Method for in vitro production of hyaline cartilage tissue

Abstract

This invention relates to a novel method for producing cartilage tissue in vitro, as well as methods for therapeutic uses and screening of the cartilage tissue produced therefrom.

Description

Technical Field

[0001] This invention relates to a novel method for producing hyaline cartilage tissue (“Cartibead”) in vitro, as well as therapeutic uses and screening methods for the cartilage tissue produced therefrom. Background Technology

[0002] Hyaline cartilage is composed of specialized cells called chondrocytes and a surrounding extracellular matrix. This matrix, synthesized and secreted by chondrocytes, is primarily composed of type II collagen fibers, glycosaminoglycans (GAG), and 60-80% water. Above the subchondral bone, hyaline cartilage has four layers: a superficial layer, an intermediate layer, a deep layer, and a calcified layer. The biomechanical properties of articular cartilage depend heavily on the composition and integrity of the extracellular matrix.

[0003] Cartilage has a very limited capacity for self-repair, and once injured, it usually develops into osteoarthritis (OA). Aging and repetitive trauma (such as during high-intensity physical activity) are major risk factors for knee cartilage degeneration. OA affects a large population and is most common in older adults (prevalence >10% in those over 60), while younger people are often affected by OA after joint injury.

[0004] Surgical treatment involves joint replacement with a prosthesis. However, the lifespan of a prosthesis is limited to 15-20 years. Furthermore, pain relief is not complete for most patients: 20% to 30% of patients with knee prostheses continue to experience discomfort or pain. Treatment is primarily palliative and aims to alleviate pain. Some biologics, such as mesenchymal stem cells, hyaluronic acid, or platelet-rich plasma injections, can slow joint degeneration, but they do not promote tissue regeneration.

[0005] Currently, strategies using external synthetic scaffolds to fill defects are unsatisfactory and cannot fully mimic the biomechanical properties of articular cartilage. Classic treatments include microfracture surgery to stimulate stem cell migration to the damaged area, or direct implantation of chondrocytes.

[0006] Microfracture surgery involves creating small openings into the subchondral bone to stimulate mesenchymal stem cells to migrate from the bone marrow to the coagulum to form new chondrocytes and replace damaged tissue.

[0007] In autologous chondrocyte implantation (ACI), chondrocytes are extracted from cartilage, cultured to a limited number of passages, and transplanted into the injury. To improve this method, matrices such as type I / III porcine collagen or hyaluronic acid can be used to culture chondrocytes (Kon E, et al., 2009, The American journal of sports medicine 37(1):33-41; Hettrich CM, Crawford D, & Rodeo SA, 2008, 16(4):230-235). These matrices are either applied directly to the injury after microfracture surgery or used in vitro to culture chondrocytes prior to reimplantation (Ekkers JE et al. 2013, Osteoarthritis Research Society 21(7):950-956).

[0008] Cell-based therapies using autologous chondrocyte transplantation (ACI) are suitable for cartilage lesions larger than 2 square centimeters (Armoiry X. et al. 2019, Pharmacoeconomics 37, 879-886). Chondrocytes represent an essential cellular source for cartilage regeneration. In fact, only this type of chondrocyte is involved in maintaining the hyaline cartilage matrix.

[0009] The main challenge is that chondrocytes often dedifferentiate into fibroblast-like cells during culture, leading to rapid loss of function. This typically occurs in the second or third cell passage in two-dimensional cell culture systems (Munirah, S. et al. 2010. Tissue & cell 42, 282-292). Dedifferentiated chondrocytes are characterized by the cessation of the production of glycosaminoglycans (GAG) and type II collagen, the main components of hyaline cartilage (Wu, L. et al. 2014. Tissue EngPart C Methods 20, 160-168; Benya, P. D et al. 1978. Cell. 15, 1313-1321), which are replaced by type I collagen in fibrocartilage. In fact, during the expansion step, chondrocytes often dedifferentiate by losing their original phenotype and become fibroblast-like cells (elongated cells) that exhibit stem cell-like characteristics in gene expression (expression of cell surface markers used to characterize mesenchymal stem cells (MSCs), such as CD73, CD90, and CD105). For example, chondrocytes extracted from patient articular cartilage rapidly lose their ability to synthesize specific hyaline matrix cartilage after several passages of in vitro culture. Fibrocartilage differs biomechanically from hyaline cartilage and is considered unsuitable for long-term treatment due to its inducing dysfunctional repair. In contrast, the hyaline properties of chondrocytes are known to be preserved in three-dimensional culture systems (Benya, PD & Shaffer, JD 1982. Cell 30, 215-224), thus mimicking the in vivo environment.

[0010] The loss of chondrogenic capacity during culture is particularly pronounced in older patients. Therefore, another challenge for chondrocyte-based cell therapies is the difficulty in expanding and differentiating cells from older patients, limiting their use in this population. Consequently, most clinical trials limit patient inclusion to 55 years of age.

[0011] If cell expansion can be performed while maintaining the original phenotype, then the idea of ​​using chondrocytes to produce cartilage would be reasonable. Therefore, there is still a need to develop a standardized method for producing cartilage to generate high-quality hyaline-like cartilage tissue (characterized by high levels of GAG detection) that mimics the intrinsic properties of patients of all ages. Summary of the Invention

[0012] The main finding of this study is a method that can reverse the loss of chondrocyte phenotype (chondrocyte dedifferentiation) during expansion. This method addresses a key issue encountered in cell therapy using chondrocytes as starting material. Current data indicate that the new three-step approach can produce high-quality cartilage with a transparent characteristic, regardless of patient age or the state of arthritis in the joint.

[0013] The Cartibead method allows for cell expansion from very small cartilage harvest samples (approximately 30 mg in our preclinical miniature pig study), compared to an average of 260 mg in conventional human chondrocyte-based cell therapy (Brittberg, M. 2018. Injury 39 Suppl 1, S40-49), thus reducing donor-site morbidity. This method showed a GAG / Cartibead ratio 20 times higher than previously reported for cartilage microtissues, suggesting lower transparency and higher fibrocartilage content in these microtissues (Bartz, C. et al. 2016. J Transl Med 14, 317). Consistent with these results, the inventors obtained an average GAG / DNA ratio at least three times higher than other published methods. Figure 2 (Mumme, M) (to the right of the right). et al. 2016. Lancet 388, 1985-1994; Dang, P. N et al. 2014. Tissue engineering. Part A 20, 3163-3175).

[0014] Redifferentiation was due to the removal of FGF-2. However, its removal in 3D culture was insufficient to induce clear matrix synthesis in a two-step process. Chondrocyte redifferentiation likely requires cell adhesion to a matrix-coated flask and the induction of specific cell signaling pathways in 2D culture.

[0015] The inventors have developed a novel method to expand chondrocytes in culture and restore their cartilage-forming capacity from a small piece of cartilage (~30 mg), regardless of patient age. In the first step, cells are expanded in a two-dimensional culture on an extracellular matrix with the support of FGF-2. Then, in the second step, pre-redifferentiation towards the original phenotype is achieved by removing FGF-2 used for cell expansion. In the final step, complete redifferentiation is achieved in a three-dimensional (3D) culture, where cells spontaneously aggregate to form microtissues. The inventors have surprisingly found that by specifically removing the FGF-2 growth factor in the second step in the 2D culture, chondrocytes begin to restore their original function by synthesizing aggrecan (a protein that binds to GAG), and by maintaining FGF-2 removal in the 3D culture in the third step, collagen II is synthesized along with GAG, resulting in a hyaline matrix specific to articular cartilage. The inventors further demonstrated that the removal of FGF-2 after the amplification step induced the inactivation of the Wnt signaling pathway (preferably the Wnt7B signaling pathway), thus allowing the generation of homogeneous hyaline cartilage highly representative of in vivo cartilage. The inventors showed that Wnt7B was activated in dedifferentiation medium (ME), reduced at least 10-fold in pre-redifferentiation medium (MR), and reduced at least 40-fold in maturation medium (MI). Transplantation of high-quality hyaline microtissue with characteristics as close as possible to natural cartilage is key to the long-term success of cartilage injury treatment (Negoro, T et al. 2018..npj Regenerative Medicine 3, 17; Madeira, C et al. 2015. Trends inbiotechnology 33, 35-42).

[0016] Therefore, engineered cartilage (“Cartibead”) has high potential to be fully integrated into cartilage lesions after transplantation.

[0017] Therefore, the present invention relates to a method for in vitro production of cartilage tissue, the method comprising: a first step of culturing chondrocytes (preferably isolated from a subject, more preferably from cartilage tissue of a human or equine subject) in an adherent culture system in a dedifferentiation medium that activates the Wnt signaling pathway to obtain fibroblast-like cells, preferably over a period of 10-15 days; a second step of culturing the fibroblast-like cells in an adherent culture system in a redifferentiation medium that inactivates the Wnt signaling pathway to obtain chondrocytes, preferably over a period of 4-8 days; and a third step of culturing the chondrocytes obtained in the second step in a maturation medium that inactivates the Wnt signaling pathway in a three-dimensional culture system, preferably over a period of 10-15 days. In a preferred embodiment, the dedifferentiation medium contains FGF-2, and preferably, the redifferentiation medium and / or maturation medium is an FGF-2-free medium. The dedifferentiation medium may contain at least one growth factor selected from the group consisting of PDGF-BB, TGF-β, and EGF. The redifferentiation and maturation media may contain TGF-β, preferably TGF-β3, more preferably TGF-β3 and FGF-7. In another embodiment, the redifferentiation media may contain platelet lysate. In particular, all the media described above may contain serum. The maturation media may contain at least one component selected from the group consisting of insulin, IGF-1, BMP-2, selenium, transferrin, and ethanolamine.

[0018] In one specific implementation, the chondrocytes are cultured in a hypoxic environment containing less than 10% O2 (v / v) in the third step.

[0019] In another aspect, the present invention relates to engineered cartilage tissue in the form of spheroids, having a GAG / double-stranded DNA ratio of at least 15. This engineered cartilage tissue can be used to treat cartilage defects and degenerative cartilage diseases in subjects with such needs, preferably through autologous transplantation.

[0020] Finally, the present invention also relates to a method for screening molecules that inhibit the degenerative process of cartilage, the method comprising contacting the engineered cartilage tissue described above with one or more candidate molecules and selecting molecules that can inhibit the degenerative process of cartilage.

[0021] Brief description of the attached figures

[0022] Figure 1: Characterization of Cartibead engineered in a three-step process. a, Schematic diagram of the three-step process for generating Cartibead derived from dedifferentiated chondrocytes: Steps 1 (amplification) and 2 (redifferentiation) were performed under 2D and atmospheric oxygen conditions (21%), and Step 3 (Cartibead formation) was performed under 3D culture and hypoxic conditions (5%). b, Figure from preclinical studies: Evaluation of the safety and efficacy of human Cartibead in SCID mice and miniature pigs. c, Histological analysis of Cartibead from fixed samples. Representative images of Cartibead obtained from three independent donors, GAG Safranin-O staining characterizing hyaline cartilage (top inset) and strong immunoassay for type II collagen (DAB staining, middle inset), and weak detection of type I collagen (bottom inset). Scale bar: 200 µm. d. Biochemical quantification of glycosaminoglycan (GAG) content in Cartibead, expressed in µg / mg tissue, determined by the dimethylmethylene blue assay (DMMB), from 15 donors (black dots) and 3 natural cartilage controls (boxes). e. Biomechanical properties of Cartibead were determined by compression tests (Young's modulus) to measure Cartibead elasticity. The stress-strain curves are representative of the three donors and show increased constraint in larger Cartibeads.

[0023] Figure 2 : GAG content in Cartibeads from all donors. Left side: Biochemical quantification of GAG content in each Cartibead by DMMB assay. Right side: Biochemical quantification of GAG content in Cartibeads normalized relative to DNA content, expressed as GAG / DNA ratio (µg / µg).

[0024] Figure 3 Comparison of Cartibead generated under hyperoxia and hypooxia levels, and in three-step and two-step methods. a) Macroscopic view of Cartibead generated from three donors under hypoxic conditions using the three-step method. b) Safranin O staining of GAG in Cartibead sections generated from one donor under four different conditions. Cartibeads were generated under hyperoxia and hypooxia levels (21% and 5%) via the three-step and two-step methods. The images are representative of the three donors. Scale bar is 200 µm. c) Assessment of GAG / Cartibead ratios from the three donors under four different conditions.

[0025] Figure 4Stability of Cartibead. a) Biochemical quantification of GAG in Cartibead after the three-step process, and in Cartibead retained for an additional 6 days in a closed recipient at 37°C (5% O2 incubator), 4°C, and room temperature (RT). b) Quantitative assessment of the number of dead cells in Cartibead by staining with red fluorescent dye. The positive control of dead cells was Cartibead pretreated with 10% triton before fluorescent staining. Scale bar: 100 µm.

[0026] Figure 5: The transparency characteristics of Cartibeads engineered by the three-step method are improved compared to the two-step method. a, The two-step protocol for generating Cartibeads. Chondrocytes are expanded in culture medium E in step 1 and directly used to generate Cartibeads in culture medium I in step 2. b, Functional annotation clustering based on gene set enrichment analysis of three-step and two-step expression. Histograms show the number of genes in each gene family that were found to be significantly enriched. Light gray represents genes with high expression levels in the three-step Cartibeads, and dark gray represents genes with low expression levels. c, Visualization of RNAseq results using a volcano plot, showing statistical significance (FDR < 0.001) and magnitude of change (> 2-fold change). The volcano plot highlights genes with statistically significant fold changes, such as... ACAN , COL2A1 (These genes are increased in the three-step method compared to the two-step method) and COL1A1 (This gene is reduced.) p<0.001; p < 0.01. d, e, and f are data from RNASeq analysis, comparing the three-step and two-step methods. ACAN (d) COL2A1 (e) and COL1A1 (f) mRNA expression level of the gene (denoted as RPKM). NA (Non Applicable) indicates that the analysis was not performed due to the absence of the step itself. g, Safranin-O staining of GAG in Cartibead produced by the three-step and two-step methods. Scale bar is 100 µm.

[0027] Figure 6MSC marker expression. a, Flow cytometry, from left to right representing CD73, CD90, and CD105. b, Differentiation study, comparing the potential of adipose-derived stem cells (ASC), mesenchymal stem cells (MSC), and chondrocytes. Alizarin Red S is a dye used for staining calcium deposition, an indicator of mature bone cells (top inset). Oil Red O staining shows neutral lipid characteristics in adipocytes (middle inset), and Safranin-O staining shows GAGs in chondrocytes (bottom inset). Scale bar: 100 µm.

[0028] Figure 7: Inhibition of WNT signaling enhances the formation of clear matrix components. a, Gene set enrichment analysis and functional grouping based on differential expression levels in medium R and medium E in a three-step method. The figure shows the enrichment of each gene family (number of differentially expressed genes). p<0.001; p<0.01. b, Volcano plot of RNA-seq results for chondrocytes in media R and E in the three-step method. c, Data from RNA-seq analysis, showing... ki67 (Proliferation markers) (top small image) TCF4 (Involved in the downstream of the WNT / β-catenin pathway) (small figure) SOX9 (mRNA expression of transcription factors involved in matrix production) (inset below) (denoted as RPKM). d, e, f, data from RNA-seq analysis showing chondrocytes in media E, R, and I in the three-step and two-step methods. WNT5A (d) WNT5B (e) and WNT7B (f) mRNA expression (denoted as RPKM). NA (Not Applicable) indicates that no analysis was performed due to the absence of the step itself. g, Immunoblotting shows the expression of the proteins shown in chondrocytes in the presence of the WNT signaling inhibitor XAV-939 (10 µM). Histograms show the density assays of β-catenin and Axin compared to controls. h, i, qPCR was used to determine the expression of proteins in chondrocytes cultured for 4 days in media E, media E+XAV-939, and in Cartibead (two-step method) from chondrocytes cultured in media E and media +XAV-939. ACAN and COL2A1mRNA expression. j, Representative diagram of Safranin O staining of GAG in Cartibead produced from chondrocytes cultured in medium R (three-step method) and medium E + XAV-939 (two-step method). Scale bar is 100 µm. k, This protocol summarizes the molecular basis of the WNT signaling pathway in medium E and the inhibition of WNT when chondrocytes are cultured in medium R or medium E + XAV939 (allowing for the production of a clear matrix containing ACAN and type II collagen).

[0029] Figure 8 Feasibility of Cartibead grafting in ex vivo human knees. a. Macroscopic view of the distal femur after total knee arthroplasty, showing manually induced damage using surgical instruments (left), Cartibead grafted on day 1 (middle), and damage using the graft one month after grafting following rotational culture in medium I (right). Scale bar: 4 mm. b. GAG Safranin-O staining of cross-sectional samples of the ex vivo specimen, showing the Cartibead fused together, remaining transparent and integrated with the natural cartilage, displayed at higher magnification (left and right insets). Data are representative of three independent experiments. Scale bar: 1 mm.

[0030] Figure 9 Human Cartibead showed no tumorigenicity after implantation into immunodeficient mice. a, Subcutaneous implantation into the back of mice (black arrow, left inset), 2 months post-implantation, Cartibead (0.2 x 10⁻⁶) showed no tumorigenicity. 6 (Top image: 0.2 x 10⁻⁶ chondrocytes / cartibead) and A549 cells (0.2 x 10⁻⁶) derived from 5 beads 6 weeks post-grafting. 6 a) Representative image of a tumor (bottom inset) with 1 A549 cell / bead. b) Tumor growth after subcutaneous implantation of 1, 1, or 5 A549 cells per Cartibead into each SCID mouse (one Cartibead donor consists of n = 10 to 14 mice, and each A549 bead is 2 x n = 4 mice). c) Safranin-O staining of GAGs in the Cartibead before (middle inset) and after (right inset). Scale bar: 200 µm.

[0031] Figure 10Integration of the hymenoplasty graft into the knee of miniature pigs following autologous Cartibead transplantation. a, The protocol describes the primary biopsy sites used to generate Cartibead and the second surgery that combined five lesions / knees with autologous Cartibead transplantation. b, Biochemical quantification of GAG (µg / Cartibead) in six miniature pigs. cd, Macroscopic view (left inset) and Safranin-O staining of GAG in miniature pig knee sections (middle inset), magnified areas showing tissue remodeling at 3 and 6 months post-transplantation. Transplantation sites are indicated by black circles, while white circles represent empty lesions as controls (right inset). Representative staining of transplanted lesions (middle inset) and empty lesions (right inset) at three months (c) and six months (d) is shown. Scale bar is 200 µm.

[0032] Figure 11 Transplantation of porcine Cartibead into miniature pigs produced transparent, integrated grafts. a. Safranin-O staining of GAGs in the Cartibead before transplantation (top inset, scale bar 200 µm), macroscopic view of the knee (medium inset), and Safranin-O staining of GAGs in the transplanted lesion (bottom inset, scale bar 200 µm) in the six miniature pigs used in this study. Invention Details

[0034] This invention relates to a method for producing cartilage tissue in vitro, the method comprising the following three steps of culturing chondrocytes.

[0035] Chondrocytes originate from mesenchymal cells and possess a characteristic phenotype primarily based on the type of extracellular matrix they produce. Precursor cells produce type I collagen, but when they orient themselves into the chondrocyte lineage, they reduce type I collagen production and begin synthesizing type II collagen, the fundamental protein constituting the clear extracellular matrix. Furthermore, oriented chondrocytes produce proteoglycans, which are aggregated proteoglycans bound to highly sulfated glycosaminoglycans.

[0036] As used herein, the term "chondrocyte" refers to a fully differentiated cell (directed chondrocyte) derived from cartilage with the ability to synthesize a clear matrix. The term "chondrocyte" also refers to cells cultured in primary culture (freshly isolated from biopsies) and expanded in vitro, including genetically modified, immortalized, selected, and preserved cells.

[0037] Isolation of chondrocytes from the subject

[0038] According to the method of the invention, in the first step, chondrocytes are isolated from the subject, preferably from the knee, ankle, hip, finger, or shoulder of the subject, and more preferably from human or equine subjects. Chondrocytes are isolated from biopsies of mature cartilage tissue using conventional methods, such as enzymatic digestion of the tissue, including trypsin, chymotrypsin, collagenase, deoxyribonuclease, elastase, and hyaluronidase. The cartilage tissue can be a very small piece, preferably less than 5 mm in diameter (equivalent to about 30 mg). The cartilage tissue is preferably mammalian, more preferably human or equine cartilage. The cartilage tissue can be collected from a healthy donor or patient, preferably from the knee, ankle, hip, finger, or shoulder of a patient, and more preferably from the knee or ankle of a patient.

[0039] Step 1: Adherent culture in dedifferentiation medium

[0040] First, the isolated chondrocytes were cultured in dedifferentiation medium on an adherent culture system.

[0041] The adherent culture system suitable for the method of the present invention can be an adherent monolayer culture system or a culture system on feeder cells, preferably an adherent monolayer culture system. The culture system can be in any form suitable for the method of the present invention, especially in the form of a flask, multiwell plate or culture dish.

[0042] According to a preferred embodiment, the adherent culture system is an adherent monolayer culture system. This system comprises a solid support, such as glass or plastic, which is typically coated with a matrix or substrate that promotes cell adhesion. The substrate can be a protein substrate composed of adhesion factors that promotes cell adhesion to the support. These adhesion factors are particularly selected from poly-L-lysine, collagen, fibronectin, laminin, or gelatin.

[0043] Matrix mimicking the extracellular matrix and suitable for the methods of the present invention are well known to those skilled in the art, and many types are commercially available. These matrices include, for example, Matrigel™, Geltrex® type, CELLstart™ matrices, or other matrices containing one or more anchoring proteins such as collagen, laminin, fibronectin, elastin, proteoglycans, glycosaminoglycans, or fibronectin. Three-dimensional hydrogel-type matrices may also be used. According to a preferred embodiment, the matrix is ​​a Matrigel™ type or a CELLstart™ substrate.

[0044] Chondrocytes are cultured in an adherent culture system in a culture medium, which enables the expansion and dedifferentiation of cells in a progenitor / undifferentiated state (referred to as the fibroblast state (fibroblast-like morphology)). Specifically, the dedifferentiation medium is a medium that allows chondrocytes to proliferate and dedifferentiate into fibroblastic-like cells, also known as fibroblast-like chondrocytes, which are dedifferentiated chondrocytes with a fibroblast-like morphology and exhibit stem cell characteristics. Preferably, the dedifferentiation medium allows chondrocytes to dedifferentiate into fibroblast-like cells expressing mesenchymal stem cell surface markers such as CD105, CD90, and / or CD73. Fibroblast-like cells are dedifferentiated chondrocytes that have lost the ability to synthesize a clear matrix (collagen 2 and GAG). The dedifferentiation medium is a basal medium containing at least one or more components that allow chondrocytes to expand and dedifferentiate in an undifferentiated state.

[0045] Many basal culture media are commercially available and well-known to those skilled in the art. These media can be minimal media, particularly containing cell-essential mineral salts, amino acids, vitamins, and carbon sources, as well as a buffering system for pH adjustment. Basal culture media that can be used in the methods of this invention include, for example, but not limited to, DMEM / F12 medium, DMEM medium, RPMI medium, Ham's F12 medium, IMDM medium, and KnockOut™ DMEM medium (Life Technologies).

[0046] Depending on the culture medium used, it may be necessary or desirable to add glutamine, vitamin C, one or more antibiotics such as streptomycin, penicillin, and / or antifungal agents such as fungizone (amphoteric B).

[0047] According to the present invention, the dedifferentiation medium activates the Wnt signaling pathway.

[0048] In its active state, the Wnt ligand binds to the frizzled receptor and their common receptor, receptor-related protein (LRP) 5 / 6. This activates disheveled protein (DSH), which subsequently inhibits GSK3β activity and β-catenin phosphorylation. β-catenin is transported to the nucleus, where it interacts with the transcription factors TCF / LEF (T cell-specific transcription factor / lymphocyte enhancer-binding factor). In its inactive state, β-catenin is phosphorylated by glycogen synthase kinase (GSK) 3β, and the phosphorylated β-catenin undergoes subsequent ubiquitination and proteasome degradation.

[0049] In a preferred embodiment, the dedifferentiation medium is a basal medium containing a compound that activates Wnt signaling, preferably Wnt7B signaling.

[0050] Activation of the Wnt signaling pathway can be determined by measuring the expression levels of Wnt RNA (especially Wnt7B, Wnt5A, or Wnt5B, preferably Wnt7B RNA) and downstream target genes (such as TCF4) in dedifferentiated chondrocytes. The Wnt signaling pathway is activated in cells when the expression levels of target genes are at least 1.5-fold, or 2, 3, 4, or 5-fold, compared to cells cultured without the compound.

[0051] The expression level of mRNA can be determined by any suitable method known to those skilled in the art. Typically, these methods involve measuring the amount of mRNA. Methods for determining the amount of mRNA are well known in the art. For example, nucleic acids contained in the sample are first extracted according to standard methods, such as using lysins or chemical solutions, or by extraction with a nucleic acid-binding resin according to the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e.g., Northern blotting analysis), amplification (e.g., RT-PCR), or sequencing (RNA-seq).

[0052] The level of the target gene protein can also be determined by any suitable method known to those skilled in the art. Typically, these methods involve contacting a cell sample, preferably cell lysate, with a binding partner capable of selectively interacting with the target gene protein present in the sample. The binding partner is typically a polyclonal or monoclonal antibody, preferably a monoclonal antibody. The amount of protein can be measured, for example, by semi-quantitative Western blotting, enzyme-labeled and mediated immunoassays such as ELISA, biotin / antibiotin assays, radioimmunoassays, immunoelectrophoresis or immunoprecipitation, or by a protein or antibody array.

[0053] In particular, compounds that can activate the Wnt signaling pathway can be Wnt proteins that bind to the Wnt receptor or small molecule GSK-3β antagonists, preferably WNT7B, WNT5A or WNT5B, more preferably WNT7B.

[0054] The compounds that can activate the Wnt signaling pathway are selected from the group consisting of: lithium chloride (CAS No. 7447-41-8), CHIR99021 (CAS No. 252917-06-9), SB-216763 (CAS No. 280744-09-4) and BIO (6-bromoindirubin-3'-oxime) (CAS No. 667463-62-9).

[0055] In a preferred embodiment, the compound is FGF2 (fibroblast growth factor-2), also known as bFGF or the basic form of fibroblast growth factor.

[0056] Activation of the Wnt signaling pathway can proliferate and dedifferentiate chondrocytes into fibroblast-like cells. In a preferred embodiment, the dedifferentiation medium contains at least 5, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 ng / mL of FGF-2. Preferably, the medium contains 5 to 100 ng / mL of FGF-2, more preferably 20 ng / mL of FGF-2.

[0057] According to the present invention, any molecule, such as a polypeptide, that acts on chondrocytes in at least one aspect in a manner similar to that of the FGF2 molecule, preferably amplifies chondrocytes in the method of the present invention, can be used.

[0058] Other components that aid in the proliferation and dedifferentiation of chondrocytes from undifferentiated cells are not particularly limited and can be appropriately selected according to the purpose. Examples of such components include bone morphogenetic protein (BMP), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), and insulin-like growth factor 1 (IGF-1). Each of the above-mentioned components can be used alone, or multiple of the above-mentioned components can be used in combination with compounds capable of activating the Wnt signaling pathway.

[0059] In one specific embodiment, the dedifferentiation medium may further comprise one or more growth factors selected from the group consisting of EGF, TGF-β, and PDGF-BB, and any combination thereof. In this embodiment, EGF, PDGF-BB, and TGF-β are each present at a concentration in the range of 1 to 100 ng / mL, preferably 5 to 50 ng / mL. In a more specific embodiment, the TGF-β is TGF-β3.

[0060] In one specific embodiment, in the method of the present invention, human chondrocytes are cultured in a dedifferentiation medium containing FGF-2, PDGF-BB, and TGF-β3, wherein the dedifferentiation medium preferably contains 5 to 100 ng / mL FGF-2, 1 to 100 ng / mL PDGF-BB, and 1 to 100 ng / mL TGF-β3, more preferably containing 5 to 50 ng / mL FGF-2, 5 to 50 ng / mL PDGF-BB, and 5 to 50 ng / mL TGF-β3.

[0061] In another specific embodiment, in the method of the present invention, equine chondrocytes are cultured in a dedifferentiation medium containing FGF-2 and EGF, wherein the dedifferentiation medium preferably contains 5 to 100 ng / mL FGF-2 and 1 to 100 ng / mL EGF, more preferably contains 5 to 50 ng / mL FGF-2 and 5 to 50 ng / mL EGF.

[0062] According to a preferred embodiment, the dedifferentiation medium further comprises animal-derived serum. In a preferred embodiment, autologous serum is used. Xenogeneic or allogeneic serum may also be used. In particular, for the culture of human chondrocytes, autologous serum or mixed human serum from human patients may be used. In a specific embodiment, for the culture of equine chondrocytes, fetal bovine serum may be used. The medium preferably contains 2 to 20%, more preferably 5 to 15%, and more preferably 10% serum.

[0063] According to a specific implementation plan, chondrocytes are contacted with dedifferentiation culture medium for 10 to 20 days, preferably 10 to 15 days.

[0064] Preferably, cells are subcultured periodically to prevent confluence, i.e., covering the entire usable surface. Confluence, in fact, leads to cessation of proliferation and unwanted metabolic changes. Cells can be subcultured using standard techniques known to those skilled in the art. In particular, they can be separated from the substrate or support by action of enzymes (e.g., collagenase IV), or by mechanical passage in PBS containing EDTA or any other enzyme-free solution (e.g., ReleSR, STEMCELL Technologies), or by action of commercial cell separation media (e.g., TrypLE™ Express, Life Technologies), collected by centrifugation, mechanically dissociated, and reseeded into a new culture system.

[0065] Step 2: Adherent culture in redifferentiation medium

[0066] By inactivating the Wnt signaling pathway used in the dedifferentiation medium during the second intermediate culture step to enable cell expansion, the inventors surprisingly found that, after culturing in a three-dimensional system, chondrocytes regained their complete original phenotype and full ability to synthesize a clear matrix.

[0067] Therefore, the method of the present invention further includes an intermediate step of culturing the fibroblast-like cells obtained in the first step in an adherent culture system as described above, using a redifferentiation medium that inactivates the Wnt signaling pathway (preferably Wnt7B, Wnt5A, or Wnt5B, preferably the Wnt7B signaling pathway). Chondrocytes are contacted with this redifferentiation medium to reverse the fibroblast phenotype and redifferentiate the fibroblast-like cells into chondrocytes with full capacity to synthesize a clear matrix. In this second step, the cells alter their morphology, becoming less elongated, larger, and exhibiting prominent granular endoplasmic reticulum in the cytoplasm under a light microscope, indicating high levels of protein synthesis activity. Chondrocytes begin to re-express aggregate proteoglycans, a proteoglycan that binds to GAGs. Collagen II is not re-expressed at the end of the second step.

[0068] Preferably, the contact is achieved by simply changing the culture medium. Alternatively, the contact can be achieved by subculturing in an adherent culture system containing a reversible culture medium as described above.

[0069] According to one embodiment, the adherent culture system is an adherent monolayer culture system as described above. According to a preferred embodiment, the substrate is Matrigel. TM Type or CELLstart TM Substrate.

[0070] In a first embodiment, the redifferentiation medium may be a redifferentiation medium containing a basal medium in which compounds that activate the Wnt signaling pathway, as previously described, have been removed.

[0071] The purpose of removing the compound is to ensure that the concentration of the compound contained in the redifferentiation medium does not activate the Wnt signaling pathway.

[0072] In a more preferred embodiment, the redifferentiation medium is an FGF-2-free redifferentiation medium. The FGF-2-free redifferentiation medium comprises a basal medium containing less than 0.5 ng / mL FGF-2, preferably less than 0.4, 0.3, 0.2, or 0.1 ng / mL FGF-2.

[0073] In another embodiment, the redifferentiation medium is a basal medium containing an inhibitor of the Wnt signaling pathway.

[0074] The Wnt signaling pathway inhibitor can be a small molecule selected from the group consisting of: compounds targeting the Dvl protein, such as NSC668036 (CAS No. 144678-63-7), 3289-8625 (CAS No. 294891-81-9), J01-017a, TMEM88, KY-02061, KY-02327, BMD4702 (CAS No. 335206-54-7), Nicolasamide (CAS No. 50-65-7), DK-520, and sulindac (CAS No. 38194-50-2); compounds targeting the β-catenin destruction complex, such as Pyrvinium (CAS No. 7187-62-4); and natural compounds, such as derricin (CAS No. 34211-25-1) and derricidin. (CAS No. 38965-74-1), oxalic acid (CAS No. 3650-09-7); compounds targeting TCF / LEF transcription reporters, such as ICG-001 (CAS No. 847591-62-2), PNU-74654 (CAS No. 113906-27-7), Windorphen (CAS No. 19881-70-0); compounds targeting Pren, such as IWP-L6 (CAS No. 1427782-89-5), Wnt-C59 (CAS No. 1243243-89-1), LGK974 (CAS No. 1243244-14-5), ETC-159 (CAS No. 1638250-96-0); compounds targeting TNKS, such as XAV939 (CAS No. 38965-74-1). 284028-89-3), E7449 (CAS No. 1140964-99-3), with XAV939 being the preferred choice.

[0075] In another embodiment, small inhibitory RNA (siRNA) can also be used to reduce the gene expression level of at least one protein involved in Wnt pathway signaling. In a preferred embodiment, the gene expression of the Wnt signaling protein can be reduced by introducing small double-stranded RNA (dsRNA) or a vector or construct that induces the production of dsRNA into the cell, thereby inactivating the Wnt signaling pathway (i.e., RNA interference or RNAi). For genes with known sequences, the methods for selecting appropriate dsRNA or dsRNA-encoding vectors are well known in the art (see, for example, Tuschl, T. et al. (1999); Elbashir, SM et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, ​​TR. et al. (2002); U.S. Patent Nos. 6,573,099 and 6,506,559; and International Patent Publications Nos. WO 01 / 36646, WO 99 / 32619 and WO 01 / 68836).

[0076] In another implementation, short hairpin RNA (shRNA) can also be used to reduce the gene expression level of Wnt signaling proteins. Short hairpin RNA (shRNA) is an RNA sequence with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). shRNA expression in cells is typically accomplished via plasmid delivery or via viral or bacterial vectors. Promoter selection is crucial for achieving robust shRNA expression. Polymerase III promoters such as U6 and HI were initially used; however, these promoters lack spatial and temporal control. Therefore, polymerase II promoters have been used instead to regulate shRNA expression.

[0077] In a preferred embodiment, siRNA is used to reduce the gene expression level of Wnt5B, Wnt7B, Wnt5A or β-catenin, preferably Wnt7B.

[0078] The basal medium can be a minimal medium, specifically containing cell-essential mineral salts, amino acids, vitamins, and carbon sources, as well as a buffer system for pH adjustment. Basal media suitable for use in the methods of this invention include, for example, but not limited to, DMEM / F12 medium, DMEM medium, RPMI medium, Ham's F12 medium, IMDM medium, and KnockOut™ DMEM medium (Life Technologies).

[0079] In one specific implementation, the redifferentiation medium is an FGF-2-free redifferentiation medium that contains a basal medium containing less than 0.5 ng / mL FGF-2 and contains at least one or more components that promote the redifferentiation of fibroblast-like cells into chondrocytes.

[0080] In one specific embodiment, the redifferentiation medium contains transforming growth factor β (TGFβ), more preferably TGF-β3. Preferably, the redifferentiation medium contains 1 to 100 ng / mL TGF-β3, more preferably 5 to 50 ng / mL TGF-β3.

[0081] In a more specific embodiment, the redifferentiation medium further comprises FGF-7, also known as keratinocyte growth factor. FGF-7 is involved in cell proliferation and cell differentiation. Preferably, the redifferentiation medium comprises 1 to 100 ng / mL FGF-7, more preferably 5 to 50 ng / mL FGF-7.

[0082] According to one specific implementation, the redifferentiation medium used further contains animal-derived serum, as described above.

[0083] In a preferred embodiment, the TGF-β, FGF-7, and / or serum are replaced with platelet lysate. Platelet lysate is a growth factor-rich cell culture supplement derived from platelets after freeze-thaw cycles. Freeze-thaw cycles cause platelet lysis, releasing a large amount of growth factors required for cell expansion. In a preferred embodiment, the redifferentiation medium contains 5% to 20%, preferably 10%, of platelet lysate. In a preferred embodiment, the platelet lysate is a mixture of human platelet lysates.

[0084] There are no particular limitations on the components that can affect chondrocyte differentiation, and they can be appropriately selected according to the purpose. Examples of such components include insulin, insulin-like growth factor (IGF-1), transforming growth factor β, bone morphogenetic protein (BMP), selenium, transferrin, ethanolamine, and platelet-derived growth factor. Each of the other components mentioned above can be used alone, or multiple of the other components mentioned above can be used in combination.

[0085] According to a specific implementation plan, chondrocytes are contacted with redifferentiation culture medium for 2 to 10 days, preferably 3 to 7 days, more preferably 7 days.

[0086] Cells can be passaged periodically to prevent the culture from confluenced, i.e., covering the entire usable surface. In fact, confluence leads to cessation of proliferation and unwanted metabolic changes. Cells can be passaged using standard techniques known to those skilled in the art as described above.

[0087] Step 3: 3D culture system in induction / maturation medium

[0088] To form cartilage tissue, the chondrocytes obtained in the second step are cultured in a maturation medium (also known as an induction medium) on a three-dimensional culture system. In this step, the chondrocytes produce their own extracellular matrix to form cartilage tissue; in particular, they begin to synthesize type II collagen. The resulting cartilage tissue is a three-dimensional tissue of varying sizes, which may be referred to as globules. These globules consist of cells contained within them and a transparent matrix formed by these cells.

[0089] A maturation medium is a basal medium that contains at least one or more components that allow chondrocytes to produce a transparent matrix and form cartilage tissue.

[0090] Specifically, the maturation medium inactivates the Wnt signaling pathway. In one embodiment, the maturation medium comprises a basal medium in which the compounds that activate the Wnt signaling pathway as described above have been removed. In another embodiment, the maturation medium is a basal medium containing an inhibitor of the Wnt signaling pathway as described above.

[0091] In a preferred embodiment, the maturation medium is a basal medium in which FGF-2 growth factor has been removed. In other words, the maturation medium is a basal medium containing less than 0.5 ng / mL FGF-2, preferably less than 0.4, 0.3, 0.2, or 0.1 ng / mL FGF-2.

[0092] There are no particular limitations on the components that can affect chondrocyte maturation, and they can be appropriately selected according to the purpose. Examples of such components include TGF-β growth factor, insulin, insulin-like growth factor (IGF-1), transforming growth factor β, bone morphogenetic protein (BMP), selenium, transferrin, ethanolamine, epidermal growth factor, and platelet-derived growth factor. Each of the other components mentioned above can be used alone, or multiple of the other components mentioned above can be used in combination.

[0093] According to a specific embodiment, the maturation medium is a basal medium as described above that further contains TGF-β, preferably TGF-β3.

[0094] Preferably, the maturation medium contains 1 to 100 ng / mL TGF-β3, more preferably 5 to 50 ng / mL TGF-β3.

[0095] According to one embodiment, the chondrocyte maturation medium is a basal medium containing TGF-β3, IGF-1, BMP-2, and insulin. Preferably, the chondrocyte maturation medium contains TGF-β3, insulin, IGF-1, BMP-2, selenium, transferrin, and ethanolamine; more preferably, it contains TGF-β3, insulin, selenium, transferrin, and ethanolamine.

[0096] Preferably, the maturation medium contains 1 to 100 ng / mL TGF-β3 (preferably 5 to 50 ng / mL TGF-β3), 1 to 100 ng / mL IGF-1 (preferably 5 to 50 ng / mL IGF-1), and / or 1 to 100 ng / mL BMP-2 (preferably 5 to 50 ng / mL BMP-2).

[0097] According to a specific implementation plan, chondrocytes are cultured in a maturation medium on a three-dimensional culture system for 10 to 20 days, preferably 10 to 15 days.

[0098] 3D culture enables cells to connect with each other, facilitating the synthesis of extracellular matrix by chondrocytes. 3D culture systems can be static or dynamic. Static methods refer to providing a way for cells to form aggregates due to static physical forces. Static methods include, but are not limited to, the hanging drop method, culture in liquid on a non-adhesive substrate (e.g., a thin coating of agar or agarose), and culture on low-adhesion plates. Dynamic methods refer to forced cell aggregation. Dynamic methods include, but are not limited to, spinner flask culture, rotating wallvessel, and pellet culture. In a preferred embodiment, the 3D culture system is pellet culture. In pellet culture, chondrocytes are dispensed into plates and centrifuged to aggregate the cells and form pellets.

[0099] In a preferred embodiment, 3D culture in the maturation medium is carried out in an anaerobic environment to improve chondrogenesis. In a preferred embodiment, chondrocytes are cultured in the maturation medium in a 3D culture system at an environment of less than 10% (v / v) O2, more preferably less than 7% O2, and even more preferably 5% O2.

[0100] Cells were maintained in chondrocyte maturation medium until cartilage tissue was obtained. As used herein, cartilage refers to hyaline cartilage. During this period, the medium was changed periodically, preferably every 2 or 3 days, in a routine manner.

[0101] The quality of the obtained cartilage tissue can be assessed by measuring the content of glycosaminoglycans (GAGs) and collagen II in the engineered cartilage tissue. The quality and quantity of the cartilage tissue can also be determined by measuring the GAG / microtissue quantity or by the GAG / double-stranded DNA ratio. The presence of GAG, collagen, or double-stranded DNA can be assessed by any method known in the art. For example, GAG can be visualized by safranin-O coloration or, more quantitatively, by using a dimethylmethylene blue assay (DMMB). Collagen II can be assessed by immunostaining or by more quantitative assays such as ELISA.

[0102] Therefore, optionally, the method of the present invention may include additional steps consisting of determining or evaluating the presence of GAG and / or collagen II in engineered cartilage tissue.

[0103] Engineered cartilage tissue (Cartibead)

[0104] The present invention also relates to engineered cartilage tissue that can be obtained by the methods of the present invention.

[0105] The present invention relates to engineered cartilage tissue in the form of spherical bodies, wherein the spherical bodies present a GAG content of at least 15, 16, 17, 18, 19 or 20 μg / spherical body, preferably between 10 and 100 μg / spherical body, and more preferably between 15 and 60 µg / spherical body.

[0106] The present invention relates to engineered cartilage tissue in the form of spherical bodies, wherein the spherical bodies exhibit a GAG / double-stranded DNA ratio of at least 10, 15 or 20, preferably 10 to 100, more preferably 10 to 80.

[0107] The spherical bodies of the cartilage tissue have a diameter of 1 to 2 mm and contain 50,000 to 250,000 cells, preferably 200,000 cells.

[0108] Pharmaceutical Composition

[0109] In another aspect, the present invention also relates to a pharmaceutical composition comprising the engineered cartilage tissue of the present invention and one or more pharmaceutically acceptable excipients.

[0110] Pharmaceutically acceptable excipients must be compatible with the cells and can be, for example, culture media, buffer solutions, or saline solutions.

[0111] In a preferred embodiment, the pharmaceutical composition is suitable for parenteral administration, preferably via a subcutaneous route, and especially for direct administration into cartilage or bone tissue. The pharmaceutical composition can be formulated according to standard pharmaceutical practices known to those skilled in the art.

[0112] In one specific embodiment, the pharmaceutical composition comprises the cartilage tissue of the present invention, encapsulated in a biocompatible matrix.

[0113] The pharmaceutical composition may also contain one or more additional active compounds, such as compounds known to improve cell survival or proliferation or prevent contamination.

[0114] Therapeutic applications

[0115] According to another aspect, the present invention relates to the therapeutic use of the engineered cartilage tissue of the present invention or a pharmaceutical composition comprising said engineered cartilage tissue in subjects in need of such treatment, particularly for the treatment of cartilage defects and degenerative cartilage diseases.

[0116] Therefore, this invention relates to engineered cartilage tissue for treating cartilage defects and degenerative cartilage diseases in subjects with such needs. It also relates to pharmaceutical compositions of this invention for treating cartilage defects and degenerative cartilage diseases in subjects with such needs.

[0117] This invention is particularly suitable for autologous cartilage tissue transplantation in subjects with this need. In the transplantation of autologous engineered cartilage tissue according to this invention, chondrocytes are first isolated from the subject, preferably from a biopsy of mature cartilage tissue from the subject. The chondrocytes are then cultured according to the method of this invention to obtain autologous engineered cartilage tissue, which is used to treat cartilage defects and degenerative cartilage diseases in the patient.

[0118] Therefore, in a preferred embodiment, the present invention relates to the use of autologous engineered cartilage tissue or pharmaceutical compositions comprising said autologous engineered cartilage tissue for treating cartilage defects and degenerative cartilage diseases in subjects with such needs.

[0119] In another implementation, allogeneic transplantation can be used for subjects who require it.

[0120] As used herein, the term “cartilage defects and cartilage degenerative disease” includes, but is not limited to, cartilage lesion, arthrosis, rheumatism, or osteoarthritis.

[0121] In one specific embodiment, the engineered cartilage tissue or pharmaceutical composition of the present invention is intended for use in patients suffering from focal articular cartilage damage or early osteoarthritis to delay or avoid total knee replacement surgery, which is generally not recommended for patients under 60 years of age due to the limited lifespan of prostheses. Therefore, in a preferred embodiment, the cartilage defects and degenerative cartilage disease are focal articular cartilage damage or early osteoarthritis.

[0122] As used in this article, the term "treatment" refers to the improvement or disappearance of symptoms, such as pain and decreased mobility, slowed disease progression, cessation of disease progression, or disappearance of the disease. The term also includes preventative and curative treatments.

[0123] As used herein, the terms “subject” or “patient” refer to a mammal. Mammal species that may benefit from the disclosed treatment methods include, but are not limited to, humans, non-human primates such as apes, chimpanzees, monkeys, and orangutans, domesticated animals including dogs and cats, and livestock such as horses, cattle, pigs, sheep, and goats, or other mammal species including, but not limited to, camels, mice, rats, guinea pigs, rabbits, hamsters, etc. In a specific embodiment, the subject is a human or a horse.

[0124] The present invention also relates to the use of the cartilage tissue or pharmaceutical composition of the present invention in the preparation of a medicament intended for the treatment or prevention of cartilage defects and degenerative cartilage diseases.

[0125] The present invention further relates to a method for treating cartilage defects and degenerative cartilage diseases, the method comprising administering a therapeutically effective amount of the engineered cartilage tissue or pharmaceutical composition of the present invention to a subject to be treated.

[0126] In other words, the present invention also relates to a method for treating cartilage defects and degenerative cartilage diseases in subjects with such needs, comprising:

[0127] i) Isolate chondrocytes from the subject;

[0128] ii) As previously described, the chondrocytes are cultured on an adherent culture system in a dedifferentiated medium that activates the Wnt signaling pathway to obtain fibroblast-like cells.

[0129] iii) As previously described, the fibroblast-like cells are cultured on an adherent culture system in a redifferentiation medium that inactivates the Wnt signaling pathway to obtain chondrocytes.

[0130] iv) As previously described, the chondrocytes obtained in step iii) are cultured on a 3D culture system in a maturation medium that inactivates the Wnt signaling pathway to obtain cartilage tissue.

[0131] v) Administer a therapeutically effective amount of cartilage tissue to the subject to be treated.

[0132] As used herein, the term “therapeutic effective dose” refers to an amount sufficient to reduce pain symptoms or improve mobility in a subject presenting with cartilage defects and / or degenerative cartilage disease.

[0133] In a preferred embodiment, 10 to 50 cartilage spheres, each approximately 1 to 2 mm in diameter, are applied to each cm² of the lesion to completely cover it. The spheres self-adhere to the subchondral bone and inner edge of the cartilage lesion.

[0134] The engineered cartilage tissue or pharmaceutical composition of the present invention can be administered by directly implanting the cartilage onto the surface of the subject's cartilage or supporting matrix, or by implanting it into the local environment of the subject's cartilage or supporting matrix.

[0135] The engineered cartilage tissue or pharmaceutical composition of the present invention can be administered to the subject via an open-joint surgery procedure or via arthroscopy, preferably via arthroscopy, to enable the patient to recover more quickly.

[0136] Once transplanted into the body, the chondrocytes within the cartilage tissue respond to mechanical load by continuing to produce matrix to fill the full-thickness defect up to the surface of the damaged tissue, and may even fuse completely in the body to form homogeneous cartilage tissue.

[0137] Reagent test kit

[0138] The present invention also relates to a kit for in vitro production of cartilage tissue. The kit comprises:

[0139] - A first container containing one or more compounds present in the dedifferentiation medium as described above, preferably FGF-2, more preferably FGF-2 and growth factors selected from the group consisting of EGF, TGF-β3 and PDGF-BB, and any combination thereof.

[0140] - A second container containing one or more compounds present in the redifferentiation medium as described above, preferably TGFβ3, more preferably TGFβ3 and FGF-7, wherein TGF-β3 and / or FGF-7 may be replaced with platelet lysate, and

[0141] - A third container containing one or more compounds present in the maturation medium as described above, preferably TGF-β3, more preferably TGF-β3 and a compound selected from the group consisting of: IGF-1, BMP-2, insulin, selenium, transferrin and ethanolamine and any combination thereof, even more preferably TGF-β3 and insulin, selenium, transferrin and ethanolamine.

[0142] Preferably, the kit comprises multiple containers, each containing one or more compounds, the concentration or amount of which facilitates the reconstitution and / or use of differentiation and / or maturation media, as well as the implementation of the methods of the present invention. The kit of the present invention may also contain a container with a basal medium as described above.

[0143] The kit of the present invention may also include an adherent culture system, particularly in the form of a flask, a multi-well plate or a petri dish.

[0144] The kit may also contain instructions on the preparation and / or use of a differentiation or maturation culture medium for the in vitro production of cartilage tissue according to the method of the present invention.

[0145] This invention also relates to the use of the kit of this invention for the in vitro production of chondrocytes according to the method of this invention.

[0146] Methods for screening molecules with therapeutic potential

[0147] According to another aspect, the present invention relates to the use of cartilage tissue of the present invention for screening molecules of therapeutic significance.

[0148] Molecules with therapeutic significance are particularly those that inhibit the degenerative process of cartilage degenerative diseases. These molecules can be used especially to treat or prevent cartilage defects or degenerative cartilage diseases as described above.

[0149] Therefore, the present invention relates to a method for screening molecules of interest, the method comprising:

[0150] i) Contact the cartilage tissue of the present invention with the candidate molecules, and

[0151] ii) Select molecules with the desired activity.

[0152] This invention particularly relates to a method for screening molecules that inhibit the degenerative process of cartilage, the method comprising:

[0153] i) Contact the cartilage of the present invention with one or more candidate molecules, and

[0154] ii) Select molecules that inhibit the degenerative process of cartilage.

[0155] The production of cartilage tissue can be assessed using techniques known to those skilled in the art, such as methods for measuring GAG or collagen II.

[0156] Depending on the nature of the molecule being sought, chondrocytes used to generate cartilage tissue can be obtained from healthy subjects or subjects with cartilage defects or degenerative cartilage diseases as defined above.

[0157] All references cited in this specification are incorporated herein by reference. Other features and advantages of the invention will become more apparent from the following embodiments given by way of non-limiting description.

[0158] Example

[0159] Materials and Methods

[0160] chemicals

[0161] XAV939 was obtained from Sigma and used at a concentration of 10 μM in medium E for 4 days in 2D culture.

[0162] Cartibead production

[0163] The following media, as described in Table 1, were used in the embodiments.

[0164] Table 1: Composition of the culture medium used in the examples

[0165]

[0166]

[0167] Sample collection

[0168] Human cartilage samples were obtained from life-consenting donors (aged 18 to 80 years) who underwent orthopedic surgery for various indications (Table 2).

[0169] Table 2: Characteristics of donors, including sex, age and pathology.

[0170]

[0171] The collected cartilage was transferred to a sterile receiving container filled with physiological saline (NaCl 0.9%) at room temperature in the laboratory. The collection of human cartilage samples was approved by the Swiss Ethics Committee (BASEC, 2016-00656).

[0172] The cartilage of miniature pigs was obtained from the lateral trochlearholder (~30 mg) acquired from the right knee of the miniature pig.

[0173] Cartilage (~30 mg) was cut into small pieces (1 mm) to facilitate the extraction of chondrocytes by enzymatic digestion. The cartilage was then placed at 37°C and rotated overnight in medium E containing antibiotics (gentamicin, 50 μg / ml) and antifungal agents (amphotericidal B or fungizone®, 0,250 μg / ml) using type II collagenase (400 U / ml, ThermoFisher).

[0174] Cell culture and Cartibead production

[0175] Wash cells and plate (p0) onto T25 cm cells pre-coated with extracellular matrix (MaxGel™, Sigma). 2 The cells were cultured in flasks and then in medium E containing gentamicin and fungizone® for 12 to 16 days, after which medium E was removed 5 days after cell expansion. All 2D cell cultures were performed on extracellular matrix-coated flasks. At confluence, cells were passaged in one T75 culture (p1) and subsequently aliquoted into 2x T75 culture media (p2) to achieve confluence. At this stage, cells could be frozen for later use (p3). Following cell expansion in medium E (step 1), cells were cultured in medium R for 7 days in step 2, during which reduced cell growth was observed before proceeding to the 3D culture in step 3. In step 3, chondrocytes were collected and aggregated in medium I to obtain 0.2 x 10⁻⁶ cells per conical 96-well plate. 6 Cells / well (~20x10) 6 (cells / plate). Centrifuge the 96-well plates at 300g for 5 minutes to allow cells to aggregate and form cartibeads after 15 days of 3D culture. Cartibeads were obtained from chondrocytes up to passage 8. These beads were removed from the 96-well plates and pooled together, and could be maintained in medium I at 4 to 23°C (room temperature) for up to 6 days with high stability.

[0176] GAG Quantitative Analysis

[0177] Glycosaminoglycan (GAG) content was assessed using the dimethyl methylene blue assay (DMMB) (Sigma, 341088). Six standards were prepared using chondroitin sulfate A (Sigma, C9819) at concentrations ranging from 0 to 50 µg / mL. Low and high internal quality controls (IQCs) were prepared using chondroitin sulfate C (Sigma, C4384) at concentrations of 15 and 35 µg / mL, respectively. Cartibead was digested with proteinase K (1 mg / mL) (Promega, V3021) at 56°C in 50 mM pH 8 Tris-HCl (Sigma) for 15 ± 2 h. Enzymatic digestion was terminated by incubation at 97°C for 15 min. The resulting samples were then diluted (1 / 5–1 / 10) in 50 mM pH 8 Tris-HCl for assay. After reacting 100 μL of standard, ICQ, and sample with 1 mL of DMMB solution for 5 minutes, the readings were taken in triplicate using a spectrophotometer (λ = 525 nm). The GAG ​​content was then normalized to the relative DNA amount, which was determined using the PicoGreen-Qubit assay. The standard and ICQ were prepared from calf thymus DNA using two separate preparations. The standard and ICQI were prepared in 200 mM Tris-HCl (TE) buffer at pH 7.5 containing 20 mM EDTA; the sample was derived from proteinase K digestion and then diluted 1 / 15 in TE buffer. For this assay, 100 μL of standard, ICQI, and sample were taken in triplicate. Then, 100 μL of a 1 / 200 dilution of PicoGreen Quant-It® (ThermoFischer, P11496) was added. The sample was then incubated for 5 minutes, during which time the intercalant complexed with the DNA. Finally, readings were taken using a Qubit 4 fluorometer (ThermoFischer, Q33238), and the excitation peak was located at 485 nm.

[0178] Cartibead's cell vitality

[0179] The viability of samples was determined using an assay using the red fluorescent dye Zombie Aqua™ (Biolegend, 423101). Zombie Aqua™ is an amine-reactive fluorescent dye that cannot penetrate live cells but can penetrate cells with damaged membranes, thus enabling the assessment of live and dead cells. Three-dimensional samples were collected at the end of step 3, washed with PBS, and then treated with Zombie dye diluted 1:100 in PBS for 20 minutes, while the samples were kept in the dark. The samples were then sectioned to a thickness of 3 µm using a cryostat and mounted on Superfrost plus slides. They were then fixed with 4% formaldehyde and treated with Hoescht (Molecular probe, H3570 Thermofisher) diluted 1:2000 in PBS for 10 minutes. A control was provided by adding PBS containing 10% Triton™ (Sigma, X100-100 mL) at room temperature (RT) before starting the Zombie dye step.

[0180] Immunohistochemical staining

[0181] For immunohistochemical staining of formalin-fixed paraffin-embedded cartilage tissue and Cartibead, paraffin blocks containing the samples were cut into 5 μm pieces using a microtome. Slides were dried overnight at 47°C. The slides were dewaxed with xylene and rehydrated via consecutive alcohol baths (100%, 95%, and 70% concentrations). Two unmasking techniques were used. For natural cartilage from miniature pigs, the inventors used a 20 mg / mL hyaluronidase solution in 0.1 M phosphate buffer, placed on slides at 37°C for 1 hour. The slides were rinsed twice in PBS for 5 minutes each time to remove the hyaluronidase. Cartibead samples were immersed in a 0.01 M citrate buffer bath at pH 6, heated three times at 620 W for 5 minutes each time in a microwave oven, and then cooled in an ice bath for 20 minutes. The slides were then rinsed in PBS for 5 minutes. Primary antibodies were then used (Collagen I Abcam, ab6308; Collagen II Abcam ab85266 and ThermoFischer MA5-12789). Notably, the two type II collagen antibodies used showed similar results. Antibodies diluted 1:100 in 0.3% triton:PBS were placed on different samples for 1 hour. After rinsing with PBS for 5 minutes, secondary antibodies were used: biotin-conjugated anti-mouse IgG (Vector lab, BA-2000), anti-rabbit IgG (Vector lab BA-1000), and an anti-biotin protein-biotin peroxidase detection system utilizing a 3,3'-diaminobenzidine substrate (Vector Labs). Samples were counterstained with hematoxylin. The slides were shaken 10-20 times in an alcohol bath (95% and 100% concentrations) for vigorous dehydration, followed by a xylene bath, until dehydration was achieved by mounting the slides in Eukitt resin (batch A1113, KiNDO1500). Imaging was performed using a Nikon Eclipse C1 confocal microscope and a Nikon Eclipse TE2000-E.

[0182] Safranin-O staining

[0183] Formalin-fixed paraffin-embedded samples of chondroids and native cartilage were stained with Safranin-O to reveal glycosaminoglycans (GAGs) on 5 µm paraffin sections. Slides were dewaxed with xylene and rehydrated via consecutive alcohol baths (100%, 95%, and 70%), followed by a 5-minute distilled water bath. Nuclear counterstaining was then performed with hematoxylin, followed by washing with running hot water for 5 minutes. Cytoplasmic staining was performed with Fast Green (Sigma F7252), followed by washing away Fast Green with acetic acid. Slides were immediately rinsed with distilled water. GAG staining was performed with 0.1% Safranin-O (Sigma S2255) in a water bath for two and a half minutes, followed by repeated washing with distilled water. The scaffolds were vigorously shaken 10–20 times in alcohol baths (95% and 100%), followed by a xylene bath, until dehydration was achieved by mounting slides with Eukitt resin (batch A1113, KiNDO1500).

[0184] Multipotency assessment

[0185] Human MSC / ASC / chondrocytes were differentiated into chondrogenic, adipogenic, and osteogenic fates to assess their pluripotency. Alizarin Red S (Merck, TMS-008-C) staining was used to assess osteogenic differentiation. Oil Red O (Sigma, O1391) staining was used to assess adipogenic differentiation, and Safranin-O (Sigma, S2255) / Fast Green staining (Sigma F7252) was used to assess chondrogenic differentiation.

[0186] Biomechanical measurement

[0187] Compression tests were performed on cartilage beads and natural cartilage with diameters ranging from 0.68 mm to 1.54 mm using an MTS standard pull-pull machine (model 42) equipped with a 1N load cell. The compression speed was set to 0.01 mm / s. -1 The maximum applied load compression was 0.2 N; a maximum of ten beads were used per test to increase the surface area probed. A custom-designed original holding system was used to prevent bead slippage and ensure isotropic compression during testing. The compressive force was determined as a function of displacement (raw data). Stress-strain curves were then calculated to estimate the Young's modulus of the cartilage (the slope of the dataset in the linear region was considered, divided by the sample surface area and the number of beads for standardization). The calculation of the Young's modulus is as follows:

[0188] E = σ / ε, where σ is the compressive stress (kPa), E is Young's modulus (kPa), and ε is the strain corresponding to the normalized elongation (unitless).

[0189] Immunoblotting

[0190] Cells were lysed on ice for 30 minutes in ice-cold RIPA buffer (Life Technologies) supplemented with phosphatase and protease inhibitors (complete anti-protease cocktail; Roche). Proteins (10 μg) were separated by SDS-PAGE (BioRad) and transferred to PVDF membranes (Amersham). The blots were detected using antiphospho-β-catenin (Cell Signaling; 5651T), TCF-4 (Cell Signaling; 2569), Axin1 (Cell Signaling; 2087), β-actin HRP (Sigma-Aldrich), and GADPH (Cell Signaling) (1:1000), followed by HRP-rabbit or mouse conjugated antibodies (1:5000).

[0191] Flow cytometry

[0192] 100,000 cells were fixed with 4% PFA and then stained for 1 hour at room temperature in FAC buffer (BSA-azide-PBS) against CD73-CFS, CD90-APC, and CD105-PerCP (Cell Sigma). At least 10,000 live cells were collected using a Gallios flow cytometer and analyzed using FlowJo software. Results represent the average of three independent experiments against CD73 and CD90. Two independent experiments were performed against CD105. For the gating strategy, live cells were first selected, and then single cells were identified based on FSC-W and FSC-A to remove doublets. Positive staining was determined based on negative controls IgG-CFS, IgG-APC, and IgG-PerCP for CD73-CFS, CD90-APC, and CD105-PerCP, respectively.

[0193] RNASeq

[0194] As mentioned earlier, SR100–Library TruSeqHT stranded–Illumina HiSeq 4000 was used, along with FastQC v.0.11.5 (Cosset, E. et al. 2016. BiomaterialsSequencing quality control was performed on reads (107, 74-87). Quality distribution along reads was assessed and validated for all samples. UCSC human hg38 reference was used to map STARaligner v.2.5.3a reads to the reference genome. The mean mapping rate was 93.54%. Transcriptome metrics were assessed using Picard tool v.1.141, and differential expression analysis was performed using statistical analysis R / Bioconductorpackage edgeR v. 3.18.1 (Gentleman, RC et al. 2004. GenomeBiol 5, R80, Huber, W. et al. 2015. Nat Methods 12, 115-121). Briefly, counts were normalized and filtered according to library size. Genes with a count greater than 1 per million reads (cpm) in at least three samples were retained for analysis. The original gene count for this group was 26,485. Genes that are poorly expressed or not expressed are filtered out. The final dataset consists of 13,884 genes. Differentially expressed genes are tested using a negative binomial distribution with an exact test. The p-values ​​for differentially expressed genes are corrected for multiple test errors, with an FDR (false detection rate) of 5%. The correction used is Benjamini-Hochberg (BH). Differentially expressed genes are tested using a negative binomial distribution with edgeR. The p-values ​​for differentially expressed genes are corrected for multiple test errors using the Benjamini-Hochberg (BH) false detection rate (FDR).

[0195] Panther analysis was used to determine the enrichment (number of entities) of each gene family (Mi, H. et al. 2017. Nucleic Acids Res 45, D183-D189).

[0196] RNA extraction and RT-PCR

[0197] Total RNA was isolated using the Qiagen RNeasy kit, as per the manufacturer's instructions, and measured using a spectrometer. cDNA was synthesized using the PrimeScript RT Reagent kit (Takara) with 500 ng of total RNA, following the manufacturer's protocol.

[0198] At the core facility of the genome platform (University of Geneva), real-time quantitative PCR was performed using a QuantStudio 12K Flex real-time PCR system (Thermo Fisher Scientific) and a PowerUp SYBRGreen Master Mix (Applied Biosystems). First, all primers were functionally tested to validate them before use. The relative levels for each sample targeted at least two housekeeping genes. ALAS1 Standardization was performed. RT-PCR reactions were conducted in at least three technically and biologically replicated samples, and the mean cycle threshold (CT) was determined.

[0199] Human Cartibead transplanted into SCID / NOD mice for safety studies

[0200] The safety of the standardized Cartibead of this invention was tested using 56 male SCID / NOD mice. 44 mice received human Cartibead via subcutaneous implantation. A control group was used, consisting of 8 animals that received aggregated A549 adenocarcinoma cells (i.e., the bead) (the number of mice in each group is summarized in Table 3).

[0201] Table 3: List of Cartibead donors transplanted into SCID mice.

[0202]

[0203] Cartibead from different donors and cell passages (3 to 6 generations) was subcutaneously injected into SCID / NOD mice (n = 8 to 14 mice per group). The adenocarcinoma cell line A549 was used as a positive control and produced 100% tumors. After euthanasia at 6 months, none of the mice showed weight loss, palpable abnormalities, or any signs of distress due to Cartibead transplantation. Furthermore, no evidence of abnormalities or tumors was observed in the lungs, heart, liver, kidneys, or spleen.

[0204] All implanted human Cartibeads were cultured according to a standardized three-step method and transplanted in the same manner. General anesthesia was achieved with 4% isoflurane followed by 2% isoflurane under a 5% oxygen mask. After shaving the back, the skin was locally disinfected with 70% alcohol. A 0.5 cm skin incision was made from the occipital pole to the tail. The generated tissues (Cartibead and adenocarcinoma bead) were subcutaneously implanted with 200,000 cells per tissue using sterile pipettes (1 bead / animal). The skin was then closed with surgical glue (Histoacryl, B. Braun Surgical SA). To locate and orient the skin samples, the inventors tattooed four azimuth points using a sterile 26-gauge needle and green tattoo ink. Mice in the control group were euthanized at 4 to 6 weeks, while mice in the Cartibead group were followed up until 6 months. At each stage, two research members harvested and examined the skin and organs. One-third of all samples were preserved in 4% formaldehyde, and two-thirds were dried at -20°C. The animal program was approved by the Swiss Federal Veterinary Office (GE / 12 / 18).

[0205] Comparative Genome Hybridization Array

[0206] Comparative Genomic Hybridization (CGH) arrays were also used to compare normal human dermal fibroblasts treated with 10% FBS or 10% PRP for 6 days to determine genomic stability. DNA was extracted using the QIAGEN QIAamp DNA Mini Kit (Qiagen, Hilden) according to the manufacturer's protocol. CGH arrays were performed using the Agilent SurePrint G3 Human CGH Microarray Kit 4_44K (design ID 014950) and a total median probe spacing of 43 kb (Agilent Technologies). The actual resolution was approximately 200 kb. Patient DNA and sex-matched control DNA (1 µg each) were labeled with Cy3-dUTP and Cy5-dUTP, respectively (Sure Tag Labeling Kit, Agilent Technologies). The labeled products were purified through an Amicon Ultra 30 K filter (Millipore). Hybridization was performed according to the protocol provided by Agilent. Patient and control DNA were combined and hybridized with 2 mg of human Cot-I DNA at 65°C under rotation for 24 hours. The arrays were analyzed using an Agilent SureScan microarray scanner and Agilent feature extraction software (v11.5), and the results were provided by Agilent Genome Workbench (v.7.0).

[0207] Efficacy studies of autologous Cartibead transplantation in miniature pigs.

[0208] Six adult female miniature pigs (37-48 kg) were used in this study. The animals were anesthetized with sevoflurane and given prophylactic antibiotics (cefazolin: 2 g / kg) intravenously 30 minutes before incision. Surgery was performed under complete anesthesia, and the tube was removed 5 minutes after skin closure. Skin disinfection was repeated three times, and aseptic covering was achieved using a removable surgical drape.

[0209] Step 1 Surgery

[0210] The first surgical procedure used a super-lateral para-patellar approach. Cartilage was harvested at the super-lateral boundary of the lateral trochlear facet to prevent potential complications with cartilage damage during subsequent surgical steps. The cartilage biopsy was further processed in culture medium E. A stepwise closure was performed. No bandages were used.

[0211] Step 2 surgery

[0212] Five to six weeks after cartilage harvesting, a second surgery is performed on miniature pigs via the medial para-patellar tendon approach (Bonadio, MB). et al. 2017. J Exp Orthop 4,11). Using this method, two lesions were created on the medial and lateral femoral trochleas in all miniature pigs (except one due to donor site limitations). The lesions were 6 mm in diameter, created with a small curette, and then completely filled with a single layer of 4–5 Cartibeads. For faster adhesion, a thin layer of Tisseel (Baxter) was added to each defect. A fifth lesion was then created on the medial femoral condyle and filled with Cartibeads in the same manner. In each animal, one lesion was left empty at a location different from the negative control lesion. Stepwise closure was performed. No bandages were used.

[0213] Rehabilitation

[0214] Full weight-bearing is permitted immediately after anesthesia ends, and no activity is restricted.

[0215] euthanasia

[0216] Three miniature pigs were euthanized three months after the second surgery, and another three were euthanized six months later. The surgically operated knee was amputated, and the distal femur was placed in formaldehyde solution for further analysis.

[0217] The animal program was approved by the Swiss Federal Veterinary Office (GE / 60 / 18).

[0218] result

[0219] Engineering and characterization of Cartibead

[0220] In this study, the inventors generated cartilage microtissue, named Cartibead, which exhibits improved hyaline cartilage characteristics (defined by GAG content and collagen II expression). The Cartibead method is a novel 3-step protocol (Figure 1A) consisting of: (Step 1) chondrocyte expansion in two-dimensional culture (2D), characterized by cell dedifferentiation; (Step 2) cell redifferentiation in 2D, defined as chondrogenic orientation; and finally (Step 3) 3D culture, allowing the formation of Cartibeads through chondrocyte aggregation. Therefore, human chondrocytes were enzymatically extracted from surgical waste and cultured in 2D under atmospheric oxygen conditions (21%) in steps 1 and 2, followed by 3D culture at a hypoxic level (5%).

[0221] Chondrocytes become "fibroblasts," exhibiting stem cell characteristics in gene expression and expressing mesenchymal stem cell surface markers (CD105, CD90, CD73). Figure 6 Within three weeks, it is possible to obtain 60 million to 100 million cells from the extracted 100,000 cells after culturing in medium 1 (medium E).

[0222] The second step corresponds to the “reversal” of the fibroblast phenotype to the chondrocyte phenotype after the mass cell expansion step. Cells are placed in redifferentiation medium (medium 2) for 3 to 7 days. On day 0, 2 million cells are plated in T75 cm² flasks to achieve confluence and are kept in confluence until day 4 to day 8, after which they are separated for 3D culture. From the T75 cm² flasks, 6 to 10 million cells are obtained in step 2. In contrast, in the dedifferentiation medium of the first step, 12 to 16 million cells are obtained in T75 when the cells reach confluence. The removal of FGF-2 growth factor in redifferentiation medium (Medium R) reduces cell growth and allows for phenotype reversal and redifferentiation to complete chondrocyte differentiation. Cells change their morphology in the second step of culture, becoming less elongated, larger, and exhibiting prominent granular endoplasmic reticulum in the cytoplasm under a light microscope, indicating high levels of protein synthesis activity. Chondrocytes began to re-express aggregated proteoglycans, a proteoglycan that binds to GAG (data from RNAseq). In this second step of 2D culture, collagen II had not yet been re-expressed.

[0223] In the third step, cells are isolated from culture medium 2 (medium R) and seeded into 3D culture. 100,000 to 200,000 cells are aliquoted into redifferentiation medium (medium 3, also known as medium I) in 96-well conical polypropylene plates. The 96-well plates are centrifuged at 300 g for 5 minutes to aggregate cells and form cell clusters. The 96-well plates are made of polypropylene material to prevent cells from sticking to the well surfaces. During the 15 days prior to use of microtissue culture, the cultures are incubated in an anaerobic environment (5% O2) to increase hyaline cartilage production.

[0224] Cartibead, engineered from chondrocytes, has the potential to be used as an ATMP for treating cartilage damage. Therefore, to assess its safety and efficacy, the inventors used animal models (SCID mice and miniature pigs) (Figure 1B). Histological qualitative analysis was performed to better characterize Cartibead. The presence of clear features of GAGs with uniformly distributed Safranin-O staining was observed, and this was strongly correlated with immunoassay of type II collagen, while type I collagen showed weak immunoassay (Figure 1C).

[0225] The inventors were able to engineer cartilage tissue (called Cartibead) of similar quality from donors up to 80 years old, including patients with osteoarthritis (OA). Quantitative analysis of the Cartibead showed high GAG content, independent of patient age and osteoarthritis status (Figure 1D, Table 2). The inventors determined an average GAG / cartibead ratio of 40 µg and an average GAG / DNA ratio of 50 (10;140). Figure 2 ).

[0226] To characterize the biomechanical properties of cartilage, the inventors used a device with a custom-fitted mold to quantify the elasticity of cartilage based on compression tests. Young's modulus was calculated by dividing the compressive stress by the strain (normalized elongation) of the elastic portion of the curve (Lee, JK et al. 2017, Nature Materials, 16:864-873). This method is commonly used to test cartilage elasticity and reflects its extracellular matrix (ECM) composition. The inventors observed increased resistance to constraint in cartilage containing more GAGs, demonstrating the benefit of increasing GAG content (Figure 1E), consistent with other published studies (Omelyanenko, NP et al. 2018, Cartilage, 1947603518798890).

[0227] Since hypoxia-inducible factor 1-α (HIF-1α) is known to induce ECM components (Madeira et al. 2015, Trends in Biotechnology, 33:35-42), the inventors evaluated the effect of hypoxia levels (5% oxygen) on cartilage bead quality during 3D culture compared to atmospheric conditions. As expected, the results showed that the hyaline quality of the cartilage bead was improved under hypoxic conditions. Figure 3 It is worth noting that the standard culture conditions under atmospheric conditions actually correspond to hyperoxia, since oxygen levels recorded in natural cartilage tissue are between 0.5% and 5%, depending on depth (Lafont, JE 2010, Int. J. exp. Pathol. 91(2):99-106).

[0228] In cell therapy, the stability of the cellular components of the material to be transplanted is crucial. Therefore, the inventors evaluated the stability of Cartibead and the proportion of viable cells under different conditions. The inventors compared the amount of GAG in Cartibead on day 0 after the ripening step with the amount of GAG after maintaining Cartibead at 4°C and 23°C for an additional 6 days. Figure 4 A). The results showed that the concentration of GAG remained constant over time under different temperature variations, averaging 55 µg / cartibead, indicating stability. Under these conditions, only a small number of dead cells (13%) were detected, suggesting good chondrocyte survival in the cartibead. Figure 4 B).

[0229] Cartibead's three-step transcriptomic analysis identified low-level expression of the WNT gene as a key pathway involved in clear matrix quality.

[0230] To identify the molecular pathways involved in increased hyaline cartilage production, a three-step method for cartibead was compared with a classic two-step method for tissue engineering (Figure 5A). Morphologically, the three-step method produced larger white cartibeads (1–2 mm in diameter) and a greater amount of GAG / bead compared to the two-step method (0.5–1 mm in diameter). Figure 2 The improvement in Cartibead transparency in the three-step method is primarily due to the introduction of an additional step following large-scale cell expansion using FGF-2. This step corresponds to the starvation step using FGF-2-free medium "R," which promotes chondrocyte redifferentiation and the production of transparent matrix. In both methods, the final step involves 2 weeks of 3D culture in maturation medium I supplemented with TGF-β3 (Figure 5A).

[0231] To understand the molecular mechanisms that allow for Cartibead production, the inventors performed RNA sequencing (RNA-seq) analysis using three donor samples at each key step of both methods. For the three-step Cartibead method, RNA was extracted at different time points: (i) at the end of Step 1 (expanded chondrocytes, medium E), (ii) at the end of Step 2 in medium R, and (iii) 15 days after Step 3 (bead, medium I). For the two-step method, two time points were selected: Step 1 (expanded chondrocytes, medium E) and the end of Step 2 (bead, medium I). The inventors first compared the cartilage microtissue produced by the two methods in medium I (Figures 5B and C).

[0232] They observed significantly increased levels of genes involved in collagen and ECM degradation / formation, as well as ECM organization, in Cartibead. COL1A1, COL2A1, COL4A1, MMP1, MMP13, MMP11 Differential gene expression analysis showed that the three-step method induced higher levels of type II collagen in Cartibead. COL2A1 ) and aggregated proteoglycans ( ACAN ), while in the two-step method, fibrocartilage-specific type I collagen ( COL1A1 The increase in GAG levels confirmed the improvement in chondrocyte differentiation achieved by Cartibead's three-step method (Fig. 5G). Figure 2 In the three-step method (M1+M2+M3), SOX9 (transcription factors involved in matrix production) also increased ( Figure 7C ).

[0233] These data confirmed the dedifferentiation of chondrocytes in culture medium E (containing FGF-2), and detected... COL1A1 Expression, and the absence of hyaline cartilage markers, such as COL2A1 and ACAN (A GAG-binding protein) (Figure 5D-F). The inventors confirmed the enrichment of dedifferentiated cells in culture medium E by flow cytometry, showing that the proportion of cells expressing mesenchymal stem cell markers CD73, CD90, and CD105, as defined by the International Society for Cell Therapy (ISCT), accounted for more than 90% of the total cell population at the end of the expansion phase. Figure 6 A; Table 4).

[0234] Table 4: Flow cytometry analysis of dedifferentiated chondrocytes

[0235]

[0236] This table represents a flow cytometry analysis of dedifferentiated chondrocytes from three donors targeting CD73, CD90, and CD105.

[0237] Furthermore, the pluripotency of this dedifferentiated chondrocyte population is confirmed by the fact that it possesses the ability to differentiate into other mesenchymal stem cell (MSC)-derived cells (osteocytes, adipocytes, and chondrocytes), even if this potential is lower than that of MSCs. Figure 6 B).

[0238] When the inventors compared gene expression in cells cultured in medium R (redifferentiation step) and medium E (amplification step), they found that genes involved in inflammatory processes (interleukin and cytokine signaling, as well as interferon signaling) were expressed at higher levels during redifferentiation. Simultaneously, the inventors also found that genes related to the cell cycle ( MI67, CDK, CCNB1 The expression level of ) was low, which suggests that the production of the transparent matrix requires a dynamic balance between cellular inflammation and tissue remodeling (associated with cell cycle exit) (Figure 7A).

[0239] By comparing the amplification phase and the redifferentiation phase, the inventors observed WNT5A, WNT5B and WNT7B High expression levels of genes during the amplification phase (step 1, medium E, both methods) (Fig. 7B, DF). These genes were strongly downregulated during the redifferentiation step (medium R, three-step method) and 3D culture (medium I, both methods), except... WNT5B In addition, WNT5B Moderate expression was observed in 3D culture (step 3, medium I) (Figure 7E).

[0240] The expression of KI67 (a proliferation marker) and TCF4 (involved in the downstream of the Wnt / β-catenin pathway) was also decreased during the redifferentiation process. Figure 7C Most importantly, KI67 and WNT7B / WNT5B decreased after the dedifferentiation and redifferentiation phases (M1+M2) before globule formation in the maturation phase (M3). When cells were used to form globules directly from the dedifferentiation phase (M1), they still highly expressed KI67 and WNT7B / WNT5B, resulting in less clear matrix production.

[0241] These results are consistent with previous reports of WNT upregulation following FGF-2 treatment (Buchtova, M. et al. 2015. Biochim Biophys Acta 1852, 839-850; Deng, Y. et al. 2019. Biomaterials 192, 569-578). Indeed, WNT signaling is involved in stem cell-like phenotypes (Buchtova, M. et al. 2015. Biochim Biophys Acta 1852, 839-850). Therefore, removal of FGF-2 in culture medium R resulted in downregulation of WNT signaling during the redifferentiation phase. Compared to the three-step method, the inventors detected lower levels in the two-step method. COL2A1 and ACAN Expression (Fig. 5C-E). However, they observed that in the 3D differentiation phase of medium I, the two-step method... WNT5A , WNT5B and WNT7B The reduction in genes is considerable (Figure 7B).

[0242] To verify the role of WNT pathway downregulation in the redifferentiation phase (medium medium R) (Figure 1A), the inventors replaced medium R with medium E supplemented with 10 μM XAV-939 (a WNT pathway inhibitor) for 4 days (Figure 7G). Following XAV-939 treatment, the inventors observed an increase in phosphorylated β-catenin and axin (Figure 7G), proteins known indicators of WNT signaling blockade (Huang, SM et al. 2009. Nature 461, 614-620). Therefore, similar to medium R, drug inhibition of WNT induced [a specific effect] after 2D and 3D culture in the two-step method. ACAN and COL2A1 Increased expression (Fig. 7H, I). Therefore, drug inhibition of WNT in the presence of XAV-939 resulted in a transparent feature on the bead obtained by the two-step method, as confirmed by GAG safranin-O staining (Fig. 7J).

[0243] In summary, transcriptomic analysis identified the involvement of the WNT signaling pathway and its regulatory role in chondrocyte dedifferentiation and redifferentiation. WNT5A, WNT5B and WNT7B It acts as a potential major mediator of this response (Fig. 7D-F). Medium E supplemented with XAV-939 mimics the function of medium R (Fig. 7K), but the three-step method naturally induces downregulation of the WNT gene within 7 days without the use of expensive drug molecules.

[0244] Feasibility study of ex vivo treatment of human cartilage defects

[0245] In the context of the clinical application of Cartibead, the inventors evaluated the ability of Cartibeads to fuse together and integrate into injuries induced in isolated human knee joints. An lesion was created using an 8 mm surgical perforation biopsy and filled with 20 to 50 Cartibeads under dry conditions. The Cartibeads were held in this condition for 20 minutes to promote adhesion between the Cartibeads and the lesion site. Figure 8 A, left small image and middle small image), and then add culture medium I. Then, the entire specimen filled with Cartibead was rotated and cultured for 1 month to promote high-quality transfer of nutrients. One month after implantation, the inventors analyzed the osteochondral tissue ( Figure 8 A, right inset), and safranin-O staining was performed at the level of the filled lesion ( Figure 8 B). The inventors observed the maintenance of the transparent features within the lesion and a degree of integration of Cartibead with the surrounding natural tissue, as well as the fusion of Cartibeads with each other. Figure 8 B).

[0246] In vitro and in vivo preclinical safety studies of Cartibead

[0247] Transplantation of in vitro expanded cells can lead to potential problems with uncontrolled proliferation. The safety of Cartibead was assessed through preclinical studies. CGH (Comparative Genomic Hybridization) array analysis showed the genetic stability of chondrocytes during cell expansion up to the 11th generation (Table 5).

[0248] Table 5: List of Cartibead donors analyzed by CGH array

[0249]

[0250] After extensive amplification in FGF-2-containing medium E, the genetic stability of chondrocytes was assessed by comparative genomic hybridization (CGH) array analysis of 10 donor samples from generations 3 to 11.

[0251] Consistent with previous studies, Y chromosome deletion is a common acquired mutation in normal male aging and has been observed in a subset (43%) of older donors (Thompson, DJ et al. 2019. Nature 575, 652-657; Stumm, M. et al. 2012. Osteoarthritis and cartilage 20, 1039-1045).

[0252] In in vivo studies, the inventors evaluated the potential tumorigenicity of transplanted human Cartibead in SCID mice. Cartibead did not amplify and was virtually undetectable 6 months after implantation in SCID mice, thus confirming the absence of potential tumorigenicity during the 6-month follow-up period. Figure 9 AC, Table 5).

[0253] In vivo preclinical efficacy studies

[0254] To advance towards clinical application, the inventors conducted preclinical efficacy studies on adult Göttingen miniature pigs, chosen for their cartilage maturity and knee anatomy similar to that of humans (Christensen, BB et al. 2015. Journal of experimental orthopaedics 2, 13; Pfeifer, CG et al. 2017. Tissue engineering. Part C, Methods 23, 745-753). Six females were used in the study. A first surgery was performed to collect cartilage biopsy (~30 mg) from the knee to produce autologous miniature pig Cartibead. A second surgery was performed to induce 4 to 5 lesions (per animal / right knee) and transplant autologous Cartibead. The miniature pigs were followed up for 3 to 6 months. Figure 10 A; Figure 11 Prior to transplantation, the amount of GAG in the cartibeads of miniature pigs was quantified, with an average range of 40 to 50 µg / bead. Figure 10 B). Three months post-transplantation, the inventors confirmed the translucency of the lesion in which miniature pig Cartibead was grafted using safranin-O staining. Figure 10 C). Similar results were obtained from graft-related injuries 6 months post-transplantation ( Figure 10 D). During the 3-month and 6-month follow-up periods, complete fusion of Cartibeads was observed in all miniature pigs, and they were integrated within the surrounding natural cartilage and subchondral bone. Figure 10 In summary, autologous Cartibeads demonstrated their efficacy in implantation within lesions while maintaining their translucent quality. They also proved safe, as no joint degenerative changes were detected in macroscopic examinations by two independent operators (CD). Figure 11 (small and medium images) No ectopic or hypertrophic tissue formation was found (data not shown).

[0255] Discussion and Conclusion

[0256] The main finding of this study is a method that can reverse the loss of chondrocyte phenotype (chondrocyte dedifferentiation) during expansion. This addresses a key issue encountered in cell therapy using chondrocytes as starting material. Current data indicate that a novel three-step approach can produce high-quality cartilage with a transparent characteristic, regardless of patient age or the state of arthritis in the joint.

[0257] The Cartibead method allows for cell expansion from very small cartilage harvest samples (approximately 30 mg in our preclinical miniature pig study), compared to an average of 260 mg in conventional human chondrocyte-based cell therapy (Brittberg, M. 2018. Injury 39 Suppl 1, S40-49), thus reducing donor-site morbidity. This method showed a GAG / Cartibead ratio 20 times higher than previously reported for cartilage microtissues, suggesting that these previously reported microtissues had lower transparency and contained more fibrocartilage (Bartz, C. et al. 2016. J TranslMed 14, 317). Consistent with these results, the inventors obtained an average GAG / DNA ratio at least three times higher than other published methods. Figure 2 (Mumme, M) (to the right of the right). et al. 2016. Lancet 388, 1985-1994; Dang, P. N et al. 2014. Tissue engineering. Part A 20, 3163-3175).

[0258] Redifferentiation was due to the removal of FGF-2. However, its removal in 3D culture was insufficient to induce clear matrix synthesis in a two-step process. Chondrocyte redifferentiation likely requires cell adhesion to a matrix-coated flask and the induction of specific cell signaling pathways in 2D culture. FGF-2 deficiency shuts down genes involved in pluripotency and stemness, including several genes involved in the WNT signaling pathway. WNT signaling is involved in both the inhibition and stimulation of chondrogenic differentiation in adult progenitor cells (Day, TF et al. 2005. Developmental cell 8, 739-750; Hill, TP, et al. 2005. Developmental cell 8, 727-738; Hu, H. et al. 2005. Development, 132, 49-60). As shown in the embryo, high levels of WNT / β-catenin signaling inhibit chondrogenic differentiation of stem cells, while downregulation of this pathway induces chondrogenesis (Hartmann, C. 2007. Molecules and cells 24, 177-184; Johnson, ML & Rajamannan, N. 2006. Reviews in endocrine & metabolic disorders 7, 41-49; Westendorf, JJ, Kahler, RA & Schroeder,TM 2004. Gene 341, 19-39).

[0259] In this study, the inventors identified WNT5A , WNT5B and WNT7B This is a WNT isoform that may be involved in this mechanism. With the downregulation of the WNT pathway, the inventors observed increased expression of genes involved in inflammatory pathways (interleukins, cytokines). In wound healing and tissue repair, the dynamic balance between pro-inflammatory and anti-inflammatory factors is crucial for effective healing (Gerhard T. Laschober et al. 2011. Rejuvenation Research 14, 119-131; Bosurgi, L. et al. 2017. Science 356, 1072-1076). Therefore, the current data suggest that inflammation is a prerequisite for tissue regeneration associated with the biosynthesis of the hyaline matrix (Karin, M. & Clevers, H. 2016. Nature 529, 307-315).

[0260] Human cartibead did not proliferate when implanted in SCID mice and even disappeared after 6 months, consistent with similar studies (Zscharnack, M. et al. 2015. J Transl Med 13, 160). Furthermore, autologous transplantation of cartibead in miniature pigs showed stable integration into the lesion, maintained high levels of GAG, and successfully repaired cartilage damage 6 months post-transplantation. In summary, the inventors demonstrated the feasibility of cartibead transplantation with high GAG levels in large animal models, supporting its potential for long-term cartilage repair. Therefore, cartibead represents a breakthrough in the field of cartilage repair and is now ready to enter Phase I clinical trials (the first time in humans) for autologous cartilage transplantation in patients with cartilage damage.

Claims

1. A method for producing hyaline cartilage tissue in vitro, the method comprising: i) Chondrocytes were cultured in an adherent culture system in FGF-2-containing dedifferentiation medium that activates the Wnt signaling pathway to obtain chondrocytes with fibroblast-like cell morphology. ii) The fibroblast-like chondrocytes were cultured for 2 to 10 days on an adherent culture system in a redifferentiation medium without FGF-2 that inactivates the Wnt signaling pathway to obtain chondrocytes with full capacity to resynthesize the clear matrix. iii) The chondrocytes obtained in step ii) are cultured in a three-dimensional culture system for 10 to 20 days in an induction / maturation medium without FGF-2 that maintains Wnt signaling pathway inactivation.

2. The method of claim 1, wherein the dedifferentiation medium further comprises at least one growth factor selected from the group consisting of PDGF-BB, TGF-β and EGF.

3. The method of claim 1, wherein the redifferentiation medium and the induction / maturation medium contain TGF-β.

4. The method of claim 1, wherein the redifferentiation medium and the induction / maturation medium contain TGF-β3.

5. The method of claim 1, wherein the redifferentiation medium and the induction / maturation medium comprise TGF-β3 and FGF7.

6. The method of any one of claims 1 to 5, wherein the redifferentiation culture medium comprises platelet lysate.

7. The method of any one of claims 1 to 5, wherein the dedifferentiation medium, redifferentiation medium and / or induction / maturation medium comprises serum.

8. The method of any one of claims 1 to 5, wherein the induction / maturation medium comprises at least one component selected from the group consisting of insulin, IGF-1, BMP-2, selenium, transferrin, and ethanolamine.

9. The method of any one of claims 1 to 5, wherein the chondrocytes are cultured in a hypoxic environment containing less than 10% O2 (v / v) in step iii).

10. The method of any one of claims 1 to 5, wherein the chondrocytes are cultured for 10 to 15 days in step i), 4 to 8 days in step ii), and / or 10 to 15 days in step iii).

11. The method of any one of claims 1 to 5, wherein the chondrocytes of step i) are isolated from the subject.

12. The method of any one of claims 1 to 5, wherein the chondrocytes of step i) are isolated from the cartilage tissue of a human or equine subject.

13. Engineered hyaline cartilage tissue in the form of spherical bodies, said spherical bodies having a glycosaminoglycan (GAG) content of 10 to 100 μg / spherical body, said hyaline cartilage tissue being obtained by the method of any one of claims 1 to 12.

14. The engineered hyaline cartilage tissue of claim 13, wherein the hyaline cartilage tissue has a spherical diameter of 1 to 2 mm and contains 50,000 to 250,000 cells.

15. Use of the engineered hyaline cartilage tissue of claim 13 or 14 in the preparation of a medicament for treating cartilage defects and degenerative cartilage diseases in subjects with such needs.

16. The use as described in claim 15, wherein the drug is used for autologous transplantation.

17. The use as described in claim 15, wherein the drug is used for allogeneic transplantation.

18. A method for screening molecules that inhibit chondrogenic processes, the method comprising: i) Contacting the engineered hyaline cartilage tissue of claim 13 or 14 with one or more candidate molecules, and ii) Select molecules that inhibit the degenerative process of cartilage.

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