sheet evaluation device according to claim 11 , wherein the negatively correlated gene is selected from the group consisting of RARRES2, APOE, and PGF.

15. The cartilage cell sheet evaluation device described in claim 11, characterized in that the estimation means estimates that the effectiveness of the cartilage cell sheet before transplantation is high when the expression level of the negatively correlated gene is below a predetermined expression level.

16. The cartilage cell sheet evaluation device described in claim 11, characterized in that the measurement means measures the expression level of the correlated gene by quantifying RNA transcribed from the correlated gene and / or quantifying the protein encoded by the correlated gene.

17. A chondrocyte sheet used for cartilage regeneration, The expression level of a positively correlated gene selected from the group consisting of ESM1, GREM1, SERPINA3, DKK1, MIA, NTN4, FABP3, PDGFA, COLEC12, CTSS, FTL, FTH1, GRN, CYCS, IL6R, THBS2, PRKCD, SLPI, IL12B, IL23A, MRC2, DKK4, UFC1, and SST in the chondrocyte sheet before transplantation is equal to or higher than a predetermined expression level, and / or RA A cartilage cell sheet characterized in that the expression level of a negatively correlated gene selected from the group consisting of RRES2, APOE, PGF, NACA, CXCL6, SMPDL3A, DKKL1, PRL, TEC, CCL11, IL1B, IFNGR1, CXCL12, MAP3K7, TAB1, TNFRSF11B, TIMP1, EEF1B2, EPHB4, KDR, ANGPTL4, STK16, and TNFSF12 is below a predetermined expression level.

18. A method for producing a cartilage cell sheet used for cartilage regeneration, comprising: The expression levels of positively correlated genes selected from the group consisting of ESM1, GREM1, SERPINA3, DKK1, MIA, NTN4, FABP3, PDGFA, COLEC12, CTSS, FTL, FTH1, GRN, CYCS, IL6R, THBS2, PRKCD, SLPI, IL12B, IL23A, MRC2, DKK4, UFC1, and SST in the chondrocyte cell sheet before implantation are set to a predetermined expression level or higher, and / or RARRES2, APOE , PGF, NACA, CXCL6, SMPDL3A, DKKL1, PRL, TEC, CCL11, IL1B, IFNGR1, CXCL12, MAP3K7, TAB1, TNFRSF11B, TIMP1, EEF1B2, EPHB4, KDR, ANGPTL4, STK16, and TNFSF12. A method for producing a cartilage cell sheet, comprising culturing the cartilage cell sheet so that the expression level of a negatively correlated gene selected from the group consisting of: PGF, NACA, CXCL6, SMPDL3A, DKKL1, PRL, TEC, CCL11, IL1B, IFNGR1, CXCL12, MAP3K7, TAB1, TNFRSF11B, TIMP1, EEF1B2, EPHB4, KDR, ANGPTL4, STK16, and TNFSF12 is below a predetermined expression level.

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