Chondrogenic human mesenchymal stem cell (MSC) sheets

Abstract

The present disclosure relates to a chondrogenic cell sheet comprising at least two layers of confluent chondrogenic differentiated cells, the cell sheet being prepared from mesenchymal stem cells (MSCs), and the chondrogenic differentiated cells on the basal side of the cell sheet express one or more adhesion molecules. The present disclosure also relates to a method for preparing a chondrogenic cell sheet, the method comprising: a) culturing MSCs in a temperature-responsive incubator until confluent to form a cell sheet; b) detaching the cell sheet by lowering the temperature and causing the cell sheet to contract to form a contracted cell sheet; c) contacting the contracted cell sheet with a culture surface; and d) treating the contracted cell sheet on the culture surface with a chondrogenic medium and culturing it to form a chondrogenic cell sheet. A method for using a chondrogenic cell sheet for repairing cartilage tissue or treating joint diseases is also disclosed.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 62 / 911,441, filed October 7, 2019, the contents of which are incorporated herein in their entirety. [Background technology]

[0002] Degenerative joint diseases, such as osteoarthritis (OA), are becoming increasingly prevalent and impairing quality of life throughout the world. OA is a systemic, poorly defined disease that is difficult to treat successfully once degeneration begins. Traumatic injuries, most often in the knee joint, can cause focal defects of articular cartilage, which account for more than 12% of OA cases. Articular cartilage lacks a direct blood supply and has minimal ability to repair itself spontaneously, so cartilage damage is unlikely to heal without intervention. Healthy articular cartilage exhibits a hyaline structure, and the goal of focal defect therapy is to promote cartilage regeneration toward a hyaline-like phenotype at the site of injury. Bone marrow stimulation techniques, such as microfracture, are the most frequently used methods in clinical practice. However, the resulting mixed fibrocartilage tissue is inferior to native hyaline cartilage, leading to reduced cartilage function. To adequately treat OA and reduce the incidence of this systemic and debilitating disease, improved treatment options need to be developed to regenerate hyaline cartilage at sites of focal articular cartilage defects before progression to OA.

[0003] In an attempt to improve the outcome of articular cartilage local defect therapy, autologous chondrocyte transplantation has been developed. This approach relies on autologous cell procurement, where the patient is both the donor and the recipient, increasing the burden on the patient due to multiple surgeries, donor-dependent cell quality, and long cell growth and preparation times. In addition, this therapy delivers cells using cell suspensions or seeded scaffolds, which have poor localization to the target tissue site and poor retention of cell functionality. Despite the promising results brought about by advances in tissue engineering, various limitations remain to be resolved, such as cell growth period (large-scale production; off-the-shelf products) and cell delivery system (low engraftment efficiency).

[0004] Increasing efforts are moving away from autologous chondrocyte sources towards "off-the-shelf" allogeneic cell procurement. Mesenchymal stem cells (MSCs) as an allogeneic cell source offer well-documented regenerative and immunomodulatory properties as well as a standard for preparing cells with specific phenotypes. The multipotency of MSCs, including the chondrogenic lineage, allows for directed differentiation in vitro to chondrocyte phenotype in 3D constructs, usually pellet cultures. Although in vitro chondrogenic differentiation of MSCs has been well documented, cells to be implanted in vivo are either enzymatically digested and delivered as cell suspensions or seeded on scaffold materials. Because the cellular environment changes during enzymatic cell collection and interfacial interaction with the scaffold construct, the chondrogenic potential of the cells is barely manifested after implantation, and thus MSC applications for cartilage local defect healing fail before reaching the clinic. Although there are many advantages to using MSCs for cartilage regeneration, no method exists for preparing constructs with differentiation potential and capable of implantation of in vitro differentiated hyaline cartilage without disrupting structural or chondrogenic properties. One of the major challenges to translating MSC cell therapy into the clinic is the ability to reliably engraft and maintain the localization of administered cells, therefore there is a need to develop chondrogenic cell sheets with hyaline-like properties that can be successfully implanted into host tissues. Summary of the Invention

[0005] In certain aspects, the present disclosure relates to a chondrogenic cell sheet comprising at least two layers of confluent chondrogenic differentiated cells, the cell sheet being prepared from mesenchymal stem cells (MSCs), and the chondrogenic differentiated cells at the basal side of the cell sheet express one or more adhesion molecules. In certain embodiments, the MSCs are human bone marrow MSCs (hBM-MSCs). In certain embodiments, the cell sheet consists essentially of chondrogenic differentiated cells. In certain embodiments, at least 50% of the cells in the cell sheet are chondrogenic differentiated cells. In certain embodiments, the cell sheet comprises an extracellular matrix.

[0006] In certain embodiments, the extracellular matrix comprises a protein selected from the group consisting of type II collagen and sulfated proteoglycans. In certain embodiments, the chondrocyte-differentiated cells express a protein selected from the group consisting of SOX9, aggrecan, COL2A1, ACAN, COMP, and BMP2. In certain embodiments, the cell sheet comprises lacunar structures. In certain embodiments, the one or more adhesion molecules are selected from fibronectin and laminin. In certain embodiments, the cell sheet exhibits physical adhesion to the cartilage surface after implantation onto the cartilage surface.

[0007] In certain aspects, the present disclosure relates to a method for preparing a chondrogenic cell sheet, comprising: a) culturing mesenchymal stem cells (MSCs) in a temperature-responsive incubator until confluent to form a cell sheet; b) detaching the cell sheet by lowering the temperature and shrinking the cell sheet to form a shrunken cell sheet; c) contacting the shrunken cell sheet with a culture surface; and d) treating the shrunken cell sheet on the culture surface with a chondrogenic medium and culturing to form a chondrogenic cell sheet.

[0008] In certain embodiments, the contracted cell sheet is grown on the culture surface for at least 3 weeks. In certain embodiments, the chondrogenic medium comprises a protein selected from the group consisting of transforming growth factor beta (TGFβ) and bone morphogenetic protein (BMP). In certain embodiments, the cell sheet in step b) is detached from the temperature-responsive culture vessel without treating the cell sheet with one or more enzymes.

[0009] In certain aspects, the present disclosure relates to chondrogenic cell sheets produced by the methods described herein.

[0010] In certain aspects, the present disclosure relates to a method of transplanting a chondrogenic cell sheet into a subject in need thereof, comprising applying a chondrogenic cell sheet as described herein to a tissue of the subject. In certain embodiments, the tissue is selected from cartilage and bone. In certain embodiments, the cartilage is articular cartilage.

[0011] In certain aspects, the present disclosure relates to a method for repairing cartilage tissue in a subject in need thereof, comprising applying a chondrogenic cell sheet as described herein to cartilage in a subject, thereby repairing cartilage tissue in the subject. In certain embodiments, the cartilage is articular cartilage. In certain embodiments, the subject has a focal cartilage defect.

[0012] In certain embodiments, the subject has a symptomatic cartilage defect. In certain embodiments, the symptomatic cartilage defect is caused by acute or repetitive trauma. In certain embodiments, the subject has a degenerative joint disease.

[0013] In certain aspects, the present disclosure relates to a method for treating or preventing a joint disease in a subject in need thereof, comprising applying a chondrogenic cell sheet as described herein to a joint of the subject, thereby treating or preventing the joint disease in the subject. In certain embodiments, the joint is selected from the group consisting of a synovial joint and a cartilaginous joint. In certain embodiments, the synovial joint is a knee joint, a wrist joint, a shoulder joint, a hip joint, an elbow joint, or a neck joint. In certain embodiments, the joint disease is a degenerative joint disease. In certain embodiments, the joint disease is selected from the group consisting of arthritis, osteoarthritis, rheumatoid arthritis, and chondromalacia patellar.

[0014] In certain aspects, the present disclosure relates to a method of preventing osteoarthritis in a subject having a symptomatic cartilage defect caused by acute or repetitive trauma, comprising applying a chondrogenic cell sheet as described herein to cartilage having the defect, thereby preventing osteoarthritis in the subject. In certain embodiments, the subject is a human. In certain embodiments, the chondrogenic cells in the cell sheet are allogeneic to the subject. In certain embodiments, the chondrogenic cells in the cell sheet are autologous to the subject. [Brief description of the drawings]

[0015] [Figure 1] 1A-1F: Cell sheet contraction promotes structural rearrangements. Representative macroscopic images of non-contracted (A) and contracted (B) whole cell sheets in 35 mm culture dishes. The edges of the cell sheets are marked with orange dotted lines. Scale bar = 5 mm. Representative cross-sectional histology of H&E for non-contracted (C) and contracted (D) cell sheets. Diameter (E) and thickness (F) of cell sheets comparing contracted and non-contracted sheets. Error bars represent mean ± SD (n = 4: *p < 0.05, **p < 0.01). [Figure 2A-F]Differentiation potential of hBM-MSCs at 3 weeks. Representative images of histological sections of pellets in control medium (A-C) and chondrogenic medium (D-F). Staining was with Alcian Blue for acidic mucins (A,D), Safranin O / Fast Green for sulfated proteoglycans (B,E), and DAPI (blue) for collagen type II (pseudo red) (C,F). Scale bar: 200 μm. [Figure 2G-I] Chondrogenic gene expression (G-I) using RT-PCR for Sox9 (G), collagen type II (H), and aggrecan (I). All gene expression was normalized to GAPDH and compared to control samples. Error bars represent the mean ± SD (n=4: *p<0.05, **p<0.01). [Diagram 3] FIG. 1 shows chondrogenic differentiation of hBM-MSC monolayers, contracted sheets, and pellets at 3 weeks as demonstrated by histology, IHC, and RT-PCR (scale bar=200 μm, *p<0.05, **p<0.01). [Figure 4] FIG. 1 shows healthy articular cartilage, or hyaline cartilage phenotype. The goal of localized cartilage defect therapy is to replace or regenerate hyaline-like tissue in vivo. See Castagnola, P. et al. JCB (1986); Moore, D. et al. Ortho B. (2019); and Akkiraju, H. et al. J Dev Biol. (2016). [Diagram 5] Figure 1 shows cell sheets as a cell delivery method. Limitations of cell delivery methods (suspension, scaffold, pellet) include poor cell retention and poor interfacial interaction with native cartilage tissue. Current tools for chondrocyte differentiation can be directly implanted into the target tissue site, but do not achieve complete differentiation to a hyaline-like cartilage phenotype in vitro. [Figure 6]Figure 14: Advantages of MSC cell sheets: By using cell sheet technology with MSCs, it is possible to generate fully scaffold-free 3D constructs that allow differentiation towards a hyaline-like phenotype during in vitro culture with chondrogenic medium, and chondrogenic constructs that can be directly implanted into target tissue sites without disrupting the structural properties or the hyaline-like phenotype. [Figure 7] Figure 14. Chondrogenic differentiation of cell sheets: hBM-MSCs exhibit hyaline-like chondrogenic potential as contracting but not non-contracting sheets. [Figure 8] Figure 14. Control of cell sheet thickness using layering In addition to shrinkage, layering can control the thickness and three-dimensionality of the cell sheet construct. [Figure 9] Figure 1 shows the in vitro engineering of transplantable, scaffold-free, 3D hyaline-like cartilage tissue from human bone marrow-derived mesenchymal stem cell (hBM-MSC) sheets. This approach uses cell sheet tissue engineering to prepare scaffold-free 3D MSC cell constructs via cell sheet contraction after spontaneous detachment from a temperature-responsive culture dish (TRCD). This technique allows in vitro differentiation towards a hyaline-like cartilage phenotype by replating the 3D contracted sheets onto cell culture inserts and inducing them with chondrogenic medium for 3 weeks. It is also possible to directly implant the sheets into target tissues after differentiation without compromising the structure or chondrogenic properties of the sheet construct. [Figure 10]10A-10F: Cell sheet contraction promotes cytoskeletal rearrangement and pro-chondrogenic signaling compared to 2D cell culture. Representative confocal images of whole dishes (top-down) of (a) 2D cultures and (b) 3D contracted sheets before chondrogenic induction on day 0 with phalloidin / F-actin (green) staining for cytoskeleton and DAPI (blue) nuclear staining. Quantitative real-time PCR gene expression for (c) cytoskeletal structure marker β-actin, (d) cell-cell marker β-catenin, and chondrogenic ECM markers (d) BMP2 and (f) COMP. All gene expression was normalized to GAPDH and compared to 2D cell culture day 0 control samples. Error bars represent mean ± SD (n=4) (*p<0.05, **p<0.01). [Figure 11A-L] FIG. 10: Cell sheet contraction stimulates enhanced chondrogenic differentiation of hBM-MSCs. Comparison of 3-week chondrogenically induced hBM-MSC sheets before (2D cultures) and after (3D cell sheets) contraction of the detached sheets. (a-f) Representative images of histological cross-sections of hBM-MSC constructs at 3 weeks in control medium and (g-l) chondrogenic medium. Staining was (a, b, g, h) Safranin O / Fast Green for sulfated proteoglycans, (c, d, i, j) Type II collagen (pseudo red) with DAPI (blue), and (e, f, k, l) Type I collagen (red) with DAPI (blue). [Fig. 11M-T]Representative confocal images of whole dishes (top-down) of 3-week chondrogenically induced (m) 2D cultures and (n) 3D cell sheets with phalloidin / F-actin (green) staining for cytoskeleton and DAPI (blue) nuclear staining. (o) Thickness and (p) linear nuclear density of cell sheets for 2D cultures and 3D cell sheets at 0 days (gray bars) and 3 weeks (black bars) of differentiation. Chondrogenic gene expression by quantitative real-time PCR for (q) SOX9, (r) aggrecan, and (s) collagen type II. (t) Ratio of collagen type II to collagen type I is shown as relative gene expression ratio. All gene expression was normalized to GAPDH and compared to 2D culture 3 weeks control sample. Error bars represent mean ± SD (n=4) (ns: p≧0.05, *p<0.05, **p<0.01). [Figure 12A-H] Figure 1. Progression of chondrogenic differentiation over time for 3D hBM-MSC cell sheets compared to standard pellet cultures. Representative images of histological cross sections of (a-d, i-l) pellets and (e-h, m-p) 3D cell sheets in chondrogenic medium from 0 days to 3 weeks stained with (a-h) Safranin O / Fast Green for sulfated proteoglycans and (i-p) MMP13 (green) with DAPI (blue). [Figure 12I-P] Figure 1. Progression of chondrogenic differentiation over time for 3D hBM-MSC cell sheets compared to standard pellet cultures. Representative images of histological cross sections of (a-d, i-l) pellets and (e-h, m-p) 3D cell sheets in chondrogenic medium from 0 days to 3 weeks stained with (a-h) Safranin O / Fast Green for sulfated proteoglycans and (i-p) MMP13 (green) with DAPI (blue). [Fig. 12Q-R] Representative images of histological cross-sections of pellets and 3D cell sheets at 3 weeks in chondrogenic medium stained for (q) collagen type II (pseudo red) and (r) collagen type I (red) with DAPI (blue). [Fig. 12S-U]Quantitative real-time PCR of pellets (blue dotted line) and 3D cell sheets (black solid line) for (s) chondrogenic gene expression: SOX9, type II collagen and aggrecan, (t) hypertrophic and fibrocartilage gene expression: type X collagen and MMP13, (u) cell-cell interaction expression: β-catenin. All gene expression was normalized to GAPDH and compared to single cell day 0 control samples. Error bars represent the mean ± SD (n=4) (*p<0.05, **p<0.01). [Figure 13A-C] Figure 1. Manipulation and secondary adhesion ability of differentiated 3D contracted hBM-MSC cell sheets and pellet cultures onto FBS-coated surfaces. Representative images of Safranin O stained histological cross sections of 3-week differentiated 3D sheets (a) before transfer and (b) 3 days after transfer onto FBS-coated surfaces. (c) Representative phase contrast images of the edge of the sheet 0-72 hours after transfer. Cell migration from the edge of the cell sheet marked by orange dotted line. [Fig. 13D-F] (d) Attachment efficacy of 3-week differentiated pellet and 3D cell sheets after 1 hour of attachment to secondary FBS-coated culture dishes and 6 hours of continued culture (n=6). Construct attachment = ((number of attached constructs) / (number of constructs attempted to migrate)) x 100. Representative IHC cross-sectional images of 3-week differentiated (e) contracted sheet and (f) pellet cultures for expression and localization of adhesion molecule laminin by DAPI nuclear staining. Adhesion surface marked by yellow dotted line (basal side of (e) cell sheet and periphery of (f) pellet culture). [Figure 14] 14A-14D show the transplantation properties of 3D contracted hBM-MSC cell sheets after differentiation into fresh ex vivo human cartilage pieces. (a) Cell sheets after 3 weeks of differentiation in 35 mm culture dishes, (b) transplanted into fresh ex vivo human articular cartilage samples. Quadrants of transferred cell sheets marked with orange dotted lines. (c) Representative cross-sectional histological and IHC staining of 3-week chondrogenic 3D hBM-MSC sheets (CS) spontaneously attached to fresh ex vivo human articular cartilage (hAC) for the cell adhesion molecule laminin counterstained with Safranin O / Fast Green and (d) DAPI. [Figure 15]15A-15F are diagrams showing the transplantation ability of chondrocyte-differentiated cell sheets in vivo. Macroscopic images of 2 mm local defects in the trochlear groove of nude (RNU) rat hind legs (A) with an implanted 3-week chondrocyte-differentiated hyaline-like human MSC-derived cell sheet (B). The cell sheet was implanted at the time of defect creation. Knee joints were harvested 2 weeks after implantation (C) to evaluate cell sheet transplantation ability. Representative cross-sectional histological analysis using Safranin O (D), Hematoxylin & Eosin (E), and human-specific vimentin immunohistochemical staining (F) of 3-week differentiated human cell sheets in rat local defects 2 weeks after implantation. Positive Safranin O staining (red) indicates retention of positive hyaline-like properties of the cell sheet in the implanted area (D). H&E staining indicates integration with host tissue (E). Transplanted human cells within the cell sheet are positive for human-specific vimentin, indicating engraftment and retention in the implanted area (F). Scale bar = 200 mm. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] This disclosure describes the preparation of mesenchymal stem cells as cell sheets in a temperature-responsive culture device and demonstrates the ability of the cell sheets to differentiate in vitro into a hyaline-like cartilage phenotype. This disclosure also demonstrates the ability of the cell sheets to be transplanted into a target tissue site without compromising the structural or chondrogenic properties of the construct.

[0017] As a next-generation cell delivery method, cell sheet tissue engineering uses temperature-responsive incubators to fabricate scaffold-free multicellular constructs. Regenerative cells are harvested as intact sheets with reproducible physiological properties and scalable fabrication methods. Cell sheets retain endogenous cell matrix, receptors, and adhesion proteins, enhancing cell viability and cell communication, retaining the endogenous cellular environment, and allowing spontaneous attachment to biomaterials and biological surfaces without sutures. Post-harvest sheet manipulation and layering are also possible without compromising cell structure or function.

[0018] The chondrogenic cell sheet described herein was demonstrated to migrate onto human cartilage and adhere onto the cartilage surface via cell adhesion proteins (e.g., laminin) aligned at the interface between the cell sheet and the cartilage. This finding is expected to allow cells to be collected without enzymes, and with the advantage of the cell sheet retaining cell adhesion proteins, differentiated cell sheets can be easily transplanted into target tissues without structural changes or damage.

[0019] The chondrogenic cell sheet described herein is prepared by culturing mesenchymal stem cells (MSCs) in a temperature-responsive incubator until confluent to form a monolayer cell sheet, detaching the cell sheet by lowering the temperature to form a contracted cell sheet with multiple cell layers, contacting the contracted cell sheet with a culture surface, and treating the contracted cell sheet on the culture surface with a chondrogenic medium. The chondrogenic cell sheet described herein is three-dimensional, which is important for cartilage differentiation. As used herein, the term "three-dimensional cell sheet" or "3D" cell sheet refers to a chondrogenic cell sheet that includes multiple cell layers, has a reorganized actin cytoskeleton compared to a monolayer cell sheet (e.g., an actin cytoskeleton that is less aligned because there is no longer tension in a monolayer culture on a cell support), and / or has cells / nuclei that are more rounded and less elongated compared to a monolayer cell sheet. In addition, the cell sheet is inhibited from hypertrophy due to its stable environment. Due to the cell adhesion proteins retained in the cell sheet, it was possible to transplant the differentiated cell sheet to the target site (i.e., cartilage).

[0020] The remarkable chondrogenic potential of human bone marrow MSCs (hBM-MSCs) as 3D cell sheets provides a strong basis for developing scaffold-free 3D chondrogenic constructs that can be used, for example, for articular cartilage local defect therapy. Cytoskeleton reorganization after cell sheet contraction may be one of the mechanisms of increased chondrogenesis. Further tailoring of 3D cell sheet structures for chondrogenesis is possible through sheet layering. This disclosure shows successful engraftment of differentiated 3D chondrogenic cell sheets into ex vivo cartilage while maintaining positive chondrogenic properties. In addition, this disclosure demonstrates that chondrogenically differentiated hyaline-like human MSC-derived cell sheets can be successfully implanted into cartilage and integrated with host tissue in vivo while maintaining hyaline-like properties.

[0021] I. Mesenchymal Stem Cells (MSCs) Mesenchymal stem cells (MSCs) have significant clinical potential for treating a wide range of debilitating diseases, primarily due to their unique immunomodulatory role and regenerative potential (Caplan and Sorrell, 2015, Immunol Lett 168(2):136-139). Suitable MSCs for use in the methods described herein include, but are not limited to, MSCs derived from bone marrow, umbilical cord, spinal blood, limb bud, dental tissue (e.g., molar), adipose tissue, muscle, and amniotic fluid. Any MSCs with chondrogenic potential may be used.

[0022] In certain embodiments, the mesenchymal stem cells are human bone marrow mesenchymal stem cells (hBM-MSCs). As used herein, the term "human bone marrow mesenchymal stem cells" or "hBM-MSCs" refers to mesenchymal stem cells isolated from human bone marrow. Methods for isolating hBM-MSCs are known in the art and described, for example, in Baghaei et al., 2017, Gastroenterol Hepatol Bed Bench. 10(3):208-213, which is incorporated herein by reference in its entirety. The most important characteristic of BM-MSCs used in the isolation and purification process is their physical adhesion to plastic cell culture plates. Various techniques have been used to isolate and enrich MSCs, including, for example, antibody-based cell sorting, low- and high-density culture techniques, positive-negative selection methods, frequent medium changes, and enzymatic digestion approaches.

[0023] Briefly, human bone marrow may be obtained from patients and collected aseptically. The buffy coat is isolated by centrifugation (450×g, 10 min), suspended in 1.5 mL of PBS, and used for culture. The isolated buffy coat is layered on an equal volume of Ficoll (GE health care, USA) and centrifuged (400×g, 20 min). The interface cells are removed and washed twice in sterile PBS. Human bone marrow progenitor cells are cultured in tissue-treated culture plates in DMEM medium supplemented with 10% FBS and penicillin / streptomycin (50 U / mL and 50 mg / mL, respectively, Gibco-Invitrogen, Carlsbad, USA). Plates are maintained for 48 hours at 37° C. in a humidified atmosphere containing 5% CO2. For medium exchange, plates are washed with PBS to remove non-adherent cells and the medium is replaced. Cultures are maintained for an additional week with one medium exchange. To characterize adherent cells, osteoblast differentiation is induced by culturing confluent human MSCs in osteoblast differentiation medium (all from Sigma) for 3 weeks and cells are stained by Alizarin after 3 weeks. To induce adipocyte differentiation, confluent MSCs are cultured in differentiation medium for 1-3 weeks and lipid droplet staining is performed by S Red Oil (Sigma). Flow cytometry is used to evaluate the immune profile of MSCs using the criteria for MSCs as described by the International Society for Cellular Therapy (ISCT). Cells (P2-3) are collected, pelleted, resuspended in 1% bovine serum albumin (BSA in PBS) and counted. Cells are directly stained with phycoerythrin (PE) and conjugated antibodies against CD14, CD34, CD45, CD90, CD105 and CD73 (ebioscience, Germany). In all analyses, appropriate isotype-matched control antibodies named Mouse IgG1K Iso Control (ebioscience, Germany) are used. Cells are analyzed by FACS flow cytometry using Cell Quest Software (Becton Dickinson, UK), see Baghaei et al., 2017 cited above.

[0024] Another convenient source for human MSCs is the umbilical cord, which is discarded after birth and thus provides an easily accessible and uncontroversial source of stem cells for therapy (El Omar et al., 2014, Tissue Eng Part B Rev 20(5): 523-544). Human umbilical cord MSCs (hUC-MSCs) have been tested for safety and efficacy as suspensions in human clinical trials (Bartolucci et al., 2017, Circ Res, 121(10), 1192-1204). Furthermore, hUC-MSCs have been successfully used in experimental animal disease models (Zhang et al., 2017, Cytotherapy 19(2): 194-199).

[0025] Methods for isolating MSCs from umbilical cord are known in the art and described, for example, in U.S. Patent No. 9,903,176, which is incorporated herein by reference in its entirety. Human umbilical cord includes umbilical artery, umbilical vein, Wharton's jelly, and subepithelial layer. In some embodiments, hUC-MSCs are isolated from the subepithelial layer of human umbilical cord. In some embodiments, hUC-MSCs are isolated from the Wharton's jelly of human umbilical cord. Various cell markers can be used to identify hUC-MSCs isolated from the subepithelial layer. For example, in some embodiments, hUC-MSCs isolated from the subepithelial layer express one or more cell markers selected from CD29, CD73, CD90, CD146, CD166, SSEA4, CD9, CD44, CD146, and CD105. In certain embodiments, hUC-MSCs express CD73. In some embodiments, the hUC-MSCs isolated from the subepithelial layer do not express one or more cell markers selected from CD45, CD34, CD14, CD79, CD106, CD86, CD80, CD19, CD117, Stro-1, HLA-DR, HLA-DP, and HLA-DQ. In certain embodiments, the hUC-MSCs do not express HLA-DR, HLA-DP, or HLA-DQ. In some embodiments, the cell sheets described herein are prepared with mesenchymal stem cells (MSCs) with low HLA expression, e.g., less than 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1% of the MSCs in the cell sheet express HLA (e.g., HLA-DR, HLA-DP, and / or HLA-DQ).

[0026] II. Chondrogenic cell sheets generated from MSCs In certain aspects, the present disclosure relates to a chondrogenic cell sheet prepared from mesenchymal stem cells (MSCs). As used herein, the term "chondrogenic cell sheet" refers to a cell sheet obtained by growing mesenchymal stem cells on a temperature-responsive cell culture support in vitro and treating the cell sheet with one or more growth factors that induce chondrogenesis (e.g., transforming growth factor beta (TGFβ) and / or bone morphogenetic protein (BMP)) to generate a cell sheet containing chondrogenically differentiated cells. As used herein, the term "chondrogenic cells" refers to cells derived from MSCs that express sox9, collagen type II, aggrecan, and sulfated proteoglycans in the extracellular matrix (ECM).

[0027] The chondrogenic cell sheet may comprise at least two layers of confluent chondrogenic differentiated cells, for example at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 layers of confluent chondrogenic differentiated cells. In some embodiments, the chondrogenic differentiated cells on the basal side of the cell sheet express laminin. The term "basal side" as used herein refers to the side of the cell sheet that is in contact with the solid cell culture support during the culture of the cell sheet. The basal side of the cell sheet is in contact with the host's cartilage tissue when the cell sheet is implanted into a host. Laminin is a high molecular weight heterotrimeric protein that is secreted into the extracellular matrix. These heterotrimeric proteins cross to form cross-like structures that can bind to other cell membranes and other extracellular matrix molecules, including laminin. Thus, laminin plays an important role in cell adhesion.

[0028] In some embodiments, the chondrogenic cell sheet exhibits physical adhesion to the cartilage surface after implantation onto the cartilage surface, for example, in some embodiments, the chondrogenic cell sheet is adhered to the cartilage surface 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days after implantation onto the cartilage surface.

[0029] In some embodiments, the chondrogenic cell sheet has a hyaline-like cartilage phenotype, including, but not limited to, an extracellular matrix (ECM) containing type II collagen and proteoglycans (allowing resistance to shear, compressive and tensile forces), expression of SOX9, aggrecan and COL2A1, low expression of type I collagen, high COL2A1 / COL1A1 ratio, lacunar nuclei, rounded cell structure and low cell density relative to the ECM.

[0030] In some embodiments, the chondrogenic differentiated cells in the chondrogenic cell sheet are aggregated or physically continuous. In some embodiments, the MSCs used to prepare the chondrogenic cell sheet are human bone marrow MSCs. The chondrogenic cell sheets described herein can be prepared by culturing MSCs on a temperature-responsive culture dish (TRCD), harvesting the MSCs as an intact sheet by temperature shift without any enzymatic treatment, shrinking the cell sheet, and subsequently treating with one or more growth factors (e.g., TGFβ and / or BMP) to induce chondrogenesis. MSC sheets maintain their integrity and shape by retaining tissue-like structures, actin filaments, extracellular matrix, intercellular proteins, and high cell viability, all of which are related to improved cell survival and cell function. Thus, the chondrogenic cell sheets described herein can include structural features that enhance cell survival and cell function, including native extracellular matrix, cell adhesion proteins, and cell junction proteins. Thus, the chondrogenic cell sheets prepared by the methods described herein have several beneficial characteristics compared to cell sheets generated by other methods. For example, chemical disruption methods cannot maintain the tissue-like structure of cells and cell-cell communication because enzymatic treatment destroys extracellular and intracellular proteins (junctions between cells and between cells and the ECM). Thus, protein cleavage by enzymes reduces cell viability and cell function. Physical disruption (i.e., by rubber policeman or medium aspiration) results in the destruction of cell-cell junctions and the disintegration of the cultured adherent sheets into cell aggregates.

[0031] In some embodiments, the extracellular matrix comprises one or more proteins selected from the group consisting of fibronectin, laminin, and collagen, hi some embodiments, the cell junction protein is selected from the group consisting of integrin-β1, connexin 43, β-catenin, and N-cadherin.

[0032] In some embodiments, the chondrogenic cell sheet consists of chondrogenic differentiated cells. In some embodiments, the chondrogenic cell sheet consists essentially of chondrogenic differentiated cells. In some embodiments, at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% of the cells in the chondrogenic cell sheet are chondrogenic differentiated cells. In some embodiments, 100% of the cells in the chondrogenic cell sheet are chondrogenic differentiated cells.

[0033] To optimize the formation or characteristics of the cell sheet, the MSCs used to prepare the chondrogenic cell sheet can be added to the culture medium on the temperature-responsive polymer in the cell culture support at various cell densities. For example, by controlling the initial cell density of the MSCs in the cell culture support (e.g., TRCD), the cytokine expression level in the MSCs can be optimized. In some embodiments, increasing the initial cell density of the MSCs in the cell culture support increases cytokine expression (e.g., HGF). In some embodiments, increasing the initial cell density of the MSCs in the cell culture support decreases cytokine expression. In some embodiments, the initial cell density of the MSCs in the cell culture support used for the preparation of the cell sheet is 1×10 3 / cm 2 From 9×10 5 / cm 2 In some embodiments, the initial cell density of the MSCs in the cell culture support is at least 1×10 3 , 5×10 3 , 1×10 4 , 2×10 4 , 3×10 4 , 4×10 4 , 5×10 4 , 6×10 4 , 7×10 4 , 8×10 4 , 9×10 4 , 1×10 5 , 2×10 5 , 3×10 5 , 4×10 5 , 5×10 5 , 6×10 5 , 7×10 5 , 8×105 Or 9 x 10 5 Cells / cm 2 Any of these values ​​can be used to define a range of initial cell densities of MSCs in the cell culture support. For example, in some embodiments, the initial cell density in the cell culture support is 1×10 3 From 5×10 3 Cells / cm 2 , 2×10 4 From 1×10 5 Cells / cm 2 , 4×10 4 From 1×10 5 Cells / cm 2 , or 1 × 10 3 From 5×10 4 Cells / cm 2 It is.

[0034] The chondrogenic cell sheets described herein can also continuously express extracellular matrix proteins and cell junction proteins after implantation into a target tissue of a host organism. For example, in some embodiments, the cell sheets express extracellular matrix proteins and / or cell junction proteins for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 days after implantation into a tissue of a host organism. In some embodiments, the cell sheets express extracellular matrix proteins and / or cell junction proteins for at least 1, 2, 3, 4, 5, or 6 months after implantation into a tissue of a host organism. In some embodiments, the extracellular matrix proteins expressed in the cell sheets after implantation are selected from fibronectin, laminin, and collagen. In some embodiments, the cell junction proteins expressed in the cell sheets after implantation are selected from vinculin, integrin-β1, connexin 43, β-catenin, integrin-linked kinase, and N-cadherin.

[0035] Current stem cell therapies often use cultured stem cells isolated from biopsies as injectable cell suspensions (Bayoussef et al., 2012, J Tissue Eng Regen Med, 6(10)). Injected cell suspensions generally show poorer engraftment and retention in diseased organs or tissues (Devine et al., 2003, Blood, 101(8), 2999-3001). Loss of intact ECM and cell-cell junctions (i.e., communication) in stem cell suspensions due to enzymatic destruction during harvesting may impair stem cell function, engraftment, and survival in vivo, limiting therapeutic efficacy in vivo. In contrast, the method for preparing chondrogenic cell sheets described herein preserves the intrinsic cell functional architecture and improves the attachment of cell sheets to target tissues after transplantation. For example, in some embodiments, the cell sheet remains attached to the target tissue of the host organism for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 days after implantation into the tissue of the host organism. In some embodiments, the cell sheet remains attached to the target tissue of the host organism for at least 1, 2, 3, 4, 5, or 6 months after implantation into the tissue of the host organism.

[0036] The chondrogenic cell sheets described herein also differ from suspension cultures in several ways. Suspension cultures contain single cells that lack ECM or intercellular junctions because their cell adhesive proteins in their intercellular junctions must be removed (e.g., by proteolytic trypsinization) from culture surfaces commonly used to prepare cell suspension cultures in order to harvest and suspend the cells. In contrast to single cell suspensions, the chondrogenic cell sheets described herein contain endogenous ECM produced by the cells and intact intercellular junctions between the cells that are generated during the formation of the cell sheet. The endogenous ECM and intrinsic intercellular junctions that are maintained during the formation, fabrication, and handling of the cell sheet facilitate the preservation of their phenotype during implantation into the host organism, properties important for cell function and adhesion of the chondrogenic cell sheet to the target tissue (e.g., articular cartilage).

[0037] III. Methods for generating chondrogenic cell sheets in vitro In certain aspects, the present disclosure relates to a method of preparing a chondrogenic cell sheet, comprising: a) culturing mesenchymal stem cells (MSCs) in a temperature-responsive incubator until confluent to form a cell sheet; b) detaching the cell sheet by lowering the temperature and shrinking the cell sheet to form a shrunken cell sheet; c) contacting the shrunken cell sheet with a culture surface; and d) treating the shrunken cell sheet on the culture surface with a chondrogenic medium and culturing to form a chondrogenic cell sheet.

[0038] In some embodiments, the contracted cell sheet is grown on the culture surface for at least 1, 2, 3, or 4 weeks. In some embodiments, the chondrogenic medium comprises a protein selected from the group consisting of transforming growth factor beta (TGFβ) and bone morphogenetic protein (BMP). In some embodiments, the chondrogenic medium comprises one or more of the following media components: insulin-transferrin-selenium (ITS-G), dexamethasone, bovine serum albumin (BSA), L-ascorbic acid 2-phosphate, L-proline, and linoleic acid.

[0039] In some embodiments, the MSCs are human bone marrow mesenchymal stem cells (hBM-MSCs). Methods for isolating hUC-MSCs are known in the art and described herein.

[0040] General methods for preparing cell sheets are known in the art and are described, for example, in U.S. Patent Nos. 8,642,338, 8,889,417, 9,981,064, and 9,114,192, each of which is incorporated by reference in its entirety.

[0041] The temperature responsive polymers used to coat the substrate of the cell culture support have a higher or lower critical solution temperature in aqueous solution, which is generally within the range of 0°C to 80°C, e.g., 10°C to 50°C, 0°C to 50°C, or 20°C to 45°C.

[0042] The temperature-responsive polymers may be homopolymers or copolymers. Exemplary polymers are described, for example, in JP 211865 / 1990. Specifically, they can be obtained by homopolymerization or copolymerization of monomers, such as (meth)acrylamide compounds ((meth)acrylamide refers to both acrylamide and methacrylamide), N-(or N,N-di)alkyl-substituted (meth)acrylamide derivatives, and vinyl ether derivatives. In the case of copolymers, any two or more monomers, such as the above-mentioned monomers, can be used. Furthermore, the monomers can be copolymerized with other monomers, one polymer can be grafted to another, two polymers can be copolymerized, or a mixture of polymers and copolymers can be used. If desired, the polymers can be crosslinked to an extent that does not impair their inherent properties.

[0043] The substrate to be coated with the polymer can be of any type commonly used in cell culture, including, for example, glass, engineered glass, silicon oxide, polystyrene, poly(methyl methacrylate), polyester, polycarbonate, and ceramic.

[0044] Methods for coating a support with a temperature-responsive polymer are known in the art and are described, for example, in JP 211865 / 1990 A. Specifically, such coating can be achieved by subjecting the substrate and the above-mentioned monomer or polymer to, for example, electron beam (EB) exposure, irradiation with gamma rays, irradiation with ultraviolet rays, plasma treatment, corona treatment, or organic polymerization reaction.

[0045] Temperature-responsive polymer coverage is 0.4-3.0μg / cm 2 , for example, 0.7 to 2.8 μg / cm 2 or 0.9 to 2.5 μg / cm 2 The form of the cell culture support may be, for example, a dish, a multiplate, a flask or a cell insert.

[0046] The cultured cells can be detached (detached) and harvested from the cell culture support by adjusting the temperature of the support to a temperature at which the polymer on the support substrate becomes hydrated, thereby allowing the cells to detach. A smooth detachment can be achieved by applying a water flow to the gap between the cell sheet and the support. The detachment of the cell sheet can be effected in the culture medium in which the cells were cultured or in another isotonic liquid, whichever is suitable.

[0047] In a particular embodiment, the temperature-responsive polymer is poly(N-isopropylacrylamide). Poly(N-isopropylacrylamide) has a lower critical solution temperature in water at 31°C. When it is in a free state, it will dehydrate in water at a temperature above 31°C, and the polymer chains will aggregate and cause turbidity. Conversely, at temperatures below 31°C, the polymer chains will hydrate and dissolve in water, thereby causing the release of the cell sheet from the polymer. In a particular embodiment, the polymer covers the surface of a substrate, such as a Petri dish, and is immobilized thereon. Thus, at temperatures above 31°C, the polymer on the substrate surface will also dehydrate, but the polymer chains will cover and immobilize the substrate surface, making the substrate surface hydrophobic. Conversely, at temperatures below 31°C, the polymer on the substrate surface will hydrate, but the polymer chains will cover and immobilize the substrate surface, making the substrate surface hydrophilic. Hydrophobic surfaces are suitable surfaces for cell attachment and proliferation, whereas hydrophilic surfaces inhibit cell attachment and cells are detached simply by cooling the culture medium.

[0048] Culture media for mesenchymal stem cells are known in the art and are described, for example, in U.S. Patent Nos. 9,803,176 and 9,782,439, each of which is incorporated herein by reference in its entirety. In some embodiments, the cell culture medium comprises human platelet lysate (hPL). In some embodiments, the culture medium comprises fetal bovine serum (FBS). In some embodiments, the culture medium comprises ascorbic acid. In some embodiments, the culture medium is a xeno-free medium, i.e., a medium that may contain products derived from humans but does not contain products derived from non-human animals. In some embodiments, the culture medium contains at least one product derived from a non-human animal (e.g., FBS). In some embodiments, the culture medium does not contain products derived from humans. In certain embodiments, the culture medium comprises one or more of Dulbecco's Modified Eagle's Medium (DMEM) (Life Technologies, CA, USA), human platelet lysate (hPL, iBiologics, Phoenix, USA), Glutamax (Life Technologies), MEM non-essential amino acid solution (NEAA) (Life Technologies) and antibiotics, such as penicillin, streptomycin.

[0049] MSCs (e.g., hBM-MSCs) can be subcultured (i.e., passaged) one or more times before culturing the cells in a culture medium on the temperature-responsive polymer coated on the substrate surface of the cell culture support. In some embodiments, MSCs (e.g., hBM-MSCs) are subcultured 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times before culturing the cells in a culture medium on the temperature-responsive polymer coated on the substrate surface of the cell culture support. Any of these values ​​can be used to define the range of the number of subcultures. For example, in some embodiments, MSCs (e.g., hBM-MSCs) are subcultured 2-10 times, 4-8 times, or 1-12 times before culturing the cells on the temperature-responsive polymer. In some embodiments, the number of subcultures is less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times. In some embodiments, the number of subcultures is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times.

[0050] Chondrogenic cell sheets can be prepared in a range of different sizes depending on their application. In some embodiments, the chondrogenic cell sheets have a diameter of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 cm. Any of these values ​​can be used to define the range of sizes of the chondrogenic cell sheets. For example, in some embodiments, the chondrogenic cell sheets have a diameter of 1 to 20 cm, 1 to 10 cm, or 2 to 10 cm. In some embodiments, the chondrogenic cell sheets have a diameter of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, or 300 cm. 2 Any of these values ​​can be used to define a range of sizes for the chondrogenic cell sheet. For example, in some embodiments, the chondrogenic cell sheet has an area of ​​1 to 100 cm 2 , 3 to 70 cm 2 Or 1 to 300 cm 2The methods described herein result in a chondrogenic cell sheet in which the surface area of ​​the chondrogenic cell sheet is much greater than its thickness. For example, in some embodiments, the ratio of the surface area of ​​the chondrogenic cell sheet to its thickness is at least 10:1, 100:1, 1000:1 or 10,000:1. The chondrogenic cell sheets described herein comprise one or more layers of confluent chondrogenic differentiated cells, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 layers of chondrogenic differentiated cells. In some embodiments, the chondrogenic cell sheet comprises less than 2, 3, 4, 5, 6, 7, 8, 9 or 10 layers of chondrogenic differentiated cells. In some embodiments, the chondrogenic cell sheet comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 layers of chondrogenic differentiated cells.

[0051] IV. Methods of Using Chondrogenic Cell Sheets In some aspects, the present disclosure relates to a method of transplanting a chondrogenic cell sheet into a subject in need thereof, comprising applying a chondrogenic cell sheet as described herein to a tissue of the subject.

[0052] In some aspects, the present disclosure relates to a method of repairing cartilage tissue in a subject in need thereof, comprising applying a chondrogenic cell sheet as described herein to cartilage in the subject, thereby repairing cartilage tissue in the subject. In some embodiments of the methods described herein, the cartilage is articular cartilage.

[0053] In some embodiments, the subject has a focal cartilage defect, e.g., a focal articular cartilage defect. As used herein, the term "focal cartilage defect" refers to damage to cartilage that is confined to a well-defined area. In some embodiments, the subject has a symptomatic cartilage defect, e.g., a symptomatic articular cartilage defect. In some embodiments, the symptomatic cartilage defect is caused by acute or repeated trauma. In some embodiments, the subject has a degenerative joint disease (e.g., osteoarthritis) or is at risk of developing a degenerative joint disease (e.g., osteoarthritis).

[0054] In some aspects, the present disclosure relates to a method of treating or preventing a joint disease in a subject in need thereof, comprising applying a chondrogenic cell sheet as described herein to a joint of the subject, thereby treating or preventing the joint disease in the subject. As used herein, the terms "treat", "treating" and "treatment" are all intended to refer to the improvement or reversal of at least one measurable physical parameter associated with a joint disease. The terms "treat", "treating" and "treatment" can also refer to preventing or at least slowing the progression of a joint disease. In one embodiment, "treat", "treating" and "treatment" refer to the reduction or complete alleviation of pain associated with a joint disease, disorder or condition. In another embodiment, "treat", "treating" and "treatment" refer to the reduction of inflammation in the joint. In yet another embodiment, "treat", "treating" and "treatment" refer to inhibiting or reducing the degradation of cartilage in the joint, such as articular cartilage in a synovial joint. In yet another embodiment, "treat", "treat" and "treatment" refer to the alleviation of one or more symptoms associated with a joint disease, such as joint pain, joint swelling, joint stiffness, inflammation, difficulty in joint movement, and reduced range of motion. In some embodiments, the subject has a joint disease at the time the chondrogenic cell sheet described herein is first applied to the subject. In some embodiments, the chondrogenic cell sheet is applied to the subject before the onset of a joint disease (e.g., osteoarthritis), for example, to prevent the onset of a joint disease.

[0055] In some embodiments, the joint is selected from a synovial joint and a cartilaginous joint. The term "synovial joint" refers to the most common and mobile type of joint in the mammalian body. Synovial joints include hinge joints (e.g., elbow and knee), pivot joints (e.g., atlas and shaft bones at the top of the neck), ball and socket joints (e.g., hip joint), saddle joints (e.g., carpal joint of thumb), condylar joints (e.g., wrist, metacarpophalangeal joint, metatarsophalangeal joint), and glenohumeral joints (e.g., intercarpal joint of wrist). Non-limiting examples of synovial joints include, but are not limited to, knee joint, wrist joint, shoulder joint, hip joint, elbow joint, metacarpal joint, carpometacarpal joint, and tarsometatarsal joint. "Cartilaginous joint" is a joint that is completely connected by cartilage, for example, an amphiassic joint such as the sternum and intervertebral disc. In certain embodiments, the joint disease affects a synovial joint. In other embodiments, the joint disease affects a cartilaginous joint.

[0056] In some embodiments, the joint disease is a degenerative joint disease, e.g., osteoarthritis. As used herein, "osteoarthritis" refers to a form of arthritis that occurs in synovial joints. It is usually a chronic condition that occurs when the protective cartilage known as articular cartilage at the ends of bones that come together to form a joint wears down and / or deteriorates. "Articular cartilage" is the tissue at the ends of bones in a joint that helps keep the bones in frictionless contact when moving the joint. Articular cartilage is made up of two main components: collagen and proteoglycans. Causes of cartilage destruction in synovial joints include, but are not limited to, proteases, aging, obesity, and genetic defects in cartilage formation. In some embodiments, the joint disease is selected from the group consisting of arthritis, osteoarthritis, rheumatoid arthritis, and chondromalacia patellar. The subject is a human.

[0057] In certain aspects, the present disclosure relates to a method of preventing osteoarthritis in a subject having a symptomatic cartilage defect caused by acute or repetitive trauma, comprising applying a chondrogenic cell sheet as described herein to cartilage having a defect, thereby preventing osteoarthritis in the subject.

[0058] In some embodiments, the tissue to which the chondrogenic cell sheet is applied is cartilage, e.g., articular cartilage. In some embodiments, the tissue to which the chondrogenic cell sheet is applied is bone, e.g., subchondral bone.

[0059] One advantage of the chondrogenic cell sheet described herein is that the extracellular matrix of the applied cell sheet acts as a natural adhesive to bind the cell sheet to the target cartilage tissue, so that no sutures or stitches are required to attach the cell sheet to the tissue. A support membrane or other device can be used to transfer the chondrogenic cell sheet to the target cartilage tissue, and then removed after sheet transfer. The support can be, for example, poly(vinylidene difluoride) (PVDF), cellulose acetate, cellulose ester, plastic, and metal. The chondrogenic cell sheet easily adheres to the target tissue and self-stabilizes without sutures after being placed directly on the target tissue for a short period of time. For example, in some embodiments, the chondrogenic cell sheet adheres to the target tissue within 5, 10, 15, 20, 25, or 30 minutes of contact with the tissue. After the chondrogenic cell sheet adheres to the uterine tissue, the support membrane can be removed.

[0060] In certain embodiments, the chondrogenic cells in the cell sheet are allogeneic to the subject, i.e., isolated from a different individual of the same species as the subject, and therefore the genes at one or more loci are not identical. In certain reported cases, cell sheets derived from MSCs appear to avoid allogeneic rejection in humans and animal models (Jiang et al., 2005, Blood, 105(10), 4120-4126). Thus, the chondrogenic cell sheets described herein can be used in allogeneic cell therapy as a ready-made product. In other embodiments, the chondrogenic cells in the cell sheet are allogeneic to the subject to which the chondrogenic cell sheet is applied. In some embodiments, the subject is a human.

[0061] The allogeneic cell source must be capable of eliciting meaningful therapy under standard immunocompetence in the host patient allogeneic tissue. This includes reliable cell homing to the tissue site of therapeutic interest and engraftment or retention there in fractional doses for a sufficient period of time (Leor et al., 2000, Circulation, 102(19 Suppl 3), III 56-61). Current expectations are that when a stem cell suspension is administered to a subject, less than 3% of the injected stem cells will be retained in the injured myocardium 3 days after injection following ischemic injury (Devine et al., 2003, Blood, 101(8), 2999-3001). Furthermore, most administered cells from the cell suspension that engraft in the target tissue die within the first few weeks (Reinecke & Murry, 2002, J Mol Cell Cardiol, 34(3), 251-253). In contrast, the chondrogenic cell sheets described herein are stably engrafted into host tissues with high division retention 3 days after implantation. Thus, the chondrogenic cell sheets described herein offer distinct advantages over injected or administered mesenchymal stem cell suspensions.

[0062] In the methods described herein, more than one chondrogenic cell sheet can be applied to the cartilage tissue of a subject. In some embodiments, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more chondrogenic cell sheets can be applied to the cartilage tissue of a subject (e.g., articular cartilage tissue). Any of these values ​​can be used to define the range of the number of chondrogenic cell sheets to be applied to the cartilage tissue of a subject. For example, in some embodiments, 2-4, 3-5, or 1-10 chondrogenic cell sheets are applied to the cartilage tissue of a subject (e.g., articular cartilage tissue). For example, the present invention encompasses the following embodiments: [Embodiment 1] A chondrogenic cell sheet comprising at least two layers of confluent chondrogenic differentiated cells, the cell sheet being prepared from mesenchymal stem cells (MSCs), and the chondrogenic differentiated cells on the basal side of the cell sheet express one or more adhesion molecules. [Embodiment 2] The chondrogenic cell sheet according to embodiment 1, wherein the MSCs are human bone marrow MSCs (hBM-MSCs). [Embodiment 3] A chondrogenic cell sheet according to embodiment 1 or 2, wherein the cell sheet essentially consists of chondrogenic differentiated cells. [Embodiment 4] A chondrogenic cell sheet according to embodiment 1 or 2, wherein at least 50% of the cells in the cell sheet are chondrogenic differentiated cells. [Embodiment 5] A chondrogenic cell sheet described in any one of embodiments 1 to 4, wherein the cell sheet comprises an extracellular matrix. [Embodiment 6] A chondrogenic cell sheet as described in embodiment 5, wherein the extracellular matrix comprises a protein selected from the group consisting of type II collagen and sulfated proteoglycans. [Embodiment 7] A chondrogenic cell sheet described in embodiment 5 or 6, wherein the chondrogenic differentiated cells express a protein selected from the group consisting of SOX9, aggrecan, COL2A1, ACAN, COMP and BMP2. [Embodiment 8] A chondrogenic cell sheet described in any one of embodiments 1 to 7, wherein the cell sheet comprises a lacunar structure. [Embodiment 9] A chondrogenic cell sheet described in any one of embodiments 1 to 8, wherein the one or more adhesion molecules are selected from fibronectin and laminin. [Embodiment 10] A chondrogenic cell sheet described in any one of embodiments 1 to 9, wherein the cell sheet exhibits physical adhesion to the cartilage surface after transplantation onto the cartilage surface. [Embodiment 11] A method for preparing a chondrogenic cell sheet, comprising: a) Mesenchymal stem cells (MSCs) are cultured in a temperature-responsive incubator until they become confluent to form a cell sheet; b) peeling off the cell sheet by decreasing the temperature, shrinking the cell sheet, and forming a shrunken cell sheet; c) contacting the contracted cell sheet with a culture surface; and d) Treating the contracted cell sheet on the culture surface with a chondrogenic medium and culturing it to form a chondrogenic cell sheet. A method comprising: [Embodiment 12] The method of embodiment 11, wherein the contracted cell sheet is grown on the culture surface for at least 3 weeks. [Embodiment 13] The method described in embodiment 11 or 12, wherein the chondrogenic medium comprises a protein selected from the group consisting of transforming growth factor beta (TGFβ) and bone morphogenetic protein (BMP). [Embodiment 14] The method according to embodiment 11, wherein the cell sheet in step b) is detached from the temperature-responsive culture vessel without treating the cell sheet with one or more enzymes. [Embodiment 15] A chondrogenic cell sheet produced by the method according to any one of embodiments 11 to 14. [Embodiment 16] A method for transplanting a chondrogenic cell sheet into a subject in need thereof, comprising applying a chondrogenic cell sheet described in any one of embodiments 1 to 10 or 15 to the subject's tissue. [Embodiment 17] The method of embodiment 16, wherein the tissue is selected from cartilage and bone. [Embodiment 18] The method of embodiment 17, wherein the cartilage is articular cartilage. [Embodiment 19] A method for repairing cartilage tissue in a subject in need thereof, comprising applying a chondrogenic cell sheet described in any one of embodiments 1 to 10 and 15 to cartilage of the subject, thereby repairing cartilage tissue in the subject. [Embodiment 20] The method of any one of embodiments 16 to 19, wherein the cartilage is articular cartilage. [Embodiment 21] The method of any one of embodiments 16 to 20, wherein the subject has a localized cartilage defect. [Embodiment 22] The method of any one of embodiments 16 to 20, wherein the subject has a symptomatic cartilage defect. [Embodiment 23] The method of embodiment 22, wherein the symptomatic cartilage defect is caused by acute or repetitive trauma. [Embodiment 24] The method of any one of embodiments 16 to 20 and 22 to 23, wherein the subject has a degenerative joint disease. [Embodiment 25] A method for treating or preventing a joint disease in a subject in need thereof, comprising applying a chondrogenic cell sheet described in any one of embodiments 1 to 10 and 15 to a joint of the subject, thereby treating or preventing the joint disease in the subject. [Embodiment 26] The method of embodiment 25, wherein the joint is selected from the group consisting of a synovial joint and a cartilaginous joint. [Embodiment 27] The method of embodiment 26, wherein the synovial joint is a knee joint, a wrist joint, a shoulder joint, a hip joint, an elbow joint, or a neck joint. [Embodiment 28] The method of embodiment 25, wherein the joint disease is a degenerative joint disease. [Embodiment 29] The method of embodiment 25, wherein the joint disease is selected from the group consisting of arthritis, osteoarthritis, rheumatoid arthritis, and chondromalacia patellar. [Embodiment 30] A method for preventing osteoarthritis in a subject having a symptomatic cartilage defect caused by acute or repetitive trauma, comprising applying a chondrogenic cell sheet described in any of embodiments 1 to 10 and 15 to cartilage having a defect, thereby preventing osteoarthritis in the subject. [Embodiment 31] The method of any one of embodiments 16 to 30, wherein the subject is a human. [Embodiment 32] The method of any of embodiments 16 to 31, wherein the chondrocyte-differentiated cells in the cell sheet are allogeneic to the subject. [Embodiment 33] The method of any of embodiments 16 to 31, wherein the chondrocytes differentiated in the cell sheet are autologous to the subject.

[0063] [Example] [Example 1] Materials and Methods cell culture Human bone marrow-derived mesenchymal stem cells (hBM-MSCs) purchased from Lonza at passage 2 and passage 4 were cultured at 3,000 cells / cm in growth medium containing Dulbecco's modified Eagle's medium (DMEM, Life Technologies, CA, USA) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific, MA, USA), 1% penicillin-streptomycin (PS, Life Technologies), and 5 ng / mL basic fibroblast growth factor (bFGF, PeproTech, NJ, USA). 2 The cells were plated at 100°C and incubated at 37°C with 5% CO2 in a humidified environment. The medium was changed on days 1 and 5, and the cells were cultured for approximately 7 days until they were 90% confluent. The cells were passaged using 0.05% trypsin-EDTA (Gibco, NY, USA), and the cell suspension was counted using a hemocytometer.

[0064] Preparation of cell sheets and pellets and chondrocyte differentiation Passage 5 hBM-MSCs were cultured at 2.5 x 10 cells in 20% FBS growth medium in a 15 mL Eppendorf tube for pellet culture. 5 Aliquot 6.7 x 10 cells into 1 µm diameter pore 6-well cell culture inserts for monolayer culture. 4 pieces / cm2 and 6.7 × 10 cells were seeded onto 35 mm UpCell dishes (Nunc, Thermo Fisher Scientific, Denmark) for cell sheet culture. 4 pieces / cm 2Cells were seeded at 100°C for 10 min. For pelleting, the tubes were spun at 500×g for 10 min. The lid was loosened and the cells were incubated at 37°C, 5% CO2 for 3 days to allow pellet formation. For cell sheet generation, the cells were cultured for 5 days. On day 5, the cell sheet was left at 20°C for 1 h and then detached with forceps. For cell sheet reseeding, 1 μm pore cell culture inserts (Falcon, NE, USA) were conditioned overnight with FBS before cell sheets were replated. The inserts were washed twice with 1× PBS (Gibco) and stored in a standard incubator. The detached cell sheet was transferred to the conditioned cell culture insert using an OHP film (Apollo, NY, USA) to ensure basal contact with the insert wells. The cell sheet was incubated in 20 μL of growth medium at 37°C, 5% CO2 for 1 h. After 1 h, fresh cell growth medium was added to the sheets and incubated for 3 days at 37°C, 5% CO2 for cell attachment. After the 3-day incubation step, chondrogenic samples were induced with chondrogenic medium, control samples were maintained in cell growth medium, and all samples were transferred to a low-oxygen incubator (37°C, 5% CO2, 5% O2). Chondrogenic medium contained Dulbecco's modified Eagle's medium (DMEM, Life Technologies) supplemented with 10 ng / mL transforming growth factor beta-3 (TGFβ3, Thermo Fisher Scientific), 200 ng / mL bone morphogenetic protein-6 (BMP6, PeproTech), 1% insulin-transferrin-selenium (ITS-G, Thermo Fisher Scientific), 1% PS (Life Technologies), 1% non-essential amino acids (NEAA, Thermo Fisher Scientific), 100 nM dexamethasone (MP Biomedicals, OH, USA), 1.25 mg / mL bovine serum albumin (BSA, Sigma-Aldrich, MO, USA), 50 μg / mL L-ascorbic acid 2-phosphate (Sigma-Aldrich), 40 μg / mL L-proline (Sigma-Aldrich), and 5.35 μg / mL linoleic acid (Sigma-Aldrich).For chondrogenic and control samples, medium was changed twice a week throughout the differentiation period (day 0 to week 4).

[0065] Histological analysis After fixation with 4% paraformaldehyde (PFA, Thermo Scientific) for 15 min, the samples were embedded in paraffin. The embedded samples were sectioned at 4 μm and stained with Alcian Blue and Safranin O. Alcian Blue is a polyvalent basic dye used to stain acidic polysaccharides, such as glycosaminoglycans, in cartilage. Safranin O is a basic dye that stains components of articular cartilage (e.g., proteoglycans, chondrocytes, and type II collagen) with various shades of red. To detect early chondrogenesis, the samples were stained with Alcian Blue (EMD Millipore, MA, USA) for 10 min. To detect mature chondrogenesis, Safranin O staining was performed according to standard methods. Briefly, samples were stained with Wiegert's iron hematoxylin (Sigma-Aldrich) for 4 min, 0.5 g / L fast green (Sigma-Aldrich) for 5 min, and 0.1% Safranin O (Sigma-Aldrich) for 8 min. All samples were dried overnight and then imaged on a BX41 widefield microscope using AmScope Software. Safranin O stained slides were used to calculate cell sheet thickness and nuclear density. For each cell sheet slide, three pictures were taken along the length of the cell sheet. Five measurements from the apical to the basal end of the sheet were taken per picture using the measurement tool built into the AmScope software, and these measurements were evenly spaced along the sheet. Nuclei counts were performed using the same three pictures / sheet. A 500 μm long cell sheet was marked in the image using the measurement tool built into the AmScope software. The number of nuclei was counted within the marked section using ImageJ software. For calculation of cell sheet diameter and volume, macroscopic images of the sheets were analyzed using ImageJ software. Five diameter measurements were taken on four cell sheets per group. Thickness measurements were taken from the Safranin O samples described above. All measurements were averaged for each sample group.

[0066] Immunohistochemical analysis For cross-sectional IHC analysis, samples were fixed with 4% PFA on insert membranes for 15 min and embedded in paraffin. Embedded samples were sectioned at 4 μm and stained for collagen type II to detect mature chondrogenesis, MMP13 to detect hypertrophy, and fibronectin and laminin to detect adhesion molecules. Briefly, antigen retrieval was performed by incubating for 15 min at low pressure in a pressure cooker with a 1:50 dilution of 50× low pH target retrieval solution (Dako, Agilent Technologies, CA, USA) for collagen type II samples and for 6 min at room temperature with proteinase K (Dako, Agilent Technologies) for MMP13, fibronectin, and laminin samples. Fibronectin and laminin samples were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 10 min at room temperature. For all samples, nonspecific binding was blocked with 10% normal goat serum (Vector Laboratories, CA, USA) for 30 min at room temperature. Collagen II, MMP13, fibronectin, and laminin samples were then incubated overnight at 4°C in a 1:200 dilution of anti-collagen II primary antibody (Thermo Fisher Scientific), a 1:200 dilution of anti-MMP13 primary antibody (Abcam, Cambridge, UK), a 1:100 dilution of anti-fibronectin primary antibody (Abcam), or a 1:100 dilution of anti-laminin primary antibody (Abcam). Samples were washed with 1× PBS and covered and incubated for 2 h at room temperature in a 1:200 dilution of goat anti-rabbit 488 secondary antibody (Thermo Fisher Scientific). After 2 h, samples were washed with 1× PBS and mounted with DAPI-containing mounting medium (Invitrogen, MA, USA). Samples were visualized with a Zeiss Axio widefield microscope and ZEN software. Phalloidin (F-actin) staining was performed to determine cytoskeletal arrangement.Briefly, samples were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 15 min and washed with 1x PBS. Samples were then covered with a 1:100 dilution of phalloidin Alexa Fluor 488 (Life Technologies) and incubated at room temperature for 30 min. Samples were then washed with 1x PBS and incubated with DAPI solution (2 drops / mL, Life Technologies) for 5 min at room temperature. Samples were washed with 1x PBS and then prepared for mounting. Samples were imaged with a confocal microscope (Nikon A1-NIS Elements AR Software). Images were analyzed and prepared using ImageJ.

[0067] RT-PCR analysis RNA from samples was extracted with 1 mL TRIzol / sample (Ambion, Life Technologies, CA, USA) in a pestle motor mixer. Total RNA was isolated using the PureLink RNA Mini Kit (Invitrogen, Thermo Fisher Scientific) according to the manufacturer's instructions. For cDNA synthesis, all samples were synthesized simultaneously. Prior to cDNA synthesis, RNA was quantified with Nanodrop and all cDNA samples were prepared from 1 μg RNA / sample. Purity (A) of >1.8 was used. 260 / A 280) were considered to be of sufficient purity to continue. cDNA synthesis was performed using a High Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems, Thermo Fisher Scientific, MA, USA) according to the manufacturer's instructions. RT-PCR analysis was performed using an Applied Biosystems Step One instrument with TaqMan Universal PCR Master Mix (Applied Biosystems, Thermo Fisher Scientific). Gene expression levels were analyzed for the following genes: 1) glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Hs99999905_m1) as a housekeeping gene, 2) SRY-box 9 (Sox9, Hs01001343_g1), 3) collagen type II alpha 1 chain (Col II, Hs00264051_m1), 4) aggrecan (ACAN, Hs00153936_m1), 5) collagen type X alpha 1 chain (ColX, Hs00166657_m1), 6) alkaline phosphatase (ALPL, Hs01029144_m1), 7) matrix metallopeptidase 13 (MMP13, Hs00942584_m1), 11) β-catenin (Hs00355049_m1), 12) bone morphogenetic protein 2 (BMP2, Hs00154192_m1), 13) cartilage oligomeric matrix protein (COMP, Hs00164359_m1). All primers were from Applied Biosystems. Relative gene expression was calculated by the quantitative comparative CT method. Gene expression was normalized to GAPDH expression levels. For chondrocyte differentiation, expression levels are relative to the 3 week monolayer control group, and time-dependent differentiation expression levels are relative to the time 0 monolayer control group.

[0068] Post-differentiation manipulation and reattachment For structural changes in transplantation potential assessment, 3-week chondrogenic contractile sheets were cut in half using a scalpel. Half of each sheet was immediately fixed in 4% PFA for 15 min and embedded in paraffin. The other half of each sheet was reseeded onto a new FBS-coated 35 mm tissue culture plastic dish. To transfer the cell sheets, they were gently nudged to dislodge them from the cell culture insert membrane and manually transferred to the new dish using forceps. After placement on the secondary surface, the sheets were allowed to attach by incubation in a small amount of chondrogenic medium for 1 h. After 1 h, fresh chondrogenic medium was added and the cell sheets were moved to a low-oxygen incubator. Bright-field images of the cells were taken daily at the edge of the sheet during the 3-day culture period. After 3 days, half of the cell sheets were fixed in 4% PFA for 15 min and embedded in paraffin.

[0069] For potential transplantation into target tissue sites, the 3-week chondrogenically differentiated contracted sheets were cut into quarters using a scalpel. One quarter of each sheet was immediately fixed in 4% PFA for 15 min and embedded in paraffin. The other quarter of each sheet was transferred to fresh ex vivo human articular cartilage pieces (approximately 2 cm 2 ) on the apical side of the cartilage. Human articular cartilage samples were collected from human hip joint cartilage during the procedure. After 3 days of co-culture, the cell sheets on the cartilage samples were fixed in 4% PFA for 3 days and embedded in paraffin.

[0070] statistical analysis All statistical analyses were completed on n=4 data sets. All quantitative values ​​are expressed as mean and standard deviation. Statistical significance was calculated using a paired two-tailed Student's T-test. Significance was defined as *p<0.05 and **p<0.01.

[0071] result Chondrogenic potential of hBM-MSCs After 3 weeks in chondrogenic medium and hypoxia, hBM-MSC as pellet cultures show the expected chondrogenic properties positive (Figure 2). 3-week differentiated pellets are positive for all chondrogenic stains (Alcian blue (Figure 2D), Safranin O (Figure 2E), collagen type II (Figure 2F)). 3-week control pellets were negative for all chondrogenic stains (Figure 2A-C). Genetic analysis using RT-PCR shows that the expression of chondrogenic markers (Sox9 (Figure 2G)), collagen type II (Figure 2H), and aggrecan (Figure 2I) was significantly increased in 3-week differentiated pellets compared to 3-week control pellets. This data supports the chondrogenic potential of hBM-MSC.

[0072] Intrinsic post-detachment contraction of hBM-MSC cell sheets Cell sheets undergo spontaneous contraction after detachment from the temperature-responsive culture dish. Intrinsic post-detachment contraction significantly alters the size and structure of contracted cell sheets (Figures 1B, 1D) compared to non-contracted conditions (Figures 1A, 1C). Contracted cell sheets show a 29.2% reduction in construct diameter (Figure 1E) and an 883% increase in thickness (Figure 1F) compared to non-contracted sheets.

[0073] Increased chondrogenic potential of hBM-MSCs as contractile sheets Chondrogenic differentiation over 3 weeks led to chondrogenic positivity in the cell sheets (Figure 11). Non-contracted cell sheets showed slight chondrogenic staining positivity with Alcian Blue (Figure 11), Safranin O (Figure 11), and collagen type II (Figure 11) compared to non-contracted 3 week control sheets (Figure 11). Positive Alcian Blue staining is marked by a dark blue color that correlates with the acidic mucin content of the sample. Safranin O stains positive sulfated proteoglycans dark red relative to GAG content and counterstains all other ECM blue. Collagen type II is indicated by red fluorescence (mock) and nuclei are counterstained with DAPI (blue). For all chondrogenic markers (Alcian Blue (Figure 11), Safranin O (Figure 11), collagen type II (Figure 11)), contracted sheets stained intensely compared to both 3 week contracted controls (Figure 11) and chondrogenic non-contracted sheets. The differentiated contractile sheets also exhibited lacunar morphology associated with mature hyaline cartilage. Gene expression of all chondrogenic markers (Sox9 (Figure 11Q), Col II (Figure 11S), and aggrecan (Figure 11R)) was significantly increased in the contractile sheets compared to the noncontractile sheets after 3 weeks of differentiation. Contractile sheets also showed a 30-fold increase in thickness after 3 weeks of differentiation versus 23-fold for the noncontractile sheets (Figure 11). This increase in thickness after differentiation did not significantly alter the number of cells in either the contractile or noncontractile sheets (Figure 11), suggesting that the increase in thickness of the chondrogenic contractile cell sheets is caused by the deposition of ECM associated with chondrogenesis.

[0074] Changes in cytoskeletal structure for chondrocyte differentiation Cytoskeletal arrangement was observed by phalloidin (f-actin) fluorescent staining, and nuclei were identified by DAPI (Figures 10 and 11). At day 0, non-contracted sheets exhibit an elongated and aligned cytoskeletal structure associated with standard monolayer cultures of MSCs (Figures 10 and 11). Conversely, cell sheets that contracted after detachment exhibited a more random and crossed fibrous structure at day 0 (Figures 10 and 11). After 3 weeks of differentiation, the majority of cells in the 3-week differentiated contracted sheet cells exhibit a reduced and rounded cytoskeletal structure associated with mature chondrocytes (Figures 10 and 11). While some cells in the non-contracted sheets exhibit this rounded cytoskeletal structure, the majority of cells in the 3-week differentiated non-contracted sheets have a more diverse MSC-like structure (Figures 10 and 11). In addition to these cytoskeletal changes, contracted sheets showed a significant increase in cell-cell interactions (β-catenin (Figures 10 and 11)) and ECM components associated with the induction and initiation of chondrogenesis (BMP2 (Figures 10 and 11), COMP (Figures 10 and 11)) compared to non-contracted sheets (Figures 10 and 11).

[0075] Time-dependent chondrocyte differentiation of cell sheets Chondrogenic differentiation of contractile cell sheets and pellets over time shows very similar chondrogenic progression, with a slightly earlier onset of sulfated GAG accumulation seen in contractile cell sheets (Figure 12). Both pellet cultures and contractile sheets are negative for Safranin O staining at day 0 (Figure 12). By 1 week of differentiation, contractile cell sheets (Figure 12) show greater Safranin O staining throughout the sample than pellet cultures (Figure 12), although staining is very faint for both samples. In conjunction with this Safranin O staining, contractile sheets showed a significant increase in ECM components associated with induction and initiation of chondrogenicity (BMP2 (Figure 12), COMP (Figure 12)) compared to pellet cultures. By 3 weeks of differentiation, both contractile cell sheets (Figure 12) and pellets (Figure 12) stain strongly for Safranin O and show lacunar morphology. Genetic analysis shows similar trends in chondrogenic marker (Sox9 (FIG. 12) and collagen type II (FIG. 12)) expression over 4 weeks of differentiation of contractile sheets and pellets. For both contractile sheets and pellets, Sox9 expression increases during early chondrogenesis and then decreases as the samples continue to differentiate. Expression of chondrogenic markers is not significantly different between contractile sheets and pellets at any time during differentiation.

[0076] Delayed hypertrophy development of contractile cell sheets Contractile sheets show a delayed development of hypertrophic characteristics compared to standard pellet cultures (Figure 12). Immunohistochemical staining for the hypertrophic marker MMP13 is negative in pellets and contractile sheets at day 0 (Figure 12). MMP13 staining in pellet cultures remains negative or low over 2 weeks (Figure 12), but is highly expressed in both 3- and 4-week samples (Figure 12). In contractile sheet samples, MMP13 staining is negative or low up to 3 weeks (Figure 12), and positive only in 4-week samples (Figure 12). Expression of collagen type X gene increases throughout chondrogenic differentiation for both contractile sheets and pellets (Figure 12). Expression of collagen type X is significantly higher in pellet cultures than contractile sheets at both 3 and 4 weeks of differentiation. In all samples, MMP13 gene expression is low during early chondrogenesis and begins to increase at later stages of differentiation (Figure 12). Increased MMP13 expression occurs earlier in pellet cultures (2-3 weeks) than in contracted sheet cultures (3-4 weeks). Expression of MMP13 at 3 weeks of differentiation is significantly higher in pellets compared to contracted cell sheets.

[0077] After differentiation, the contracted cell sheet can be manipulated and transferred to a secondary biological surface without losing its structural properties. Contractile cell sheets differentiated for 3 weeks could be manipulated and transferred to new culture dishes and allowed to adhere strongly for 3 days (Figure 13). After 3 days of secondary culture on FBS-coated dishes, the cell sheets were harvested, fixed, and stained with Safranin O. This staining did not reveal any discernible changes in the structure and content of the cell sheets after transfer (Figure 13). Conversely, pellet cultures transferred to secondary culture dishes showed a decrease in Safranin O staining and some changes in pellet shape (Figure 13). After 3 days of secondary culture, contractile sheets maintained laminin cell adhesion staining localized to the basal side of the cell sheets (Figure 13). In laminin staining of pellets, staining was localized to the periphery of the pellet and weaker at the interface surface compared to cell sheet cultures (Figure 13). During secondary culture, cell migration could be seen at the edge of the cell sheets as early as 24 hours after transfer of differentiated contractile sheets (Figure 13), whereas cell migration was minimal and proceeded later in pellet cultures (Figure 13). This indicates greater surface attachment and cell viability in the shrink sheets than in the pellet cultures.

[0078] The differentiated cell sheets adhere strongly to fresh ex vivo cartilage surfaces for 3 days (Figure 14). Laminin cell adhesion staining is strongest at the interface between the sheet and the cartilage surface (Figure 14), indicating strong adhesion and interfacial interactions between the cell sheet and the target tissue.

[0079] [Example 2] Many stem cells used in articular cartilage defect healing therapies demonstrate chondrogenic potential using in vitro pellet culture, but this potential is rarely retained when the cells are seeded onto a biomaterial scaffold. Cell sheet tissue engineering removes the scaffold requirement and allows for the transplantation of cells directly from culture. To develop human MSC sheets for cartilage therapy, it is important to maintain the chondrogenic potential of the cells in the sheet morphology.

[0080] This study aims to generate ready-to-use hyaline-like cartilage constructs from MSCs in vitro using cell sheet technology. This study further demonstrates that the cell sheets retain transplantation capacity after chondrogenic differentiation to a hyaline-like phenotype, allowing spontaneous adhesion and interfacial interaction with the target tissue site without compromising the structural or chondrogenic properties of the construct (Figure 9). This study describes the development of pre-differentiated hyaline-like cell sheet constructs that can reduce the time it takes for transplanted cells to establish hyaline cartilage in vivo for regenerative therapy.

[0081] 1. Materials and Methods 1.1 Cell culture hBM-MSCs purchased from Lonza at passage 2 were cultured at 3,000 cells / cm in growth medium containing high glucose (4.5 g / L) Dulbecco's modified Eagle's medium (HG-DMEM) (Life Technologies, CA, USA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, MA, USA), 1% penicillin-streptomycin (PS) (Gibco, NY, USA), and 5 ng / mL basic fibroblast growth factor (bFGF) (PeproTech, NJ, USA). 2 The cells were plated at 100°C and incubated in a humidified environment (37°C, 5% CO2). The medium was changed on days 1 and 5 and the cells were cultured for approximately 7 days until they were 90% confluent. The cells were passaged using 0.05% trypsin-EDTA (Gibco) and the cell suspension was counted using a hemocytometer. The cells were expanded and deposited for the 3rd and 4th passages, and the 5th passage was used for the experiments.

[0082] 1.2 Preparation of cell sheets and pellets and chondrocyte differentiation Passage 5 hBM-MSCs were cultured at 2.5 x 10 cells in 20% FBS growth medium in a 15 mL conical tube for pellet culture. 5 Aliquot 6.7 x 10 cells into 1.0 µm diameter pore 6-well cell culture inserts for monolayer culture. 4 pieces / cm 2The cells were seeded at 100°C for 1 h and seeded in 35 mm UpCell dishes (CellSeed Co., Ltd., Tokyo, Japan) for cell sheet culture. For pelleting, the conical tubes were centrifuged at 500 × g for 10 min. The lid was loosened and the cells were transferred to a standard incubator (37°C, 5% CO2) for 3 days to form a pellet. For cell sheet culture, the cells were cultured for 5 days. On the fifth day, the cell sheet was transferred for 1 h at 20°C and then detached with forceps. To re-seed the cell sheet, 6-well cell culture inserts with 1.0 μm pores were conditioned overnight with FBS before re-seeding the cell sheet. The inserts were washed twice with 1× phosphate-buffered saline (PBS) (Gibco) before transferring the sheet. The detached cell sheets were transferred to conditioned cell culture inserts using an overhead projector polyester film (Apollo, NY, USA) to ensure basal contact with the insert well culture surface and incubated in 20 μL of growth medium for 1 h in a standard incubator. After 1 h, fresh cell growth medium was added to the sheet and incubated for an additional 3 days to ensure sheet attachment and mirror the pellet culture incubation period. After the 3-day incubation step, chondrogenic samples were induced with chondrogenic medium. Control samples were maintained in 10% FBS cell growth medium. All samples were transferred to a low-oxygen incubator (37 °C, 5% CO2, 5% O2).Chondrogenic medium contained HG-DMEM supplemented with 10 ng / mL transforming growth factor beta-3 (TGFβ3) (Thermo Fisher Scientific), 200 ng / mL bone morphogenetic protein-6 (BMP6, PeproTech), 1% insulin-transferrin-selenium (ITS-G) (Thermo Fisher Scientific), 1% PS (Life Technologies), 1% non-essential amino acids (NEAA) (Thermo Fisher Scientific), 100 nM dexamethasone (MP Biomedicals, OH, USA), 1.25 mg / mL bovine serum albumin (BSA) (Sigma-Aldrich, MO, USA), 50 μg / mL L-ascorbic acid 2-phosphate (Sigma-Aldrich), 40 μg / mL L-proline (Sigma-Aldrich), and 5.35 μg / mL linoleic acid (Sigma-Aldrich). For chondrogenic and control samples, medium was changed twice a week throughout the differentiation period (day 0 to week 3).

[0083] 1.3 Histological analysis After fixation with 4% paraformaldehyde (PFA) (Thermo Scientific) for 15 min, samples were embedded in paraffin. Embedded samples were sectioned at 4 μm. To identify cell morphology, hematoxylin and eosin (H&E) staining was performed according to standard methods. Briefly, samples were stained with Mayer's hematoxylin (Sigma-Aldrich) for 4 min and eosin (Thermo Scientific) for 4 min. To detect mature cartilage formation, Safranin O staining was performed according to standard methods. Briefly, samples were stained with Wiegert's iron hematoxylin (Sigma-Aldrich) for 4 min, 0.5 g / L fast green (Sigma-Aldrich) for 5 min, and 0.1% Safranin O (Sigma-Aldrich) for 8 min. All samples were dried overnight and then imaged with a BX41 wide-field microscope (Olympus Corporation, Japan) using AmScope Software (USA). Safranin O stained slide cross sections were used to calculate the thickness and nuclear density of the cell sheets. For each cell sheet slide, three photographs were taken along the length of the cell sheet. Using the measurement tool built into the AmScope software, five measurements from the apical to the basal end of the sheet were taken per photograph, and these measurements were evenly spaced along the sheet. Nuclei counts were performed using the same three photographs / sheet. Using the measurement tool built into the AmScope software, 500 μm of the cell sheet length was marked. The number of nuclei within the marked sections was counted using ImageJ software (NIH, USA). For calculation of cell sheet diameter, macroscopic images of the sheets were analyzed using ImageJ software. Five diameter measurements were taken for four cell sheets per group. All measurements were averaged for each sample group.

[0084] 1.4 Immunohistochemical analysis For cross-sectional IHC analysis, samples were fixed on insert membranes with 4% PFA for 15 min and embedded in paraffin. Embedded samples were sectioned at 4 μm and stained for collagen types II and I to detect mature chondrogenesis, MMP13 to detect hypertrophy, and laminin to detect adhesion molecules. Briefly, antigen retrieval was performed for collagen type II samples by incubation at 106–110 °C for 15 min at low pressure in a pressure cooker with a 1:50 dilution of 50× low pH target retrieval solution (Dako, Agilent Technologies, CA, USA), and for collagen type I, MMP13, and laminin samples by incubation with proteinase K (Dako, Agilent Technologies) for 6 min at room temperature (RT). Laminin samples were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 10 min at room temperature. Non-specific binding was blocked with 10% normal goat serum (Vector Laboratories, CA, USA) for collagen type II, MMP13, and laminin samples, and with 5% normal donkey serum (Abcam) for collagen type I samples for 30 min at room temperature. Collagen type II, collagen type I, MMP13, and laminin samples were then incubated overnight at 4°C in a 1:200 dilution of anti-collagen type II primary antibody (Thermo Fisher Scientific), a 1:200 dilution of anti-collagen type I primary antibody (Thermo Fisher Scientific), a 1:200 dilution of anti-MMP13 primary antibody (Abcam), or a 1:100 dilution of anti-laminin primary antibody (Abcam). Samples were washed with 1x PBS and covered with either a 1:200 dilution of goat anti-rabbit 488 secondary antibody (Thermo Fisher Scientific) for collagen type II, MMP13 and laminin samples, or a 1:200 dilution of goat anti-donkey 594 secondary antibody (Thermo Fisher Scientific) for collagen type I samples, and incubated for 2 hours at room temperature.After 2 hours, samples were washed with 1x PBS and mounted with DAPI-containing mounting medium (Invitrogen, MA, USA). Samples were visualized with a Zeiss Axio widefield microscope and ZEN software. Samples were visualized with a BX41 widefield microscope using AmScope Software. Phalloidin (F-actin) staining was performed to determine cytoskeleton arrangement. Briefly, samples were permeabilized with 0.1% Triton X-100 (Sigma-Aldrich) for 15 min and washed with 1x PBS. Samples were then covered and incubated with a 1:100 dilution of phalloidin AlexaFluor488 (Life Technologies) for 30 min at room temperature. Samples were then washed with 1x PBS and incubated with DAPI solution (2 drops / mL, Life Technologies) for 5 min at room temperature. Samples were then washed with 1x PBS and prepared for mounting. Samples were imaged with a confocal microscope (Nikon A1-NIS Elements AR Software). Images were prepared using ImageJ software.

[0085] 1.5. Real-time quantitative PCR analysis RNA from samples was extracted with 1 mL of TRIzol / sample (Ambion, Life Technologies, CA, USA) in a pestle motor mixer. Total RNA was isolated using the PureLink RNA Mini Kit (Invitrogen, Thermo Fisher Scientific) according to the manufacturer's instructions. For cDNA synthesis, all comparative samples were synthesized simultaneously. Prior to cDNA synthesis, RNA was quantified using a NanoDrop spectrophotometer (Thermo Scientific, USA) and all cDNA samples were prepared from 1 μg of RNA / sample. Purity (A) of >1.8 was used. 260 / A 280) were considered to be sufficiently pure for use. cDNA synthesis was performed using a High Capacity cDNA Reverse Transcriptase Kit (Applied Biosystems, Thermo Fisher Scientific, MA, USA) according to the manufacturer's instructions. Real-time qPCR analysis was performed using TaqMan Universal PCR Master Mix (Applied Biosystems, Thermo Fisher Scientific) using an Applied Biosystems Step OnePlus instrument. Gene expression levels were analyzed for the following genes: 1) glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Hs99999905_m1) as a housekeeping gene, 2) β-actin (Hs99999903_m1), 3) β-catenin (Hs00355049_m1), 4) bone morphogenetic protein 2 (BMP2, Hs00154192_m1), 5) cartilage oligomeric matrix protein (COMP, Hs00164359_m1), 6) SRY-box9 (SOX9, Hs01001343_g1), 7) aggrecan (ACAN, Hs00153936_m1), 8) collagen type II alpha 1 chain (COL2A1, Hs00264051_m1), 9) collagen type I alpha 1 chain (COL1A1, Hs00164004_m1), 10) collagen type X alpha 1 chain (COL1, Hs0016665_m1), 11) matrix metallopeptidase 13 (MMP13, Hs00942584_m1). All primers were from Applied Biosystems. Relative gene expression was calculated by the quantitative comparative CT method. Gene expression was normalized to GAPDH expression levels. For cytoskeleton analysis and time-comparative differentiation, expression levels are relative to the 2D monolayer control group at day 0, and for chondrocyte differentiation, expression levels are relative to the 2D monolayer 3 weeks control group.

[0086] 1.6 Post-differentiation manipulation and reattachment To evaluate structural changes and adhesive ability during post-differentiation manipulation, 3-week chondrogenically differentiated contracted hBM-MSC sheets were cut in half using a scalpel. Half of each sheet was immediately fixed in 4% PFA for 15 min and embedded in paraffin. The other half of each sheet was reseeded onto an FBS-coated 35 mm tissue culture plastic dish. To transfer the cell sheets, half of the cell sheets were gently poked to dislodge them from the cell culture insert membrane and manually transferred to a new FBS-coated culture dish using forceps. After placement on the secondary surface, the sheets were incubated in chondrogenic medium for 1 h to allow attachment. After 1 h, fresh chondrogenic medium was added and the cell sheets were moved to a low-oxygen incubator for further culture. Bright-field images of the cells were taken at the edge of the sheet from 0 to 72 h during the 3-day culture period using a Zeiss Axio wide-field microscope and ZEN software. After 3 days, half of the cell sheets were fixed in 4% PFA for 15 min and embedded in paraffin. For comparison, 3-week chondrogenic pellets were also re-seeded onto FBS-coated 35 mm tissue culture plastic dishes. The pellets were manually transferred to a new culture surface by gentle pipetting. After placement on the secondary surface, the pellets were incubated in a small amount of chondrogenic medium for 1 hour to allow attachment. After 1 hour, fresh chondrogenic medium was added and the pellets were moved to a low-oxygen incubator and cultured for an additional 6 hours. The attachment efficacy of the constructs was quantified as the ratio of attached sheets or pellets after this additional 6 hours of culture to the total number of transferred sheets or pellets (n=6).

[0087] 1.7 Transplantation potential after differentiation To determine the transplantation potential for targeting cartilage tissue, 3-week chondrogenically differentiated contractile sheets were cut into quarters using a scalpel. One quarter of each sheet was immediately fixed in 4% PFA for 15 min and embedded in paraffin. The other quarter of each sheet was cut into quarters using fresh (same day of collection) ex vivo human articular cartilage fragments (approximately 2 cm) collected as anonymized tissue waste from human hip cartilage during routine hip arthroscopy procedures. 2 After co-culture for 3 days in a low-oxygen incubator, the cartilage samples on the cell sheets were fixed in 4% PFA for 3 days and embedded in paraffin.

[0088] 1.8 Statistical analysis All statistical analyses were completed on data sets of n≧4 biological replicates. All quantitative values ​​are expressed as mean±standard deviation. Statistical significance was assessed using a paired two-tailed Student's T-test, assuming normal distribution of the data. Sufficient normal distribution for this statistical analysis was defined as a skewness of the data of ≧−0.8 and ≦+0.8. Statistical significance was defined as not significant (ns) p≧0.05, *p<0.05, and **p<0.01.

[0089] 2 Results 2.1 Spontaneous post-detachment contraction of hBM-MSC sheets Cultured human bone marrow-derived mesenchymal stem cell (hBM-MSC) sheets contracted spontaneously after detachment from the temperature-responsive culture dish (Figure 15). Spontaneous post-detachment contraction significantly altered the size and structure of the resulting 3D contracted cell sheet compared to 2D monolayer cell culture conditions (Figure 15). hBM-MSC sheets showed a significant decrease in diameter (3.5-fold decrease, p=1.77E-5) (Figure 15) and increase in thickness (7-fold increase, p=3.63E-6) (Figure 15) after contraction of the post-detachment cell sheet compared to 2D monolayer cell culture conditions.

[0090] 2.2 Sheet cytoskeletal changes for chondrogenic potential The cytoskeletal arrangement of cultured hBM-MSC sheets was observed by phalloidin (F-actin) fluorescent staining, and nuclei were identified by DAPI (Figure 21a, b). At day 0, cells in 2D cultures exhibited elongated and aligned cytoskeletal structures associated with standard adherent cultures of MSCs (Figure 10A). Conversely, harvested hBM-MSC cell sheets that were contracted after detachment exhibited a more random crossed fibrous structure with more rounded nuclei (Figure 10B). These cytoskeletal changes were confirmed to be significant based on the relative β-actin gene expression between contracted 3D sheets and 2D monolayer cultures (Figure 10C) (p=0.0194). In addition to these cytoskeletal changes, contracted 3D sheets showed a significant increase in cell-cell interactions, β-catenin (p=.00865), and the pro-chondrogenic signaling molecules BMP2 (p=.0000457) and COMP (p=.000947) compared to 2D monolayer cultures prior to chondrogenic induction (Figure S10D-F).

[0091] 2.3 Increased chondrogenic potential of hBM-MSCs as 3D contracted sheets hBM-MSCs were selected as a MSC source with demonstrated chondrogenic potential, as confirmed in standard 3D pellet cultures (see Figure 2). Three-week chondrogenic differentiation of hBM-MSCs collected as 3D contracted cell sheets led to positive hyaline-like chondrogenesis (Figure 11). Positive Safranin O and collagen type II staining was identified in the 3-week differentiated samples compared to control samples (Figures 11a-d, g-j). Safranin O staining stains sulfated proteoglycans red (red intensity is relative to GAG content) and fast green counterstains other ECM proteins blue. Collagen type II is indicated by red fluorescence (pseudo red immunostaining) and nuclei are counterstained with DAPI (blue). Monolayer 2D hBM-MSC cultures showed slightly positive chondrogenic staining with Safranin O (Figure 11G) and collagen type II (Figure 11I) after 3 weeks of differentiation. 3D contractile sheets after 3 weeks of differentiation stained more intensely for all chondrogenic markers (Safranin O (FIG. 11H) and type II collagen (FIG. 11J)) compared to chondrogenic 2D cultures (FIG. 11G,I). Differentiated 3D contractile sheets also developed lacunar structures associated with mature hyaline cartilage (FIG. 11H). After 3 weeks of differentiation, most cells in the 3-week differentiated contractile sheets exhibited a reduced, rounded cytoskeletal structure associated with mature chondrocytes (FIG. 11N). While some cells in the 2D monolayer cultures exhibited this rounded cytoskeletal structure, the majority of cells in the 3-week differentiated monolayer had a more diverse spindle-like or fibroblastic shape (FIG. 11M). Gene expression of all chondrogenic markers was significantly increased in 3D contracted cell sheets compared to 2D cell cultures after 3 weeks of differentiation (SOX9 (Figure 11Q) (p=0.0044), ACAN (Figure 11R) (p=0.0242), and COL2A1 (Figure 11S) (p=0.0126)). 3D contracted cell sheets also expressed significantly higher collagen type II to collagen type I ratio (Figure 11T) (p=.0107) compared to 2D cultures (Figure 11K) after 3 weeks of differentiation, with minimal staining for collagen type I (Figure 11L). Furthermore, 3D contracted sheets showed a 30-fold increase in thickness after 3 weeks of differentiation, significantly greater than the 23-fold increase in thickness of 2D cell cultures (Figure 11O) (p=4.26E-6). This increase in thickness after differentiation did not significantly change cell numbers in either the 3D contracted cell sheets or the 2D cell cultures (FIG. 11P) (p=0.422 (3D); 0.997 (2D)), suggesting that the increased thickness of the chondrogenic cell sheets results from the deposition of chondrogenic-inductive ECM.

[0092] 2.4 Chondrogenic differentiation of cell sheets over time Chondrogenic differentiation of 3D contracted harvested cell sheets and pellets over time showed very similar progression in chondrogenesis, with a slightly earlier onset of sulfated GAG accumulation and a delayed onset of hypertrophic and fibrocartilage phenotype in 3D contracted hBM-MSC sheets (Figure 12). Both pellet cultures and 3D sheets were negative for Safranin O staining at day 0 (Figure 12A, E). After 1 week of differentiation, 3D contracted cell sheets (Figure 12F) showed greater Safranin O staining throughout the sample than pellet cultures (Figure 12B), although staining was faint in both samples. By 3 weeks of differentiation, both 3D cell sheets (Figure 12H) and pellet cultures (Figure 12D) showed lacunar structures while staining strongly for Safranin O (Figure 12H, D) and type II collagen (Figure 12Q). Genetic analysis showed similar trends of chondrogenic marker expression (SOX9, COL2A1, ACAN) over 3 weeks of differentiation for contracted sheets and pellets (Figure 12S). Chondrogenic marker expression was not significantly different between 3D sheet and pellet cultures at any time during differentiation (Sox9: p=0.435 (0 days), 0.754 (1 week), 0.189 (2 weeks), 0.222 (3 weeks); COL2A1: p=0.169 (0 days), 0.158 (1 week), 0.265 (2 weeks), 0.131 (3 weeks); ACAN: p=0.773 (0 days), 0.681 (1 week), 0.213 (2 weeks), 0.724 (3 weeks)).

[0093] Although chondrogenic expression was similar, 3D contracted sheets showed delayed development of hypertrophic and fibrocartilaginous properties compared to standard pellet cultures. 44,45Immunohistochemical staining for MMP13 was negative in pellet cultures and 3D sheets at day 0 (Figure 12I,M). MMP13 staining in hBM-MSC pellet cultures remained negative or minimal over 2 weeks (Figure 12I-K), but was strongly present in 3-week samples (Figure 12L). MMP13 staining in 3D sheet samples remained negative or minimal over 3 weeks (Figure 12M-P). By 3 weeks of differentiation, pellet cultures stained strongly for the fibrocartilage marker collagen I, whereas 3D contracted sheets showed minimal collagen I staining positivity (Figure 12R). Hypertrophic collagen X gene expression increased throughout chondrocyte differentiation in both 3D sheets and pellets (Figure 12T), but collagen X expression after 3 weeks of differentiation was significantly higher in pellet cultures than 3D sheets (p=0.00596). Expression of the MMP13 gene was low in both constructs during early chondrogenesis, but was significantly higher in pellets compared to 3D contracted cell sheets at 3 weeks (Figure 12T) (p=0.0114). The cell-cell adhesion marker β-catenin is associated with chondrogenic commitment at early stages of chondrogenesis, but also with chondrocyte hypertrophy when overexpressed at later stages of chondrocyte differentiation. This expression of β-catenin was significantly higher in 3D cell sheets compared to hBM-MSC pellet cultures at induction, but was then significantly lower throughout differentiation (Figure 12U) (p=0.00248 (0 days); 0.0374 (1 week); 0.00591 (2 weeks); 0.0464 (3 weeks)).

[0094] 2.5 Engineering hyaline-like cell sheets without affecting sheet properties 3D contracted hBM-MSC sheets differentiated for 3 weeks could be manipulated and transferred as intact sheets to new culture surfaces (Figure 13). After 3 days of secondary culture on FBS-coated surfaces, cell sheets were harvested, fixed, and stained with Safranin O. This staining showed no discernible changes in the structure or GAG composition of the cell sheets after transfer (Figure 13A,B). During secondary culture, cell migration / proliferation was observed at the edges of the cell sheets as early as 6 hours after transfer in harvested differentiated 3D contracted sheets (Figure 13C), indicating rapid and spontaneous surface attachment and cell viability. In contrast to 100% successful attachment of 3-week differentiated cell sheets (6 / 6 sheets fully attached), none of the reseeded 3-week differentiated pellets were able to attach and remained attached to the secondary culture dishes (0 / 6 pellets attached) after adding fresh medium and culturing the constructs for an additional 6 hours (Figure 13D). Immunohistochemical analysis of 3-week differentiated 3D contractile sheets and pellets showed that staining for the adhesion molecule laminin appeared strong along the basal side of the cell sheets (Figure 13E), whereas laminin staining of pellet cultures showed minimal positive staining around the periphery (interface) of the pellet (Figure 13F).

[0095] 2.6 Transplant potential of hyaline-like cell sheets 3D contracted hBM-MSC sheets differentiated for 3 weeks were able to spontaneously and strongly adhere to fresh ex vivo human articular cartilage pieces (Figure 14). Differentiated 3D hBM-MSC sheets (Figure 14A) strongly adhered to fresh ex vivo human articular cartilage surfaces within 1 h (Figure 14B) and remained adherent for at least 3 days during continued culture. Safranin O staining after 3 days of coculture showed tight physical adhesion between the sheet and the cartilage surface, with little or no gaps along the interface (Figure 14C). Laminin staining after 3 days of coculture was strongest at the interface between the sheet and the cartilage surface (Figure 13E), supporting the possibility of continuous adhesion and biological coupling between the cell sheet and the target cartilage tissue.

[0096] 3. Discussion Many studies have demonstrated that MSCs exhibit increased chondrogenic potential in 3D structures compared to 2D structures. Unlike 2D cell cultures, 3D cultures allow cells to adopt a rounded cell morphology associated with the cytoskeletal organization of chondrocytes. Contracted cell sheets spontaneously generate this 3D environment in which cells adopt a more rounded and less elongated cytoskeletal structure. This directly relates to the chondrogenic potential of the cells. The cytoskeletal reorganization and 2D to 3D transition seen in contracted cell sheets upon temperature-mediated detachment (Figures 1 and 10) is most likely caused by a change in stromal cell tensegrity. In this change, cell release from fixed / adherent cultures allows spontaneous contraction of actin filaments, encouraging contraction of cells within the cell sheet.

[0097] The cell sheet technology spontaneously detaches cells as confluent sheets without collecting enzymes or compromising endogenous cell-cell and cell-ECM interactions, and maintains endogenous cell contractile forces due to collective interactions along actin filaments, which stimulates sheet contraction as a continuous unit. This post-detachment cell sheet contraction spontaneously creates multinucleated, thick, scaffold-free 3D cell sheet structures (Figure 1) and induces cytoskeletal reorganization (Figure 10). These changes in cytoskeletal structure may also stimulate mechanotransduction and mimic early chondrogenic condensation by altering both cell shape and ECM structure, leading to increased cell-cell interactions via β-catenin, as well as upregulation of the pro-chondrogenic signaling molecules BMP2 (a regulator of cell condensation) and COMP (a regulator of collagen accumulation and ECM assembly) prior to chondrogenic induction.

[0098] Structural transition and upregulation of pro-chondrogenic signaling prior to chondrogenic induction results in 3D contractile cell sheets that achieve a chondrogenic phenotype after induction with chondrogenic medium (Figure 11). Healthy articular cartilage has a hyaline chondrogenic phenotype that includes, among other things, an ECM rich in collagen type II and proteoglycans (allowing resistance to shear, compressive, and tensile forces), expression of SOX9 and aggrecan, low expression of collagen type I, nuclei in lacunar structures, and low cell density compared to the ECM. The harvested 3D contractile sheets successfully achieve the standard and accepted benchmarks of their hyaline-like phenotype after differentiation, namely, prominent collagen type II and proteoglycan content in the ECM, high expression of common hyaline cartilage markers (SOX9, COL2A1, ACAN), low expression of collagen type I resulting in a high COL2A1 / COL1A1 ratio, and rounded cell structure with nuclei in lacunar structures at relatively low cell density. In addition to ECM composition (i.e., proteoglycan, aggrecan, and type II collagen content), ECM deposition (as seen in differentiating 3D contractile sheets) is also associated with hyaline cartilage differentiation. Cytoskeletal reorganization within 3D contractile cell sheets prior to chondrogenic induction upregulates COMP and BMP2, which are directly associated with ECM assembly and collagen accumulation, resulting in significantly more ECM deposition in 3D cell sheets than in 2D cultures following chondrogenic induction. 3D cell sheets generated from spontaneous contraction upon temperature-mediated detachment from the culture surface are initially highly cell-dense structures. However, ECM deposition, which significantly increases the thickness of 3D cell sheets during chondrogenesis, reduces the overall cell density of the construct. This reduction in overall cell density from ECM deposition results in a hyaline-like tissue construct that more closely matches native hyaline cartilage structure and cell distribution.

[0099] The main limitation of MSC chondrocyte differentiation towards a hyaline-like phenotype is the inevitable progression of MSC-derived chondrocytes towards hypertrophy and fibrocartilage, both in vitro and in vivo. It is clear that a 3D structure is necessary for proper hyaline-like chondrocyte differentiation, but a specific threshold must be determined, as construct thickness and cell density affect medium diffusion, and the generation of low oxygen pressure regions and increased nutrient diffusion gradients in thick tissues have been shown to affect chondrocyte differentiation and hypertrophy. In the present study, 3D cell sheets showed a similar chondrogenic developmental progression in vitro compared to control 3D pellet cultures, but the development of hypertrophic characteristics was delayed (Figure 12). Although the driving mechanism of hypertrophy of MSC-derived chondrocytes in vitro is still largely unknown, the delayed development of hypertrophic characteristics of MSC sheets observed in this study is most likely due to the structural properties of the sheets. In particular, the 3D cell sheets used in this study were thinner and had reduced cell density compared to the control 3D pellet cultures, which promoted more substantial and uniform medium diffusion throughout the construct, allowing chondrogenesis to be driven primarily by medium supplementation rather than cell signaling. Cell signaling during chondrogenic differentiation, for example, continuous overexpression of β-catenin, has been linked to chondrocyte hypertrophy induction in vitro via activation of the canonical Wnt pathway. The thicker 3D pellet cultures, combined with the higher cell density, present a barrier to sufficient nutrient diffusion, which may increase the reliance on cell-cell interactions (i.e., upregulation of β-catenin (Figure 12)) to propagate chondrogenic cues to cells in the core of the pellet that are poorly exposed to free medium. Together, these data suggest that tuning the thickness and cell density of the 3D cell sheet constructs may modulate cell interactions during chondrogenesis and delay the occurrence of MSC-derived chondrocyte hypertrophy in vitro. It is important to assess and monitor any transition towards hypertrophic or fibrocartilage phenotypes during in vitro differentiation, as those phenotypes would be detrimental to establishing long-term therapeutic benefits in future in vivo studies that require stable hyaline-like tissue.Further in vitro studies are required to clarify the optimal construct thickness and the specific mechanisms driving this hypertrophic transition, and to identify the most effective sheet preparation parameters to generate hyaline cartilage in vitro while preventing hypertrophy of MSC-derived chondrocytes.

[0100] Although various differentiation platforms have been successful in promoting chondrogenic differentiation of MSCs to a hyaline-like phenotype in vitro, none of these differentiation products have been successfully translated into human applications. For example, pellet cultures have been used primarily for in vitro validation of differentiation potential rather than in vivo therapeutic applications due to limited adhesion capacity and limited homogeneity of the regenerated tissue. Clusters of pellet cultures have been used to fill cartilage defects in vivo, showing some ability to assemble into the negative space left by the constraints of the pellet's spherical shape. However, these pellet clusters do not generate homogenous tissue, do not adhere strongly to biological surfaces without additional adhesives, or support the membrane containing them at the defect site.

[0101] One unique advantage of cell sheet technology is its ability to directly and spontaneously implant cells into target tissue sites without scaffolds or support materials, and also provide a stable cell culture environment for interaction with native tissues, through the retention of endogenous ECM, cellular interactions, and intact adhesion proteins. Our data show that cell sheets can be migrated and attached to biological surfaces after differentiation, and that this migration does not affect structural or chondrogenic properties (Figure 13). The maintenance of chondrogenic and structural properties of the sheets after manipulation and migration is promising for rapid replacement of damaged or missing hyaline cartilage. These differentiated cell sheets were also able to strongly adhere to fresh ex vivo human cartilage and could potentially initiate interfacial interactions with native chondrocytes within 3 days (Figure 13). These endogenous adhesive capabilities are likely due to the retention of adhesion molecules along the basal surface of the differentiated sheets that are not abundantly present along the periphery of pellet cultures, contributing to the disparity in transplantation potential. The retention of surface adhesion molecules is predicted to aid in the engraftment and localization of cell sheets at the cartilage defect site. It is expected that the close physical and biochemical interfacial interactions between the hyaline-like cell sheet and native cartilage will help maintain the hyaline properties of the sheet in vivo through direct chondrogenic signaling from the host cartilage. This ex vivo experiment represents an ideal interaction between the cell sheet and healthy articular cartilage in the absence of all other defect microenvironment factors. In order to fully verify the sheet transplantation ability and therapeutic effect of the chondrogenic cell sheet in localized articular cartilage defects, in vivo studies with long-term follow-up and detailed focus on regeneration and immune responses will be required.

[0102] In this study, we demonstrate that: 1) MSC sheets are capable of chondrogenic differentiation into hyaline cartilage in vitro without scaffold material after spontaneous contraction of the cell sheets after detachment via structural transformation and cytoskeleton reorganization. 2) These 3D MSC sheets provide suitable initial thickness and cell density that delay hypertrophy while maintaining a hyaline-like chondrogenic phenotype in vitro. 3) After differentiation, these 3D cell sheets can spontaneously adhere directly to cartilage surfaces, maintain structural and chondrogenic properties, and potentially interact interfacially with native tissues via retained adhesive proteins. Based on these findings, 3D MSC sheets represent a unique platform to develop allogeneic, implantable, scaffold-free hyaline cartilage constructs for articular cartilage regeneration therapy.

[0103] 4. Conclusion The cell sheet-based technique presented in this study represents an improved strategy to fabricate scaffold-free cartilage constructs with hyaline-like properties in vitro. Furthermore, the hyaline-like chondrogenic differentiated 3D MSC sheets initiate spontaneous adhesion and interfacial interactions with cartilage tissue without compromising the structural or chondrogenic properties. This cell sheet-based technique using MSCs should provide a compatible platform to generate implantable hyaline-like constructs in vitro to rapidly and directly replace damaged hyaline articular cartilage.

[0104] [Example 3] The transplantation ability of the chondrocyte-differentiated cell sheet described in Examples 1 and 2 was evaluated in vivo in a rat model of local cartilage defect. A 2 mm local defect was created in the trochlear groove of the hind leg of nude (RNU) rats (see FIG. 15A). Successful defect creation removes the full thickness of the cartilage without destroying the subchondral bone (little or no bleeding from the subchondral bone is observed). A 3-week chondrocyte-differentiated hyaline-like human MSC-derived cell sheet was transplanted into the defect at the time of defect creation (see FIG. 15B). The sheet was allowed to adhere for about 30 minutes in a slightly humidified environment before closing the joint. Two weeks after transplantation, the knee joint was harvested (FIG. 15C) to evaluate the cell sheet transplantation ability. Cross-sectional histological analysis of the 3-week differentiated human cell sheet in the rat local defect after two weeks of transplantation was performed using Safranin O (FIG. 15D), Hematoxylin & Eosin (FIG. 26E), and human-specific vimentin immunohistochemical staining (FIG. 15F). Positive Safranin O staining indicated retention of the hyaline-like properties of the cell sheet in the transplanted area (Figure 15D). H&E staining indicated integration with the host tissue (Figure 15E). Vimentin is a type III intermediate filament protein expressed in mesenchymal derived cells, therefore human specific vimentin can be used to specifically identify human cells within a xenogeneic host. Human cells transplanted within the cell sheet were positive for human specific vimentin, indicating engraftment and retention in the transplanted area (Figure 15F). Scale bar = 200 μm. Overall, these results indicate that chondrocyte-differentiated hyaline-like human MSC-derived cell sheets can be successfully transplanted into cartilage and integrated with host tissue while maintaining hyaline-like properties.

Claims

1. A chondrogenic cell sheet comprising at least two layers of confluent chondrogenic differentiated cells, the cell sheet being prepared from mesenchymal stem cells (MSCs), the chondrogenic differentiated cells on the basal side of the cell sheet express one or more adhesion molecules, and the cell sheet comprising an extracellular matrix comprising type II collagen and proteoglycans, with low expression of type I collagen.

2. The chondrogenic cell sheet of claim 1, wherein the MSCs are human bone marrow MSCs (hBM-MSCs).

3. The chondrogenic cell sheet according to claim 1 or 2, wherein the cell sheet essentially consists of chondrogenic differentiated cells.

4. 3. The chondrogenic cell sheet according to claim 1, wherein at least 50% of the cells in the cell sheet are chondrogenic differentiated cells.

5. The chondrogenic cell sheet according to claim 1, wherein the chondrogenic differentiated cells express a protein selected from the group consisting of SOX9, aggrecan, COL2A1, ACAN, COMP and BMP2.

6. The chondrogenic cell sheet according to any one of claims 1 to 5, wherein the cell sheet comprises a lacunar structure.

7. The chondrogenic cell sheet according to any one of claims 1 to 6, wherein the one or more adhesion molecules are selected from fibronectin and laminin.

8. The chondrogenic cell sheet according to any one of claims 1 to 7, wherein the cell sheet exhibits physical adhesion to the cartilage surface after transplantation onto the cartilage surface.

9. A method for preparing a chondrogenic cell sheet, comprising: a) Mesenchymal stem cells (MSCs) are cultured in a temperature-responsive incubator until they become confluent to form a cell sheet; b) peeling off the cell sheet by decreasing the temperature, shrinking the cell sheet, and forming a shrunken cell sheet; c) contacting the contracted cell sheet with a culture surface; and d) Treating the contracted cell sheet on the culture surface with a chondrogenic medium and culturing it to form a chondrogenic cell sheet. wherein the cell sheet comprises an extracellular matrix comprising type II collagen and proteoglycans, with low expression of type I collagen.

10. 10. The method of claim 9, wherein the contracted cell sheet is grown on the culture surface for at least 3 weeks.

11. 11. The method according to claim 9 or 10, wherein the chondrogenic medium comprises a protein selected from the group consisting of transforming growth factor beta (TGFβ) and bone morphogenetic protein (BMP).

12. The method of claim 9, wherein the cell sheet in step b) is detached from the temperature-responsive culture vessel without treating the cell sheet with one or more enzymes.

13. A chondrogenic cell sheet produced by the method according to any one of claims 9 to 12.

14. A pharmaceutical comprising the chondrogenic cell sheet according to any one of claims 1 to 8 or 13, for transplantation into the tissue of a subject in need of transplantation.

15. The pharmaceutical composition of claim 14, wherein the tissue is selected from cartilage and bone.

16. The method according to claim 15, wherein the cartilage is articular cartilage.

17. 14. A pharmaceutical composition for repairing cartilage tissue in a subject in need thereof, comprising the chondrogenic cell sheet according to any one of claims 1 to 8 and 13.

18. The pharmaceutical composition according to any one of claims 14 to 17, wherein the cartilage is articular cartilage.

19. The pharmaceutical composition of any one of claims 14 to 18, wherein the subject has a localized cartilage defect.

20. The pharmaceutical composition of any one of claims 14 to 18, wherein the subject has a symptomatic cartilage defect.

21. The pharmaceutical composition of claim 20, wherein the symptomatic cartilage defect is caused by acute or repetitive trauma.

22. The medicament according to any one of claims 14 to 18 and 20 to 21, wherein the subject has a degenerative joint disease.

23. 14. A pharmaceutical composition comprising the chondrogenic cell sheet according to any one of claims 1 to 8 and 13, for treating or preventing a joint disease in a subject in need thereof.

24. The pharmaceutical composition of claim 23, wherein the joint is selected from the group consisting of a synovial joint and a cartilaginous joint.

25. 25. The pharmaceutical composition of claim 24, wherein the synovial joint is a knee joint, a wrist joint, a shoulder joint, a hip joint, an elbow joint, or a neck joint.

26. The method according to claim 23, wherein the joint disease is a degenerative joint disease.

27. The pharmaceutical composition according to claim 23, wherein the joint disease is selected from the group consisting of arthritis, osteoarthritis, rheumatoid arthritis, and chondromalacia patellar.

28. 14. A pharmaceutical comprising the chondrogenic cell sheet according to any one of claims 1 to 8 and 13 for preventing osteoarthritis in a subject having symptomatic cartilage defects caused by acute or repetitive trauma.

29. The pharmaceutical composition according to any one of claims 14 to 28, wherein the subject is a human.

30. The pharmaceutical agent according to any one of claims 14 to 29, wherein the chondrocyte-differentiated cells in the cell sheet are allogeneic to the subject.

31. The pharmaceutical agent according to any one of claims 14 to 29, wherein the chondrocytes in the cell sheet are autologous to the subject.

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