Methods for the in vitro production of hyaline cartilage tissue.

Abstract

The present invention relates to a novel method for the in vitro production of cartilage tissue, as well as therapeutic uses and screening methods using the cartilage tissue so produced.

Description

[Technical field]

[0001] The present invention relates to a novel method for the in vitro production of hyaline cartilage tissue ("Cartibeads"), as well as therapeutic uses and screening methods that employ the cartilage tissue so produced. [Background technology]

[0002] Hyaline cartilage is composed of specialized cells called chondrocytes surrounded by an extracellular matrix. This matrix is ​​synthesized and secreted by chondrocytes and consists mainly of type II collagen fibers, glycosaminoglycans (GAGs), and 60-80% water. Hyaline cartilage has four layers on top of the subchondral bone: superficial, intermediate, deep, and mineralized. The biomechanical properties of articular cartilage depend primarily on the composition and integrity of the extracellular matrix.

[0003] Cartilage has a very limited ability to repair itself, and after injury, osteoarthritis (OA) often develops. Aging and repeated trauma (e.g., during intensive sports practice) are major risk factors for the degeneration of knee cartilage. OA affects many people, most commonly in the elderly (prevalence of more than 10% in people over 60 years of age), but younger people also commonly suffer from OA due to joint damage.

[0004] Surgical treatments include joint replacement with an artificial joint. However, the lifespan of an artificial joint is limited to 15-20 years. Furthermore, pain relief is not completely attenuated in the majority of patients: 20-30% of patients with knee artificial joints still continue to experience discomfort or pain. Treatments are mostly symptomatic and aimed at pain relief. Injections of some biological agents, such as mesenchymal stem cells, hyaluronic acid, or platelet-rich plasma, can slow the deterioration of the joint, but they do not promote tissue regeneration.

[0005] Currently, strategies using external synthetic scaffolds to fill this defect are unsatisfactory and imperfectly mimic the biomechanical properties of articular cartilage. Conventional treatments include microfracture surgery to stimulate stem cell migration to the diseased area, or direct transplantation of chondrocytes.

[0006] The microfracture technique consists of creating small holes in the subchondral bone with the aim of stimulating the migration of mesenchymal stem cells from the bone marrow within the clot to form new chondrocytes and replace the damaged tissue.

[0007] In autologous chondrocyte implantation (ACI), chondrocytes are extracted from cartilage, cultured for a limited number of passages, and implanted into the lesion. To improve this method, matrices such as porcine collagen type I / III or hyaluronic acid for culturing chondrocytes can be used (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 lesion after the microfracture procedure or used in vitro to culture chondrocytes before reimplantation (Ekkers JE, et al. 2013, Osteoarthritis Research Society 21(7):950-956).

[0008] Cell-based therapy using autologous chondrocyte implantation (ACI) is indicated for cartilage lesions larger than 2 cm2 (Armoiry X. et al. 2019, Pharmacoeconomics 37, 879-886). Chondrocytes represent a rational cell source for cartilage regeneration. In fact, only this type of chondrocyte was involved in maintaining the hyaline cartilage matrix.

[0009] A major challenge is the tendency of chondrocytes to dedifferentiate into fibroblast-like cells in culture, resulting in a rapid loss of function that usually occurs at the second or third cell passage in 2D cell culture systems (Munirah, S. et al. 2010. Tissue & cell 42, 282-292). Dedifferentiated chondrocytes are characterized by a loss of production of glycosaminoglycans (GAGs) and type II collagen, the main components of hyaline cartilage, which replace the type I collagen present in fibrocartilage (Wu, L. et al. 2014. Tissue Eng Part C Methods 20, 160-168; Benya, P. D et al. 1978. Cell. 15, 1313-1321). Indeed, during the amplification step, chondrocytes tend to dedifferentiate by losing their original phenotype and become fibroblast-like cells (elongated cells) with stem cell-like properties in gene expression (expression of cell surface markers such as CD73, CD90, CD105, used to characterize mesenchymal stem cells (MSCs)). For example, chondrocytes extracted from articular cartilage of patients rapidly lose their chondrogenic potential to synthesize cartilage in a specific hyaline matrix after several passages in in vitro culture. Fibrocartilage is not considered an effective long-lasting treatment since it is biomechanically different from hyaline cartilage and fibrocartilage results in a 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), mimicking the in vivo environment.

[0010] Loss of chondrogenic potential in culture is especially true for elderly patients. Another challenge for chondrocyte-based cell therapy is the difficulty in expanding and differentiating cells from elderly patients, limiting their use in this population. As a result, most clinical trials limit patient inclusion to age 55.

[0011] The idea of ​​using chondrocytes to produce cartilage makes sense if cell expansion can be performed while maintaining the original phenotype. Thus, there remains a need to develop standardized methods for the production of cartilage that result in high-quality hyaline-like cartilage tissue (characterized by high levels of GAG detection) that mimic the inherent characteristics of patients of all ages. Summary of the Invention

[0012] The main outcome of this study is a method that can reverse the loss of chondrocyte phenotype during proliferation (chondrocyte dedifferentiation). This solves an important problem facing cell therapies that use chondrocytes as starting material. The data show that the novel three-step method can result in high-quality cartilage with hyaline characteristics, regardless of the patient's age and the arthritic state of the joint.

[0013] The Cartibead method allows cell expansion from very small cartilage sample harvest (approximately 30 mg in our preclinical minipig studies) as opposed to an average of 260 mg for conventional human chondrocyte-based cell therapy (Brittberg, M. 2018. Injury 39 Suppl 1, S40-49), reducing donor site morbidity. The method demonstrated a 20-fold higher GAG / Cartibead ratio than previously reported cartilage microtissues, which have been suggested to contain less hyaline and more fibrocartilage (Bartz, C. et al. 2016. J Transl Med 14, 317). Consistent with these results, we obtained GAG / DNA ratios that were on average at least 3-fold greater than other published methods (Mumme, M. et al. 2016. Lancet 388, 1985-1994; Dang, P. N et al. 2014. Tissue engineering. Part A 20, 3163-3175) (Figure 2B).

[0014] Redifferentiation was due to removal of FGF-2. However, this removal in 3D culture was not sufficient to induce the synthesis of hyaline matrix in a two-step process. Chondrocyte redifferentiation most likely requires cell attachment to matrix-coated flasks and induction of specific cell signaling pathways in 2D culture.

[0015] The inventors developed a novel method for expanding chondrocytes in culture and recovering their chondrogenic potential from small pieces of cartilage (approximately 30 mg) regardless of the patient's age. In a first step, the cells are grown in an extracellular matrix with the support of FGF-2 in a two-dimensional culture, and then in a second step, they are pre-differentiated to their original phenotype by removing the FGF-2 used for cell growth. In a final step, complete redifferentiation is achieved in a three-dimensional (3D) culture with spontaneous cell aggregation forming microtissues. Surprisingly, the inventors showed that by removing the FGF-2 growth factor, especially in the second step in 2D culture, chondrocytes start to recover their original function through the synthesis of aggrecan, a protein associated with GAG, and that by continuing to remove FGF-2 in the third step in 3D culture, collagen II is synthesized together with GAG, thus producing the hyaline matrix characteristic of articular cartilage. Furthermore, the inventors have shown that removing FGF-2 induces the inactivation of the Wnt signaling pathway, preferably Wnt7B signaling, after the amplification step, thus allowing the production of homogeneous hyaline cartilage that is highly representative of cartilage in vivo. The inventors have shown that Wnt7B is activated in the dedifferentiation medium (ME) and reduced at least 10-fold in the pre-redifferentiation medium (MR) and at least 40-fold in the maturation medium (MI). The implantation of high-quality hyaline microtissues with characteristics as close as possible to those of native cartilage is important to predict the long-term success of the treatment of cartilage lesions (Negoro, T et al. 2018.. npj Regenerative Medicine 3, 17; Madeira, C et al. 2015. Trends in biotechnology 33, 35-42).

[0016] Thus, the engineered cartilage ("Cartibeads") are likely to fully integrate into the cartilage lesion following implantation.

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

[0018] In certain embodiments, in the third step, the chondrocytes are cultured in a hypoxic atmosphere containing less than 10% (v / v) O 2 .

[0019] In another aspect, the present invention relates to engineered cartilage tissue in the form of spheroids (Cartibeads) that present a GAG / double-stranded DNA ration of at least 15. Said engineered cartilage tissue can be used in the treatment of cartilage defects and cartilage degenerative diseases in subjects in need thereof, preferably in autologous transplantation.

[0020] Finally, the present invention also relates to a method for screening for molecules that inhibit the cartilage degeneration process, comprising the steps of contacting the engineered cartilage tissue described above with one or more candidate molecules and selecting a molecule that inhibits the cartilage degeneration process. [Brief description of the drawings]

[0021] [Figure 1] Characterization of Cartibeads engineered with the three-step method. a, Scheme of the three-step method for the generation of Cartibeads from dedifferentiated chondrocytes: step 1 (proliferation), step 2 (redifferentiation) were performed in 2D in atmospheric conditions of oxygen (21%) and step 3 (Cartibead formation) was performed in 3D culture in hypoxic conditions of oxygen (5%). b, Illustrative diagram of the preclinical study: Use of human Cartibeads to evaluate their safety in SCID mice and efficacy in minipigs. c, Histological analysis of Cartibeads from fixed samples. Representative images of Cartibeads from three independent donors with Safranin-O staining of GAGs characterizing hyaline cartilage (upper panel) and strong immunodetection of collagen type II (DAB staining, middle), as well as weak detection of collagen type I (lower panel). Scale bar 200 μm. d, Biochemical quantification of glycosaminoglycan (GAG) content in Cartibeads expressed in μg / mg tissue as determined by dimethylmethylene blue assay (DMMB) from 15 donors (black dots) and 3 native cartilage controls (squares). e, Biomechanical properties of Cartibeads are determined by compression testing (Young's modulus) which measures the elasticity of Cartibeads. Stress-strain curves are represented from 3 donors and show increasing constraint with larger Cartibeads. [Diagram 2] Amount of GAG in Cartibeads from all donors. a, Biochemical quantification of GAG content per Cartibead by DMMB assay. b, Biochemical quantification of GAG content of Cartibeads normalized to DNA content, expressed as GAG / DNA ratio (μg / μg). [Diagram 3] Comparison of Cartibeads produced at high and low oxygen levels and with the three-step and two-step methods. a, Macroscopic view of Cartibeads produced with the three-step method under low oxygen conditions in three donors. b, Safranin O staining of GAGs in sections of Cartibeads produced under four different conditions in one donor. Cartibeads were produced by the three-step and two-step methods under culture conditions with high and low oxygen levels (21% and 5%). Pictures are representative of three donors. Scale bar 200 μm. c, Evaluation of the amount of GAG / Cartibead under four different conditions in three donors. [Figure 4] Stability of Cartibeads. a, Biochemical quantification of GAGs in Cartibeads after the 3-step method was completed and in Cartibeads left in closed recipients for an additional 6 days at 37°C (incubator with 5% O2) and at 4°C and room temperature (RT). b, The number of dead cells in Cartibeads was quantitatively assessed by red fluorescent dye staining. Positive control for dead cells was Cartibeads pre-treated with 10% Triton before fluorescent staining. Scale bar 100 μm. [Diagram 5]Improved hyaline characteristics of Cartibeads engineered from the 3-step method compared to the 2-step method. a, Scheme of the 2-step method for the generation of Cartibeads. For the generation of Cartibeads, chondrocytes were grown in medium E in step 1 and used directly in medium I in step 2. b, Clustering of functional annotations of gene set enrichment analysis based on expression in 3-step vs. 2-step. The histogram shows the number of genes in each family of genes where significant enrichment was found. In light grey, genes are with high levels of expression in Cartibeads from the 3-step method and in dark grey, genes are with lower expression above. c, Visualization of RNAseq results using Volcano plot showing statistical significance (FDR<0.001) vs. magnitude of change (fold difference >2). The Volcano plot highlights genes with statistically significant fold difference such as ACAN, COL2A1, which are increased in the 3-step method compared to the 2-step, as well as COL1A1, which is decreased. ***p<0.001; **p<0.01 d, e, f Data from RNASeq analysis comparing the mRNA expression levels (in RPKM) of ACAN (d), COL2A1 (e), and COL1A1 (f) genes in the 3-step and 2-step methods. NA (not applicable) represents the absence of analysis due to the absence of the step itself. g, Safranin-O staining of GAGs in Cartibeads resulting from the 3-step and 2-step methods. Scale bar 100 μm. [Figure 6] MSC marker expression. a, Flow cytometry representing CD73, CD90, and CD105 from left to right, respectively. b, Differentiation study comparing adipose tissue stem cells (ASCs), mesenchymal stem cells (MSCs), and chondrocyte potential to provide osteocytes, adipocytes, and chondrocytes. Alizarin Red S is the dye used to stain calcium deposits indicative of mature bone cells (top panel), Oil Red O stains neutral lipids characteristic of adipocytes (middle panel), and Safranin-O stained GAGs in chondrocytes (bottom panel). Scale bar 100 μm. [Figure 7] Inhibition of WNT signaling enhances the production of hyaline matrix components. a, Gene set enrichment analysis and functional grouping based on differential expression levels in medium R vs. medium E in the three-step method. Graphs show enrichment for each gene family (number of differentially expressed genes). ***p<0.001; **p<0.01 b, Volcano plot of RNAseq results from chondrocytes in medium R vs. medium E in the three-step method. c. Data from RNAseq analysis show mRNA expression in RPKM for ki67 (proliferation marker) (top panel); TCF4 (involved downstream of the wnt / β-catenin pathway) (middle panel), and SOX9 (transcription factor involved in matrix production) (bottom panel). d, e, f Data from RNAseq show mRNA expression in RPKM of WNT5A (d), WNT5B (e), and WNT7B (f) of chondrocytes in medium E, R, and I in the 3-step and 2-step methods. NA (not applicable) represents the absence of analysis due to the absence of the step itself. g, Immunoblot shows the expression of the indicated proteins in the presence of the WNT signaling inhibitor XAV-939 (10 μM) in chondrocytes. The histogram shows the densitometric analysis of β-catenin and axin compared to the control. h, i, mRNA expression of ACAN and COL2A1 was determined by qPCR in chondrocytes after 4 days in culture in medium E, medium E + XAV-939, and in Cartibeads derived from chondrocytes cultured in medium E and medium + XAV-839 (2-step method). j, Representative images of Safranin-O staining of GAGs in Cartibeads made from chondrocytes cultured in medium R (three-step method) and medium E+XAV-939 (two-step method). Scale bar 100 μm. k, Scheme summarises the molecular basis of the WNT signalling pathway in medium E and WNT inhibition when chondrocytes are cultured in medium R or medium E+XAV939, allowing the production of hyaline matrix containing ACAN and type II collagen. [Figure 8]Feasibility of grafting Cartibeads in ex vivo human knees. a, Macroscopic view of a distal femur after total knee arthroplasty showing the lesion created manually with surgical instruments (left), the grafted Cartibeads on day 1 (middle), and the lesion with beads one month after grafting after rotational culture in Medium I (right). Scale bar 4 mm. b, Safranin-O staining of GAGs of cross-sectional samples of ex vivo specimens shows that the fused together Cartibeads retain their hyaline and are integrated with the native cartilage, as shown by higher magnification (left and right panels). Data are representative of three independent experiments. Scale bar 1 mm. [Figure 9] Absence of tumorigenicity of human Cartibeads after grafting in immunodeficient mice. a, Representative pictures of tumors derived from Cartibeads (0.2 × 106 chondrocytes / cartibead) 2 months after implantation (top panel) and from 5 A549 beads (0.2 × 106 A549 cells / bead) 6 weeks after grafting after subcutaneous grafting on the back of mice (black arrows, left panel). b, Tumor development after subcutaneous implantation of A549 cells, 1 or 5 beads of Cartibead, 1 per SCID mouse (n = 10–14 mice for 1 Cartibead donor and 2 × n = 4 mice per A549 bead). c, Safranin-O staining of GAGs in Cartibeads before (middle panel) and after (right panel) grafting. Scale bar 200 μm. [Figure 10]Integration of hyaline grafts in minipig knees after implantation of autologous Cartibeads. a, Scheme describes the main sites of biopsies used for the creation of Cartibeads and the second surgical procedure combining the creation of 5 lesions / knee with the implantation of autologous Cartibeads. b, Biochemical quantification of GAGs (μg / Cartibead) in 6 minipigs. cd, Macroscopic view (left panel) and Safranin-O staining of GAGs (middle panel) of minipig knee sections, where magnified segments show tissue remodeling 3 and 6 months after implantation. The implantation sites are circled in black, and white circles represent empty lesions as controls (right panel). Representative staining of implanted lesions (middle panel) and empty lesions (right panel) is shown at 3 months (c) and 6 months (d). Scale bar 200 μm. [Figure 11] Implantation of porcine Cartibeads in minipigs results in grafts with hyaline integration. a, Safranin-O staining for GAGs in Cartibeads before implantation (top panel, scale bar 200 μm) and macroscopic views of the knee (middle panel) and grafted lesion (bottom panel, scale bar 200 μm) for the six minipigs used in this study. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] The present invention relates to a method for the in vitro production of cartilage tissue, comprising three steps of culturing chondrocytes as described below.

[0023] Chondrocytes arise from mesenchymal cells that have a characteristic phenotype based primarily on the type of extracellular matrix they produce. Precursor cells produce type I collagen, but as they become committed to the chondrocyte lineage, they reduce production of type I collagen and begin to synthesize type II collagen, which constitutes the substantial protein of the hyaline extracellular matrix. In addition, committed chondrocytes produce proteoglycans, which are aggrecans bound to glycosaminoglycans that are highly sulfated.

[0024] The term "chondrocytes" as used herein refers to differentiated cells derived from cartilage (committed chondrocytes) that have the full ability to synthesize hyaline matrix. The term chondrocytes refers to primary cultures (freshly isolated from biopsies) and cells grown in vitro, including those that have been genetically modified, immortalized, selected, preserved, etc.

[0025] Isolation of target chondrocytes In the method of the present invention, in the first step, chondrocytes are isolated from a subject, preferably from the knee, ankle, hip, finger, or shoulder of a subject, more preferably from a human or equine subject. The chondrocytes are isolated from a biopsy of mature cartilage tissue by conventional methods, for example using enzyme digestion of the tissue, such as trypsin, chymotrypsin, collagenase, deoxyribonuclease, elastase, and hyaluronidase. The cartilage tissue can be in very small pieces, preferably less than 5 mm in diameter (corresponding to about 30 mg). The cartilage tissue is preferably mammalian, more preferably human cartilage or equine cartilage. The cartilage tissue can be collected from a healthy donor or from a patient, preferably from the knee, ankle, hip, finger, shoulder, more preferably from the knee or ankle of the patient.

[0026] First step: Adherent culture in dedifferentiation medium First, isolated chondrocytes are cultured in an adherent culture system in a dedifferentiation culture medium.

[0027] An adherent culture system suitable for use in the method according to the invention may be an adherent monolayer culture system or a culture system on feeder cells, preferably an adherent monolayer culture system. The culture system may be in any form suitable for the method according to the invention, in particular in the form of a flask, a multi-well plate or a dish.

[0028] In a preferred embodiment, the adherent culture system is an adherent monolayer culture system. This system comprises a solid support, for example glass or plastic, which is usually coated with a matrix or substrate that promotes the adhesion of the cells. The substrate may be a protein substrate consisting of adhesion factors that promote the adhesion of the cells to the support. These adhesion factors may be selected in particular from poly-L-lysine, collagen, fibronectin, laminin or gelatin.

[0029] Matrices that mimic the extracellular matrix and are suitable for use in the method according to the invention are well known to those skilled in the art and are commercially available in many types. These matrices include, for example, Matrigel™, Geltrex® type, CELLstart™ matrices, or other matrices that contain one or more anchoring proteins, such as collagen, laminin, fibronectin, elastin, proteoglycans, aminoglycans, or vitronectin. Three-dimensional hydrogel type matrices may also be used. In a preferred embodiment, the matrix is ​​Matrigel™ type or CELLstart™ Substrate.

[0030] Chondrocytes are cultured in an adherent culture system in a medium that allows the cells to expand and dedifferentiate in a precursor / undifferentiated state, referred to as a fibroblastic state (fibroblast-like morphology). In particular, the dedifferentiation medium is a culture medium that allows the expansion and dedifferentiation of chondrocytes into fibroblast-like cells, also referred to as fibroblast-like chondrocytes, which are dedifferentiated chondrocytes with a fibroblast-like cell morphology and exhibit stem cell characteristics. Preferably, the dedifferentiation medium allows the chondrocytes to dedifferentiate into fibroblast-like cells that express mesenchymal stem cell surface markers such as CD105, CD90, and / or CD73. Fibroblast-like cells are dedifferentiated chondrocytes that have lost the property of synthesizing hyaline matrix (collagen 2 and GAGs). The dedifferentiation medium is a basal medium that contains at least one or more components that allow the chondrocytes to expand and dedifferentiate in an undifferentiated state.

[0031] Many basal media are commercially available and well known to those skilled in the art. The medium may be a minimal medium that contains, among other things, inorganic salts, amino acids, vitamins, and a carbon source important to the cells, as well as a buffer system for adjusting pH. Basal media that can be used in the method of the present invention include, but are not limited to, DMEM / F12 medium, DMEM medium, RPMI medium, Ham'sF12 medium, IMDM medium, and KnockOut™ DMEM medium (Life Technologies).

[0032] Depending on the medium used, it may be necessary or desirable to add glutamine, vitamin C, one or more antibiotics, such as streptomycin, penicillin, and / or an antimycotic, such as fungizone (amphotericin B).

[0033] In the present invention, the de-differentiation medium activates the Wnt signaling pathway.

[0034] In the active state, Wnt ligands bind to frizzled receptors and their co-receptors, lipoprotein-receptor-related protein (LRP)5 / 6. This activates DSH (disheveled), which then inhibits the activity of GSK3β and phosphorylation of β-catenin. β-catenin is translocated to the nucleus and interacts with the transcription factor TCF / LEF (T-cell-specific transcription factor / lymphoid enhancer-binding factor). In the inactive state, β-catenin is phosphorylated by glycogen synthase kinase (GSK)3β, and the phosphorylated β-catenin subsequently undergoes ubiquitinylation and proteasomal degradation.

[0035] In a preferred embodiment, the de-differentiation medium is a basal medium containing one compound that activates Wnt signaling, preferably Wnt7B signaling.

[0036] Activation of the Wnt signaling pathway can be determined by measuring the expression levels of wnt RNA, particularly wnt7B, wnt5A or wnt5B, preferably wnt7B RNA, and downstream target genes of the Wnt signaling pathway, such as TCF4, in dedifferentiated chondrocytes. Activation of the Wnt signaling pathway in a cell occurs when the expression level of the target gene is at least 1.5-fold higher, or 2, 3, 4, 5-fold higher than in cells cultured without the compound.

[0037] The expression level of mRNA can be determined by any suitable method known to those skilled in the art. Typically, these methods include measuring the amount of mRNA. Methods for determining the amount of mRNA are well known in the art. For example, the nucleic acid contained in the sample is first extracted by standard methods, for example using a lytic enzyme or chemical solution, or extracted by a resin that binds nucleic acid according to the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e.g., Northern blot analysis), amplification (e.g., RT-PCR), or sequencing (RNA-seq).

[0038] 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 a cell lysate, with a binding partner capable of selectively interacting with the target gene protein present in the sample. This binding partner is generally a polyclonal or monoclonal antibody, preferably a monoclonal antibody. The amount of protein can be measured, for example, by semi-quantitative Western blot, enzyme-labeled immunoassay and enzyme-mediated immunoassay, such as ELISA, biotin / avidin type assay, radioimmunoassay, immunoelectrophoresis, or immunoprecipitation, or by protein or antibody arrays.

[0039] In particular, a compound capable of activating the Wnt signaling pathway may be a Wnt protein or a small molecule GSK-3β antagonist that binds to a Wnt receptor, preferably WNT7B, WNT5A, or WNT5B, more preferably WNT7B.

[0040] The compound capable of activating the Wnt signaling pathway can be 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), BIO (6-bromoindirubin-3'-oxime) (CAS No. 667463-62-9).

[0041] In a preferred embodiment, the compound is FGF2 (fibroblast growth factor-2), also known as bFGF, or the basic form of fibroblast growth factor.

[0042] Activation of the Wnt signaling pathway allows chondrocytes to expand and dedifferentiate 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, 100 ng / mL of FGF-2. Preferably, the medium contains 5-100 ng / mL of FGF-2, preferably 20 ng / mL of FGF-2.

[0043] In the present invention, in at least one embodiment, any molecule, such as a polypeptide, that acts on chondrocytes in a manner similar to that of the FGF2 molecule may be used, preferably for expanding chondrocytes in the methods of the present invention.

[0044] The additional component that supports chondrocyte to expand and dedifferentiate undifferentiated cells is not particularly limited and can be appropriately selected according to purpose.Examples of this component include bone morphogenetic protein (BMP), epidermal growth factor (EGF), platelet-derived growth factor (PDGF), transforming growth factor β (TGF-β), insulin growth factor 1 (IGF-1).Each of the above-mentioned other components can be used alone, or a plurality of them can be used in combination with a compound that can activate Wnt signaling pathway.

[0045] In a particular 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 1-100 ng / mL, preferably in the range of 5-50 ng / mL. In a more particular embodiment, the TGF-β is TGF-β3.

[0046] In a 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, preferably 5-100 ng / mL FGF-2, 1-100 ng / mL PDGF-BB and 1-100 ng / mL TGF-β3, more preferably 5-50 ng / mL FGF-2, 5-50 ng / mL PDGF-BB, and 5-50 ng / mL TGF-β3.

[0047] In another specific embodiment, in the method of the present invention, equine chondrocytes are cultured in a dedifferentiation medium containing FGF-2 and EGF, preferably 5-100 ng / mL FGF-2 and 1-100 ng / mL EGF, and more preferably 5-50 ng / mL FGF-2 and 5-50 ng / mL EGF.

[0048] In a preferred embodiment, the dedifferentiation medium further comprises serum of animal origin. In a preferred embodiment, autologous serum is used. It is also possible to use xenogeneic or allogeneic serum. In particular, in the culture of human chondrocytes, autologous serum from a human patient or collected human serum may be used. In a particular embodiment, in the culture of equine chondrocytes, fetal bovine serum may be used. The medium preferably comprises 2-20%, preferably 5-15%, more preferably 10% serum.

[0049] In a particular embodiment, the chondrocytes are contacted with the dedifferentiation medium for 10 to 20 days, preferably 10 to 15 days.

[0050] The cells are preferably subcultured periodically so that the culture does not reach confluence, i.e. does not cover the entire available surface. In fact, confluence induces proliferation arrest and undesirable metabolic changes. The cells can be subcultured using standard techniques well known to those skilled in the art. They can be detached from the matrix or carrier by the action of enzymes such as collagenase IV, or by mechanical passage in PBS or any other enzyme-free solution containing EDTA (e.g. ReleSR (STEMCELL Technologies)), or by the action of a commercially available cell detachment medium such as TrypLE™ Express (Life Technologies), and can be collected by centrifugation, mechanically dissociated, and reseeded in a new culture system.

[0051] Second step: Adherent culture in regeneration medium By inactivating the Wnt signaling pathway in the second intermediate step of the culture, used in the dedifferentiation medium for cell proliferation, the inventors surprisingly showed that culture in a 3D system allowed chondrocytes to recover their complete original phenotype and the full ability to synthesize hyaline matrix.

[0052] Thus, the method according to the invention further comprises an intermediate step of culturing the fibroblast-like cells obtained in the first step in the above-mentioned adherent culture system with a redifferentiation medium that inactivates the Wnt signaling pathway, preferably the Wnt7B, Wnt5A or Wnt5B, preferably the Wnt7B signaling pathway. The chondrocytes, in contact with this redifferentiation medium, reverse the fibroblast phenotype and redifferentiate the fibroblast-like cells into chondrocytes with the full properties of synthesizing hyaline matrix. In this second step, the cells change their morphology, becoming less elongated and larger, with granular endoplasmic reticulum evident in the cytoplasm under light microscopy, signaling a high level of protein synthesis activity. The chondrocytes start to reexpress aggrecan, a proteoglycan linked to GAG. Collagen II is not reexpressed at the end of the second step.

[0053] Preferably, the contacting is performed by simply changing the culture medium, or alternatively, it can be performed by subculturing in the above-mentioned adherent culture system containing the reversion medium.

[0054] In one embodiment, the adherent culture system is an adherent monolayer culture system as described above. In a preferred embodiment, the matrix is ​​of the Matrigel™ type or CELLstart™ Substrate.

[0055] In a first embodiment, the re-differentiation medium may be a re-differentiation medium comprising a basal medium from which the above-mentioned compounds that activate the Wnt signaling pathway have been removed.

[0056] By omitting a compound, it is intended that the re-differentiation medium contains a compound at a concentration that does not allow activation of the Wnt signaling pathway.

[0057] 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 of FGF-2, preferably less than 0.4, 0.3, 0.2, or 0.1 ng / mL of FGF-2.

[0058] In another embodiment, the re-differentiation medium is a basal medium containing an inhibitor of the Wnt signaling pathway.

[0059] The above-mentioned Wnt signaling pathway inhibitors include compounds that target Dvl proteins, such as NSC668036 (CAS number 144678-63-7), 3289-8625 (CAS number 294891-81-9), J01-017a, TMEM88, KY-02061, KY-02327, BMD4702 (CAS number 335206-54-7), and niclosamide (CAS number 5 0-65-7), DK-520, Sulindac (CAS No. 38194-50-2); compounds targeting the β-catenin destruction complex, such as Pyrvinium (CAS No. 7187-62-4); natural compounds, such as derricin (CAS No. 34211-25-1), derricidin (CAS No. 38965-74-1), carnosic acid (CAS No. 3650-09-7); compounds targeting TCF / LEF transcriptional 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 (C The compound may be a small molecule selected from the group consisting of TNKS targeting compounds, such as XAV939 (CAS No. 284028-89-3), E7449 (CAS No. 1140964-99-3), preferably XAV939.

[0060] In another embodiment, small inhibitory RNA (siRNA) can also be used to reduce the gene expression level of at least one protein involved in the signaling of Wnt pathway.In a preferred embodiment, the gene expression of the Wnt signaling protein can be reduced by inactivating Wnt signaling pathway by introducing small double-stranded RNA (dsRNA) or vector or construct that causes the production of small double-stranded RNA into cells (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or a vector encoding a dsRNA for a gene whose sequence is known are well known in the art (see, e.g., 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 Publication Nos. WO 01 / 36646, WO 99 / 32619, and WO 01 / 68836).

[0061] In another embodiment, small hairpin RNA (shRNA) can also be used to reduce the gene expression level of Wnt signaling protein. Small hairpin RNA (shRNA) is a sequence of RNA that forms a tight hairpin turn that can be used to silence the expression of target genes via RNA interference (RNAi). The expression of shRNA in cells is usually achieved by delivery of plasmid or via viral or bacterial vectors. The selection of promoter is important to achieve strong shRNA expression. Initially, polymerase III promoters such as U6 and HI were used; however, these promoters lack spatial and temporal control. Thus, there has been a shift to the use of polymerase II promoters to regulate the expression of shRNA.

[0062] In a preferred embodiment, siRNA is used to reduce the gene expression levels of Wnt5B, Wnt7B, Wnt5A, or β-catenin, preferably Wnt7B.

[0063] The basal culture medium may be a minimal medium that contains, among other things, inorganic salts, amino acids, vitamins, and a carbon source important to the cells, as well as a buffer system to regulate pH. Basal media that can be used in the methods of the present invention include, but are not limited to, DMEM / F12 medium, DMEM medium, RPMI medium, Ham's F12 medium, IMDM medium, and KnockOut™ DMEM medium (Life Technologies).

[0064] In certain embodiments, the redifferentiation medium is an FGF-2-free redifferentiation medium comprising a basal medium containing less than 0.5 ng / mL FGF-2 and at least one or more components that promote fibroblast-like cells to redifferentiate into chondrocytes.

[0065] In certain embodiments, the re-differentiation medium comprises transforming growth factor β (TGFβ), more preferably TGF-β3, and preferably the re-differentiation medium comprises 1-100 ng / mL TGF-β3, more preferably 5-50 ng / mL TGF-β3.

[0066] 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 of FGF-7, more preferably 5 to 50 ng / mL of FGF-7.

[0067] In certain embodiments, the regeneration medium used further comprises serum of animal origin as described above.

[0068] In a preferred embodiment, the TGF-β, FGF-7, and / or serum are replaced with platelet lysate. Platelet lysate is a cell culture supplement rich in growth factors derived from platelets after freeze / thaw cycles. The freeze / thaw cycles lyse the platelets, releasing large amounts of growth factors required for cell proliferation. In a preferred embodiment, the redifferentiation medium comprises 5-20%, preferably 10%, platelet lysate. In a preferred embodiment, the platelet lysate is a lysate of pooled human platelets.

[0069] The components that can affect the differentiation of chondrocytes are not particularly limited and can be appropriately selected according to the purpose. Examples of the 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 above-mentioned other components may be used alone or in combination.

[0070] In a particular embodiment, the chondrocytes are contacted with the re-differentiation medium for 2 to 10 days, preferably 3 to 7 days, more preferably 7 days.

[0071] Cells may be subcultured periodically to prevent the culture from reaching confluence, i.e., covering the entire available surface. In fact, confluence induces growth arrest and undesirable metabolic changes. Cells may be subcultured using standard techniques well known to those skilled in the art, as described above.

[0072] Third step: 3D culture system in induction / maturation medium To allow the formation of cartilage tissue, the chondrocytes obtained in the second step are then cultured in a three-dimensional culture system in a maturation medium, also called induction medium. In this step, the chondrocytes produce their own extracellular matrix to form cartilage tissue, and in particular start to synthesize collagen type II. The cartilage tissue obtained is a three-dimensional tissue of various sizes, which can be called spheroids. The spheroids are composed of the cells contained in the spheroids and the hyaline matrix formed by these cells.

[0073] A maturation medium is a basal medium that contains at least one component that induces chondrocytes to produce hyaline matrix and form cartilage tissue.

[0074] In particular, the maturation medium inactivates the Wnt signaling pathway. In one embodiment, the maturation medium comprises a basal medium from which the above-mentioned compounds that activate the Wnt signaling pathway are removed. In another embodiment, the maturation medium is a basal medium that comprises the above-mentioned inhibitor of the Wnt signaling pathway.

[0075] In a preferred embodiment, the maturation medium is a basal medium from which the FGF-2 growth factor has been removed. In other terms, 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, 0.1 ng / mL FGF-2.

[0076] The component that can affect the maturation of chondrocytes is not particularly limited and can be appropriately selected according to the purpose. Examples of this component 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 above-mentioned other components may be used alone, or a plurality of them may be used in combination.

[0077] In a particular embodiment, the maturation medium is the basal culture medium described above further comprising TGF-β, preferably TGF-β3.

[0078] Preferably, the maturation medium contains 1 to 100 ng / mL of TGF-β3, more preferably 5 to 50 ng / mL of TGF-β3.

[0079] In one embodiment, the chondrocyte maturation medium is a basal culture medium comprising TGF-β3, IGF-1, BMP-2, and insulin. Preferably, the chondrocyte maturation medium comprises TGF-β3, insulin, IGF-1, BMP-2, selenium, transferrin, and ethanolamine, more preferably TGF-β3, insulin, selenium, transferrin, and ethanolamine.

[0080] 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, preferably 5 to 50 ng / mL BMP-2.

[0081] In a particular embodiment, the chondrocytes are cultured in a three-dimensional culture system in a maturation medium for 10 to 20 days, preferably 10 to 15 days.

[0082] 3D culture can make cells bond with each other to support the synthesis of extracellular matrix by chondrocytes. 3D culture system can be static or dynamic. Static method implies providing cells with a mode to form aggregates by static physical force. Static method includes but is not limited to hanging drop method, liquid culture on non-adhesive substrate such as agar or agarose thin coating, culture on low-adhesive surface plate. Dynamic method implies forced cell aggregation. Dynamic method includes but is not limited to culture in spinner flask, rotating wall vessel, and pellet. In a preferred embodiment, the 3D culture system is pellet culture. In pellet culture, chondrocytes are dispersed in a plate and centrifuged to aggregate the cells and form a pellet.

[0083] In a preferred embodiment, the 3D culture in maturation medium is performed in a low oxygen atmosphere to enhance chondrogenesis. In a preferred embodiment, the chondrocytes are cultured in the 3D culture system in maturation medium in an atmosphere of less than 10 (v / v)% O2, more preferably less than 7% O2, more preferably 5% O2.

[0084] The cells are maintained in chondrocyte maturation medium until cartilage tissue is obtained. As used herein, cartilage refers to hyaline cartilage. During this period, the culture medium may be changed regularly, preferably every 2 or 3 days, in a conventional manner.

[0085] The quality of the resulting cartilage tissue can be tested by measuring the content of glycosaminoglycans (GAG) and collagen II in the engineered cartilage tissue. The quality and quantity of cartilage tissue can also be determined by measuring the amount of GAG / microtissue or by the ratio of GAG / double-stranded DNA. The presence of GAG, collagen, or double-stranded DNA can be evaluated by any method known in the art. For example, GAG can be revealed by coloring with Safranin-O or more quantitatively by the amount of GAG using dimethylmethylene blue assay (DMMB). Collagen II can be evaluated by immunostaining or by more quantitative assays such as ELISA.

[0086] Thus, optionally, the method according to the invention may comprise a further step consisting of measuring or assessing the presence of GAGs and / or collagen II in the engineered cartilage tissue.

[0087] Engineered Cartilage Tissue (Cartibeads) The present invention also relates to engineered cartilage tissue obtainable by the method according to the invention.

[0088] The present invention relates to engineered cartilage tissue in the form of spheroids, said spheroids presenting a GAG content per spheroid of at least 15, 16, 17, 18, 19, or 20 μg / spheroid, preferably 10-100 μg / spheroid, more preferably 15-60 μg / spheroid.

[0089] The present invention relates to engineered cartilage tissue in the form of spheroids, said spheroids presenting a GAG / double-stranded DNA ratio of at least 10, 15, or 20, preferably 10-100, more preferably 10-80.

[0090] The cartilage tissue spheroid has a diameter of 1 to 2 mm and contains 50,000 to 250,000 cells, preferably 200,000 cells.

[0091] Pharmaceutical Compositions 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 pharma- ceutically acceptable excipients.

[0092] The pharma- ceutically acceptable excipient must be compatible with the cells and may be, for example, a culture medium, a buffer solution, or saline.

[0093] In a preferred embodiment, the pharmaceutical composition is suitable for parenteral administration, preferably by the subcutaneous route, and in particular for direct administration into cartilage or bone tissue. The pharmaceutical composition may be formulated according to standard pharmaceutical practices known to those skilled in the art.

[0094] In certain embodiments, the pharmaceutical composition comprises the cartilage tissue of the present invention encapsulated in a biocompatible matrix.

[0095] The pharmaceutical composition may also contain one or more additional active compounds, such as compounds known to enhance cell survival or proliferation or to prevent contamination.

[0096] Therapeutic Indications In yet 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 particular for the treatment of cartilage defects and cartilage degenerative diseases in a subject in need thereof.

[0097] Thus, the present invention relates to an engineered cartilage tissue of the present invention for use in treating cartilage defects and cartilage degenerative diseases in a subject in need thereof, and it also relates to a pharmaceutical composition according to the present invention for use in treating cartilage defects and cartilage degenerative diseases in a subject in need thereof.

[0098] The present invention is particularly suitable for the transplantation of autologous cartilage tissue in a subject in need thereof. In the transplantation of the autologous engineered cartilage tissue of the present invention, chondrocytes are first isolated from the subject, preferably from a biopsy of the subject's mature cartilage tissue. The chondrocytes are then cultured by the method of the present invention to obtain autologous engineered cartilage tissue, which is used to treat cartilage defects and cartilage degenerative diseases in the patient.

[0099] Thus, in a preferred embodiment, the present invention relates to the use of the autologous engineered cartilage tissue of the present invention or a pharmaceutical composition comprising said autologous engineered cartilage tissue for the treatment of cartilage defects and cartilage degenerative diseases in a subject in need thereof.

[0100] In another embodiment, allogeneic transplants may be used in subjects in need thereof.

[0101] As used herein, the term "cartilage defects and cartilage degenerative diseases" includes, but is not limited to, cartilage lesions, arthropathy, rheumatism, or osteoarthritis.

[0102] In a particular embodiment, the engineered cartilage tissue of the present invention or a pharmaceutical composition thereof is indicated for patients suffering from focal articular cartilage lesions or early osteoarthritis, to delay or avoid total knee replacement, which is generally not proposed for patients under the age of 60 due to the limited life span of artificial joints. Thus, in a preferred embodiment, the cartilage defect and cartilage degenerative disease is focal articular cartilage lesion or early osteoarthritis.

[0103] The term "treatment" as used herein refers to the improvement or elimination of symptoms, such as reduced pain and mobility, slowing the progression of a disease, halting the development of a disease, or eliminating a disease, and includes both preventative and curative treatments.

[0104] As used herein, the term "subject" or "patient" refers to a mammal. Mammalian species that can 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 animals such as horses, cows, pigs, sheep, and goats, or other mammalian species including, but not limited to, camels, mice, rats, guinea pigs, rabbits, hamsters, and the like. In certain embodiments, the subject is a human or a horse.

[0105] The present invention also relates to the use of the cartilage tissue or pharmaceutical composition of the present invention for the preparation of a drug intended for the treatment or prevention of cartilage defects and cartilage degenerative diseases.

[0106] The present invention further relates to a method for treating cartilage defects and cartilage degenerative diseases, comprising the step of administering to a subject to be treated a therapeutically effective amount of the engineered cartilage tissue or pharmaceutical composition according to the present invention.

[0107] In other words, the present invention also provides a method for treating cartilage defects and cartilage degenerative diseases in a subject in need thereof, comprising: i) isolating chondrocytes from said subject; ii) culturing the chondrocytes in an adherent culture system in a dedifferentiation culture medium that activates the Wnt signaling pathway to obtain fibroblast-like cells; iii) culturing the fibroblast-like cells in an adherent culture system in a redifferentiation culture medium that inactivates the Wnt signaling pathway to obtain chondrocytes; iv) culturing the chondrocytes of step iii) in a 3D culture system in a maturation medium that inactivates the Wnt signaling pathway as described above to obtain cartilage tissue; v) administering a therapeutically effective amount of cartilage tissue to the subject to be treated; The present invention relates to a method comprising the steps of:

[0108] The term "therapeutically effective amount" as used herein refers to an amount sufficient to reduce symptoms of pain or increase mobility in a subject exhibiting a cartilage defect and / or a cartilage degenerative disease.

[0109] In a preferred embodiment, a 1 cm 2 Ten to fifty spheroids of cartilage tissue, approximately 1-2 mm in diameter, are administered per mouse. The spheroids self-adhere to the subchondral bone and the inner edge of the cartilage lesion.

[0110] The engineered cartilage tissue or pharmaceutical compositions of the present invention may be administered by grafting the cartilage onto the surface of a cartilage or support matrix or directly into the local environment of the cartilage or support matrix of the subject.

[0111] The engineered cartilage tissue or pharmaceutical compositions of the present invention may be administered to a subject by open-joint surgical procedures or via arthroscopy, preferably via arthroscopy to allow for faster recovery for the patient.

[0112] After being implanted in vivo, chondrocytes in the cartilage tissue respond to mechanical loading by continuing to produce matrix to fill full-thickness defects up to the surface of the damaged tissue and potentially fuse together in vivo to form homogenous cartilage tissue.

[0113] kit The present invention also relates to a kit for the in vitro production of cartilage tissue, comprising: a first container comprising one or more compounds present in the dedifferentiation medium described above, preferably FGF-2, more preferably FGF-2 and a growth factor selected from the group consisting of EGF, TGF-β3, and PDGF-BB, and any combination thereof; 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 said TGF-β3 and / or FGF-7 can be replaced by a platelet lysate; a third container containing one or more compounds present in the maturation medium as described above, preferably TGF-β3, more preferably TGF-β3 and one compound selected from the group consisting of IGF-1, BMP-2, insulin, selenium, transferrin, and ethanolamine, and any combination thereof, again more preferably TGF-β3 and insulin, selenium, transferrin, and ethanolamine; Includes.

[0114] Preferably, the kit comprises multiple containers, each containing one or more compounds at concentrations or amounts that facilitate the use of the reconstitution and / or differentiation and / or maturation medium and the practice of the methods of the invention. The kit of the invention may also comprise a container containing the basal medium described above.

[0115] The kit according to the invention may also comprise an adherent culture system, in particular in the form of a flask, a multiwell plate or a dish.

[0116] The kit may also include instructions describing how to prepare and / or use the differentiation or maturation medium for the in vitro production of cartilage tissue according to the methods of the invention.

[0117] The present invention also relates to the use of a kit according to the invention for the in vitro production of chondrocytes according to the method of the invention.

[0118] Methods for screening molecules of therapeutic interest - Patents.com In another aspect, the present invention relates to the use of the cartilage tissue of the present invention for screening molecules of therapeutic interest.

[0119] Molecules of therapeutic interest may in particular be molecules that inhibit the degenerative processes of cartilage degenerative diseases. These molecules may in particular be usable for the treatment or prevention of the above-mentioned cartilage defects or cartilage degenerative diseases.

[0120] The present invention thus provides a method for screening for a molecule of interest, comprising the steps of: i) contacting the cartilage tissue of the present invention with a candidate molecule; ii) selecting a molecule having a desired activity; The present invention relates to a method comprising the steps of:

[0121] The present invention relates in particular to a method for screening molecules that inhibit cartilage degeneration processes, comprising the steps of: i) contacting the cartilage of the invention with one or more candidate molecules; ii) selecting a molecule that inhibits the cartilage degeneration process; The present invention relates to a method comprising the steps of:

[0122] Cartilage tissue production can be assessed by techniques well known to those of skill in the art, such as methods including, for example, measuring GAGs or collagen II.

[0123] Depending on the nature of the molecule sought, the chondrocytes used to produce cartilage tissue may be obtained from healthy subjects or from subjects with a cartilage defect or cartilage degenerative disease as defined above.

[0124] All references cited in this description are incorporated by reference in the present application. Other characteristics and advantages of the invention will become more apparent on reading the following examples, given by way of non-limiting illustration.

[0125] Working Example material and method chemicals XAV939 was obtained from Sigma and used at a concentration of 10 μM in medium E for 4 days in 2D culture.

[0126] Production of Cartibeads The following media, as listed in Table 1, were used in the examples. [Table 1] TIFF0007645553000002.tif51162Table 1: Composition of the medium used in the examples

[0127] Sample collection Human cartilage samples were obtained from life consenting donors (aged 18-80 years) following orthopedic procedures for a variety of indications (Table 2). [Table 2] Table 2: Donor characteristics including gender, age, and medical condition

[0128] The harvested cartilage was transferred to a sterile recipient laboratory in normal saline (NaCl 0.9%) at room temperature. The collection of human cartilage samples was approved by the Swiss Ethics Committee (BASEC, 2016-00656).

[0129] Minipig cartilage was obtained from the lateral trochlear holder (approximately 30 mg) and harvested from the right knee of the minipig.

[0130] Cartilage (approximately 30 mg) was sliced ​​into small pieces (1 mm) to facilitate extraction of chondrocytes by enzymatic digestion, followed by overnight rotation at 37° C. using collagenase type II (400 U / ml, ThermoFisher) in medium E containing antibacterial (gentamicin, 50 ug / ml) and antimycotic (amphotericine B or Fungizone®, 0.250 ug / ml).

[0131] Cultivation of cells and production of Cartibeads Cells were washed and plated onto T25cm plates pre-coated with extracellular matrix (MaxGel™, Sigma). 2 2D cell cultures were performed in flasks coated with extracellular matrix. Upon reaching confluence, cells were passaged into 1 T75 (p1) and then split into 2x T75 (p2) and allowed to reach confluence. At this stage cells can be frozen for backup (p3). After cell growth in medium E (step 1), in step 2 cells were cultured in medium R for 7 days, where a decrease in their proliferation was observed before proceeding to 3D culture in step 3. In step 3, chondrocytes were harvested, aggregated in medium I and plated at 0.2 × 10 cells in conical 96-well plates. 6 Cells / well (approximately 20 x 10 6 100 cells / plate) were obtained. The 96-well plates were centrifuged at 300g for 5 min to allow cell aggregation and formation of Cartibeads after 15 days in 3D culture. Cartibeads were obtained from chondrocytes up to passage 8. The beads were removed from the 96-well plates and pooled together, and they can be maintained in Medium I at 4-23°C (room temperature) for up to 6 days with high stability.

[0132] Quantification of GAGs Glycosaminoglycan (GAG) content was assessed by dimethylmethylene blue assay (DMMB) (Sigma, 341088). Chondroitin sulfate A (Sigma, C9819) was used to generate six standards with concentrations ranging from 0 to 50 μg / mL. Chondroitin sulfate C (Sigma, C4384) was used to generate low and high Internal Quality Controls (IQCs) with concentrations of 15 and 35 μg / mL, respectively. Cartibeads were digested with proteinase K (1 mg / ml) (Promega, V3021) in Tris-HCl (50 mM, pH 8) (Sigma) for 15 ± 2 hours at 56 °C. Digestion with this enzyme was stopped by incubation at 97 °C for 15 minutes. The resulting samples were then diluted (1 / 5 to 1 / 10) in Tris-HCl (50 mM, pH 8) for the assay. 100 μL of standards, ICQs, and samples were read in triplicate using a spectrophotometer (λ=525 nm) after 5 min reaction with 1 mL of DMMB solution. GAG content was then normalized to the amount of DNA, which was measured with the PicoGreen-Qubit assay. Standards and ICQs were prepared from calf thymus DNA in two separate preparations. Standards and ICQs were prepared in 200 mM Tris-HCl, 20 mM EDTA, pH 7.5 (TE) buffer; these samples were from proteinase K digestion and then diluted 1 / 15 in TE buffer. In this assay, 100 μL of standards, IQCs, and then samples were taken and divided into triplicates. Then, 100 μL of 1 / 200 diluted PicoGreen Quant-It® (ThermoFischer, P11496) was added. The samples were then incubated for 5 min, during which time the intercalant was complexed with the DNA. Finally, readings were taken on a Qubit 4 Fluorometer (ThermoFischer, Q33238) with an excitation peak of 485 nm.

[0133] Cartibeads Cell Viability Samples were tested for viability via an assay using the red fluorescent dye Zombie Aqua™ (Biolegend, 423101), an amine-reactive fluorescent dye that is non-permeable to live cells but permeable to cells with defective membranes, allowing for the assessment of live versus dead cells. 3D samples were collected by the end of step 3, then washed with PBS, then Zombie dye (1:100) diluted in PBS was added for 20 minutes, and samples were stored in the dark. Samples were then cut at a thickness of 3 μm via a cryostat microtome and mounted on Superfrost Plus glass slides. They were then fixed in 4% formol and then treated with Hoescht (Molecular probe, H3570 Thermofisher) diluted in PBS (1:2000) for 10 minutes. A control for this assay was achieved by adding 10% Triton™ (Sigma, X100-100ML) in PBS for 1 hour at room temperature (RT) before starting the Zombie dye step.

[0134] Immunohistochemical staining For immunohistochemical staining of formalin-fixed paraffin-embedded cartilage tissue and Cartibeads, paraffin blocks containing samples were sectioned at 5 μm using a microtome. The slides were dried overnight at 47°C. Slides were deparaffinized with Xylol and rehydrated with successive alcohol baths (100%, 95%, and 70% concentrations). Two techniques of unmasking were used. For native cartilage from miniature pigs, we used a solution of 20 mg / mL hyaluronidase in 0.1 M phosphate buffer and placed on the slides for 1 hour at 37°C. Slides were rinsed twice with PBS for 5 minutes to remove the hyaluronidase. Cartibeads samples were immersed in 0.01 M citrate buffer baths, pH=6, and heated three times in a microwave oven at 620 w for 5 minutes each, then cooled in an ice bath for 20 minutes. Slides were then rinsed with PBS for 5 minutes. Primary antibodies were then used (Collagen I Abcam, ab6308; Collagen II Abcam ab85266 and ThermoFischer MA5-12789). Of note, the two collagen type II antibodies used showed similar results. These antibodies were placed on different samples and diluted 1:100 in 0.3% Triton:PBS for 1 hour. After rinsing with PBS for 5 minutes, secondary antibodies biotin-conjugated anti-mouse (Vector lab, BA-2000), anti-rabbit IgG (Vector lab BA-1000), and avidin-biotin peroxidase detection system with 3,3'-diaminobenzidine substrate (Vector Labs) were used. Samples were counterstained with hematoxylin. Dehydration was achieved by vigorous dehydration in alcohol baths (95% and 100% concentrations), followed by shaking the rack 10-20 times in a Xylol bath until the slides were assembled with Eukitt resin (Batch A1113, KiNDO1500). A Nikon Eclipse C1 confocal microscope and a Nikon Eclipse TE2000-E were used for imaging.

[0135] Safranin-O staining Safranin-O staining of formalin-fixed paraffin-embedded samples of cartilage spheres and native cartilage was performed to reveal glycosaminoglycans (GAGs) on 5 μm paraffin slices. Slides were deparaffinized with Xylol and rehydrated by successive alcohol baths (100%, 95%, and 70% concentrations) and finally by using a distilled water bath for 5 min. Hematoxylin stain was then used for nuclear counterstaining, followed by a 5 min hot water wash. Fast Green (Sigma F7252) was used to stain the cytoplasm, followed by a washout with acetic acid. Slides were immediately washed with distilled water. 0.1% Safranin-O (Sigma S2255) in a water bath was applied for staining of GAGs for 2.5 min, followed by repeated washes with distilled water. Dehydration was achieved by vigorously shaking the rack 10-20 times in alcohol baths (95% and 100% concentrations) and then in a Xylol bath until the slides were assembled with Eukitt resin (Batch A1113, KiNDO1500).

[0136] Assessment of pluripotency Human MSC / ASC / chondrocytes grown in monolayer were differentiated into chondrogenic, adipogenic, and osteogenic fates to assess their multipotential differentiation. Osteogenic differentiation was assessed using Alizarin Red S (Merck, TMS-008-C) staining. Adipogenic differentiation was assessed using Oil Red O (Sigma, O1391) staining, and chondrogenic differentiation was assessed using Safranin-O (Sigma, S2255) / Fast Green staining (Sigma F7252).

[0137] Biomechanical measurements Compression tests were performed on cartilage beads ranging from 0.68 mm to 1.54 mm in diameter and on native cartilage using an MTS criterion pull-push machine (model 42) equipped with a 1 N load cell. The compression speed was 0.01 mm.s. -1The strain curve was then calculated to estimate the Young's modulus of cartilage, which was divided by the surface area of ​​the sample and the number of beads for normalization and to account for the slope of the data set in the linear region. Calculation of Young's modulus was performed as described below. E=σ / ε, where σ is the compressive stress (kPa), E is Young's modulus (kPa), and ε is the strain (unitless) corresponding to the normalized elongation.

[0138] Immunoblotting Cells were lysed in ice-cold RIPA buffer (Life Technologies) supplemented with phosphatase and protease inhibitors (Complete anti-protease cocktail; Roche) for 30 min on ice. Proteins (10 μg) were separated by SDS-PAGE (BioRad) and transferred to PVDF membranes (Amersham). Blots were probed with anti-phospho-β-catenin (Cell Signaling; 5651T), TCF-4 (Cell Signaling; 2569), axin 1 (Cell Signaling; 2087), β-actin HRP (Sigma-Aldrich), and GADPH (Cell Signaling; 2118) (1:1000), followed by HRP-rabbit or mouse conjugated antibodies (1:5000).

[0139] Flow cytometry 100,000 cells were fixed with 4% PFA and then stained for CD73-CFS, CD90-APC, and CD105-PerCP (Cell Sigma) in FACs buffer (BSA-Azide-PBS) for 1 h at RT. A minimum of 10,000 live cells were acquired using a Gallios flow cytometer and analysis was performed using FlowJo software. Results represent the average of three independent experiments for CD73 and CD90. For CD105, two independent experiments were performed. 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 defined based on the negative controls IgG-CFS, IgG-APC, and IgG-PerCP for CD73-CFS, CD90-APC, and CD105-PerCP, respectively.

[0140] RNASeq Sequencing quality control was performed with FastQC v.0.11.5 (Cosset, E. et al. 2016. Biomaterials 107, 74-87) using SR100-library TruSeqHT stranded-Illumina HiSeq 4000 as previously described. The distribution of quality along the reads was assessed and verified for all samples. Reads were mapped with STAR aligner v.2.5.3a against the reference genome using UCSC human hg38 reference material. The average mapping percentage was 93.54%. Transcriptomic metrics were evaluated with Picard tools v.1.141 and differential expression analysis was performed with the statistical analysis R / Bioconductor package edgeR v. 3.18.1 (Gentleman, RC et al. 2004. Genome Biol 5, R80, Huber, W. et al. 2015. Nat Methods 12, 115-121). Briefly, counts were normalized and filtered by library size. Genes with a count of reads greater than 1 cpm (counts per million) in at least three samples were kept for analysis. The raw gene count for this set was 26,485. Poorly or not expressed genes were excluded. The final data set consisted of 13,884 genes. Tests for differentially expressed genes were performed with an exact test using the negative binomial distribution. The p-values ​​of differentially expressed genes were corrected for multiple testing error using a 5% FDR (false positive rate). The correction used was Benjamini-Hochberg (BH). Testing of differentially expressed genes was performed with edgeR using a negative binomial distribution. The p-values ​​of differentially expressed genes were corrected for multiple testing error using the Benjamini-Hochberg (BH) false positive rate (FDR).

[0141] Panther analysis was used to determine enrichment (number of entities) in each family of genes (Mi, H. et al. 2017. Nucleic Acids Res 45, D183-D189).

[0142] RNA extraction and RT-PCR Total RNA was isolated using the RNeasy kit from Qiagen according to the manufacturer's instructions and measured using a spectrophotometer. cDNA was synthesized using 500 ng of total RNA and the PrimeScript RT Reagent Kit (Takara) according to the manufacturer's protocol.

[0143] Real-time PCR was performed at the Genomic platform core facilities (University of Geneva) using the QuantStudio 12K Flex Real-Time PCR System (Thermo Fisher Scientific) with PowerUp SYBR Green Master Mix (Applied Biosystems). Initial validity testing was performed on all primers for prior use validation. Relative levels in each sample were normalized to at least two housekeeping genes (ALAS1). RT-PCR reactions were performed in at least technical and biological triplicates, and the mean cycle threshold (CT) was determined.

[0144] Implantation of Human Cartibeads in SCID / NOD Mice for Safety Studies Fifty-six male SCID / NOD mice were used to test the safety of the standardized Cartibeads of the present invention. Forty-four mice received human Cartibeads by subcutaneous implantation. One control group was used, in which eight animals received aggregated A549 adenocarcinoma cells, designated as beads (number of mice per group is summarized in Table 3). [Table 3] Table 3. List of donor Cartibeads implanted in SCID mice. Cartibeads from different donors and cell passages (3–6) were subcutaneously injected into SCID / NOD mice (n=8–14 mice per group). The adenocarcinoma cell line A549 was used as the positive cell line, which resulted in 100% tumors. After euthanizing the animals at 6 months, the mice showed no weight loss, abnormalities by palpation, or signs of distress due to Cartibead implantation. Furthermore, no abnormalities or evidence of tumor presence was observed in the lungs, heart, liver, kidneys, and spleen.

[0145] All implanted human Cartibeads were cultured by a standardized three-step method and implanted in the same way. General anesthesia was achieved with 4% isoflurane, then 2% isoflurane, while shielding with 5% oxygen. After shaving the back, the skin was topically disinfected with 70% alcohol. A skin incision was made 0.5 cm caudal to the occipital pole. 200,000 cells of the resulting tissues (Cartibeads and adenocarcinoma beads) were subcutaneously implanted (1 bead / animal) using a sterile pipette, respectively. The skin was then closed using surgical glue (Histoacryl, B. Braun Surgical SA). To indicate the site and orientation of the samples in the skin, we tattooed the four cardinal points using a sterile 26-gauge needle and green tattoo ink. Mice in the control group were euthanized at 4-6 weeks, and mice in the Cartibeads group were followed up to 6 months. Skin and organs were collected and examined by two study members at each stage. All samples were preserved 1 / 3 in 4% formol and 2 / 3 desiccated at -20°C. Animal procedures were approved by the Swiss Federal Veterinary Office (GE / 12 / 18).

[0146] Comparative genomic hybridization arrays Genomic stability was also determined by comparing normal human dermal fibroblasts treated with 10% FBS or 10% PRP for 6 days using array comparative genomic hybridization (CGH). DNA was extracted using the QIAGEN QIAamp DNA Mini Kit (Qiagen, Hilden) according to the manufacturer's protocol. Array CGH was performed using the Agilent SurePrint G3 Human CGH Microarray kit 4_44K (design ID 014950) (Agilent Technologies) with an overall median probe distance of 43 Kb. The actual resolution was approximately 200 kb. Patient DNA and gender-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 using Amicon Ultra 30 K filters (Millipore). Hybridization was performed according to the protocol provided by Agilent. DNA from patients and controls was pooled and hybridized with 2 mg of human Cot-I DNA for 24 h with rotation at 65°C. Arrays were analyzed using an Agilent SureScan microarray scanner and Agilent Feature Extraction software (v11.5), and results were presented by Agilent Genomic Workbench (v.7.0).

[0147] Implantation of Autologous Cartibeads in Minipigs for Efficacy Studies Six adult female minipigs (37-48 kg) were used in this study. The animals were anesthetized with sevoflurane and received prophylactic antibiotics (cefazolin: 2 g / kg) intravenously 30 min before incision. They were operated on under total coma and extubated 5 min after skin closure. Skin sterilization was repeated three times and sterile drapes were performed using disposable surgical drapes.

[0148] Step 1 Surgery A super-lateral para-patellar approach was chosen for the first surgical procedure. Cartilage harvesting was performed at the superolateral border of the lateral trochlear facette to prevent possible overlap with the cartilage lesion during the second surgical step later. Cartilage biopsies were placed in culture medium E for further processing. Staged closure was performed. No dressing was applied.

[0149] Step 2 Surgery Five to six weeks after cartilage harvest, the minipigs underwent a second surgical procedure via the medial para-patellar tendon approach (Bonadio, MB et al. 2017. J Exp Orthop 4, 11). With this technique, two lesions each on the trochlear groove of the medial and lateral femur were created in all but one minipigs due to the extent of the donor site. The lesions were 6 mm in diameter and created with a small curette and then completely filled with a single layer of 4 to 5 Cartibeads. For faster integration, 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 an identical manner. In each animal, one lesion was left empty in a different position as a negative control lesion. A staged closure was performed. No dressing was applied.

[0150] Rehabilitation As soon as the coma ended, full weight bearing was possible without any restriction of activity.

[0151] Euthanasia Three minipigs were euthanized 3 months after the second surgery and the remaining 3 were euthanized at 6 months. The operated knee was amputated and the distal femur was placed in formaldehyde solution for further analysis.

[0152] Animal procedures were approved by the Swiss Federal Veterinary Office (GE / 60 / 18).

[0153] result Engineering and Characterization of Cartibeads In this study, we created cartilage microtissues, named Cartibeads, with improved hyaline cartilage characteristics, defined by GAG content and collagen II expression. The Cartibeads method is an innovative three-step protocol (Figure 1A) consisting of (step 1) chondrocyte proliferation in two-dimensional culture (2D), characterized by cell dedifferentiation, followed by (step 2) cell redifferentiation in 2D, defined as chondrogenic commitment, and finally (step 3) three-dimensional culture (3D), allowing chondrocyte aggregation to form Cartibeads. Thus, human chondrocytes were enzymatically extracted from surgical waste and cultured in 2D in atmospheric oxygen conditions (21%) in steps 1 and 2, followed by 3D culture at low oxygen levels (5%).

[0154] Chondrocytes become "fibroblasts" with stem cell characteristics in gene expression and express mesenchymal stem cell surface markers (CD105, CD90, CD73) (Figure 6). After culturing in medium 1 (medium E) for 3 weeks, it is possible to obtain 60 to 100 million cells from the extracted 100,000 cells.

[0155] The second step corresponds to the "reversion" of the fibroblast phenotype to the chondrocyte phenotype after a large cell expansion step. The cells are placed in the redifferentiation medium (medium 2) for 3-7 days. On day 0, 2 million cells are placed in a T75 cm2 flask until confluence is reached, and the cells are kept confluent until days 4-8, after which they are detached for 3D culture. From a T75 cm2 flask, 6 to 10 million cells are obtained in step 2. In contrast, in the first step in dedifferentiation medium, 12 to 16 million cells are obtained in a T75 upon reaching confluence. The removal of the FGF-2 growth factor in the redifferentiation medium (medium R) reduces the proliferation of the cells and allows the reversion of the phenotype and redifferentiation towards the full differentiation of chondrocytes. The cells change their morphology in the second step of culture, becoming less elongated and larger, with granular endoplasmic reticulum evident in the cytoplasm under light microscopy, thus signaling high levels of protein synthesis activity. Chondrocytes start to re-express aggrecan, a proteoglycan linked to GAGs (data RNAseq). In this second step in 2D culture, collagen II is not yet re-expressed.

[0156] In the third step, cells are detached from the culture in medium 2 (medium R) and seeded in 3D culture. 100000-200000 cells are dispersed in redifferentiation culture medium (medium 3, also called medium I) in polypropylene conical 96-well plates. The 96-well plates are centrifuged at 300 g for 5 min to aggregate the cells and form a pellet. The 96-well plates are made of polypropylene material to avoid cell sticking at the surface of the wells. The cultures were incubated in a low oxygen atmosphere (5% O2) to increase the production of hyaline cartilage for 15 days before using the microtissues.

[0157] Cartibeads engineered from chondrocytes held potential as ATMPs to treat cartilage lesions. Therefore, to evaluate their safety and efficacy, we used animal models (SCID mice and minipigs) (Figure 1B). To better characterize Cartibeads, a histological qualitative analysis was performed. The presence of hyaline characteristics with a homogenous distribution of GAGs stained with Safranin-O was shown, which correlated with strong immunodetection of collagen type II, but faint collagen type I (Figure 1C).

[0158] We were able to engineer similar quality cartilage tissue (called cartibeads) from donors up to 80 years old, including from osteoarthritis (OA) patients. Quantitative analysis of the cartibeads revealed high GAG content, which was independent of the patient's age and the osteoarthritic status of the joint (Figure 1D, Table 2). We measured an average of 40 μg GAG / cartibead and an average of 50 (10:140) GAG / DNA (Figure 2).

[0159] To characterize the biomechanical properties of Cartibeads, we used a device with a custom-made mold adapted to quantify the elasticity of Cartibeads by compression testing. Young's modulus was calculated by dividing the compressive stress by the strain (normalized elongation) in the elastic part of the curve (Lee, JK et al. 2017, Nature Materials, 16:864-873). This method is commonly used to test the elasticity of cartilage, reflecting the composition of its extracellular matrix (ECM). We observed an increased resistance to constraint in Cartibeads containing more GAGs, suggesting an advantage of increased amounts of GAGs (Figure 1E), consistent with other published studies (Omelyanenko, NP et al. 2018, Cartilage, 1947603518798890).

[0160] Since HIF-1α (Hypoxia-Inducible Factor 1-alpha) is known to induce components of the ECM (Madeira et al. 2015, Trends in Biotechnology, 33:35-42), we evaluated the effect of low oxygen levels (5% oxygen) during 3D culture on the quality of Cartibeads compared to atmospheric conditions. As expected, the results showed an improvement in the hyalinity quality of the cartilage beads in hypoxic conditions (Figure 3A-C). Of note, standard culture conditions in atmospheric conditions actually correspond to hyperoxia, since the level of oxygen recorded in native cartilage tissue is 0.5-5% depending on the depth (Lafont, JE 2010, Int. J. exp. Pathol. 91(2):99-106).

[0161] In cell therapy, the stability of the cellular components of the implanted material is important. Therefore, we evaluated the stability of Cartibeads in different conditions and viable cell ratios. We compared the amount of GAG in Cartibeads at day 0 after the end of the maturation step, where Cartibeads were stored at 4°C and 23°C for an additional 6 days (Figure 4A). These results showed that the concentration of GAG remained unchanged over time and in the variation of insensitive temperature, on average 55 μg / cartibead, indicating stability. Only a small number of dead cells were detected in these conditions (13%), suggesting good survival of chondrocytes in Cartibeads (Figure 4B).

[0162] Transcriptome analysis of the three-step Cartibead method identified low-level expression of WNT genes as a key pathway involved in hyaline matrix quality. In an attempt to identify the molecular pathways responsible for the increased production of hyaline cartilage, the three-step Cartibead method was compared to the two-step method traditionally used in tissue engineering (Figure 5A). From the morphology point of view, the three-step method resulted in larger white-pigmented Cartibeads (1-2 mm diameter) and a higher amount of GAG / bead than the two-step method (0.5-1 mm diameter) (Figure 2A-C). The improvement in the hyaline quality of Cartibeads in the three-step method was mainly due to the introduction of a further step after extensive cell expansion in FGF-2. This step corresponds to a starvation step, using medium "R" without FGF-2, thus facilitating the redifferentiation of chondrocytes and the production of hyaline matrix. In both methods, the final step consists in 2 weeks of 3D culture in maturation medium I with supplementation of TGF-β3 (Figure 5A).

[0163] To understand the molecular mechanisms that allow the production of Cartibeads, we performed RNA sequencing (RNAseq) analysis using three donor samples at each critical step of both methods. RNA was extracted at different time points of the three-step Cartibead method: (i) at the end of step 1 (amplified chondrocytes, medium E), (ii) at the end of step 2 in medium R, and (iii) after 15 days at step 3 (beads, medium I). For the two-step method, two time points were selected: at the end of step 1 (amplified chondrocytes, medium E) and step 2 (beads, medium I). First, we compared the cartilage microtissues produced by both methods in medium I (Figures 5B and C).

[0164] We observed significantly higher levels of genes involved in collagen and ECM degradation / formation along with ECM organization on Cartibeads (COL1A1, COL2A1, COL4A1, MMP1, MMP13, MMP11). Differential gene expression analysis showed that the three-step method induced higher levels of type II collagen (COL2A1) and aggrecan (ACAN) in Cartibeads, whereas type I collagen (COL1A1), specific to fibrocartilage, was higher in the two-step method (Figure 5C-F). Increased levels of GAGs confirmed improved cartilage differentiation in the three-step Cartibead method (Figure 5G; Figure 2A-C). SOX9, a transcription factor involved in matrix production, was also increased in the three-step method (M1+M2+M3) (Figure 7C).

[0165] These data confirmed the dedifferentiation of chondrocytes in medium E (containing FGF-2) with detection of COL1A1 expression and the absence of hyaline cartilage markers such as COL2A1 and ACAN (GAG-binding protein) (Figure 5D-F). We confirmed by flow cytometry that medium E contained a high proportion of dedifferentiated cells, which represents a proportion of cells expressing mesenchymal stem cell markers CD73, CD90, and CD105, as defined and proposed by ISCT (International Society for Cell Therapy), of more than 90% of the total cell population at the end of the proliferation phase (Figure 6A, Table 4).

[0166] [Table 4] Table 4: Flow cytometry analysis of dedifferentiated chondrocytes. The table represents the flow cytometry analysis of dedifferentiated chondrocytes for CD73, CD90, and CD105 from three donors.

[0167] Furthermore, the multipotent character of this dedifferentiated chondrocyte population was confirmed by the fact that this population possessed the properties to differentiate into other mesenchymal stem cell (MSC) derived cells (osteocytes, adipocytes, and chondrocytes), even though this potential was still lower compared to MSCs (Figure 6B).

[0168] When comparing gene expression of cells cultured in medium R (redifferentiation step) to medium E (proliferation step), we found higher levels of expression of genes involved in inflammatory processes during redifferentiation (interleukin and cytokine signaling as well as interferon signaling). In parallel, we found lower levels of expression of genes involved in the cell cycle (MI67, CDK, CCNB1), indicating that the production of hyaline matrix requires a dynamic balance between cellular inflammation and tissue remodeling associated with cell cycle exit (Figure 7A).

[0169] By comparing the proliferation phase with the redifferentiation phase, we observed high expression levels of the genes WNT5A, WNT5B, and WNT7B during the proliferation phase (step 1, medium E, both methods) (Figure 7B, D-F). These genes were strongly downregulated during the redifferentiation step (medium R, 3-step method) and during 3D culture (medium I, both methods), except for WNT5B, which showed intermediate expression in 3D culture (step 3, medium I) (Figure 7E).

[0170] Also, the expression of KI67 (proliferation marker) and TCF4 (involved downstream of the wnt / β-catenin pathway) was decreased during the redifferentiation step (Figure 7C). Most importantly, after the dedifferentiation and redifferentiation phases (M1+M2), KI67 and WNT7B / WNT5B were decreased before the formation of spheres at the maturation phase (M3). When cells were used to form spheroids directly from the dedifferentiation phase (M1), they still significantly expressed KI67 and WNT7B / WNT5B and produced less hyaline matrix.

[0171] These results were consistent with previous studies reporting upregulation of WNT after 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-like phenotype (Buchtova, M. et al. 2015. Biochim Biophys Acta 1852, 839-850). Thus, removal of FGF-2 in medium R led to downregulation of WNT signaling during the redifferentiation phase. We detected lower levels of COL2A1 and ACAN expression in the two-step method compared to the three-step method (Figure 5C-E). However, we observed a comparable decrease in WNT5A, WNT5B, and WNT7B genes in the two-step method during the 3D differentiation phase in medium I (Figure 7B).

[0172] To verify the role of downregulation of the WNT pathway during the redifferentiation phase (medium R) (Figure 1A), we replaced medium R with medium E supplemented with 10 μM of XAV-939, an inhibitor of the WNT pathway, for 4 days (Figure 7G). After treatment with XAV-939, we observed an increase in phospho-β-catenin and axin, known indicators of WNT signaling inhibition (Figure 7G) (Huang, SM et al. 2009. Nature 461, 614-620). Thus, similar to medium R, pharmacological inhibition of WNT induced an increase in the expression of ACAN and COL2A1 in a two-step manner after 2D and 3D culture (Figure 7H, I). As a result, pharmacological inhibition of WNTs resulted in the presence of hyaline characteristics in beads obtained by the two-step method in the presence of XAV-939, as confirmed by Safranin-O staining of GAGs (Figure 7J).

[0173] Taken together, transcriptome analysis identified the involvement of the WNT signaling pathway in chondrocyte de- and re-differentiation and its regulation, with WNT5A, WNT5B, and WNT7B acting as potential key mediators of this response (Figure 7D-F). Medium E supplemented with XAV-939 mimics the effect of medium R (Figure 7K), while the three-step method naturally induced downregulation of WNT genes in 7 days without the use of costly pharmacological molecules.

[0174] Ex vivo feasibility study of human cartilage defects In view of the clinical application of Cartibeads, we evaluated the properties of Cartibeads to fuse together and integrate within lesions created in ex vivo human knee joints. Lesions were created with 8 mm surgical punch biopsies and filled with 20-50 Cartibeads in dry conditions. The Cartibeads were left in these conditions for 20 min to promote adhesion of the Cartibeads to the lesion site and to each other (Figure 8A, left and center panels), after which Medium I was added. The entire specimen with its lesion filled with Cartibeads was then cultured for one month with rotation to promote the bulk transfer of nutrients. One month after implantation, we analyzed the osteochondral tissue (Figure 8A, right panel) and performed Safranin-O staining at the level of the filled lesion (Figure 8B). We observed preservation of hyaline features within the lesions and some level of integration of the Cartibeads with the native surrounding tissue and fusion of the Cartibeads with each other (FIG. 8B).

[0175] Preclinical safety testing of Cartibeads in vitro and in vivo Implantation of in vitro expanded cells raises the issue of potential uncontrolled growth. The safety of Cartibeads was evaluated through preclinical studies. Comparative genomic hybridization (CGH) array analysis demonstrated genetic stability of chondrocytes during cell expansion up to passage 11 (Table 5).

[0176] [Table 5] Table 5: List of donor Cartibeads analyzed by CGH-array. Genetic stability of chondrocytes was assessed after extensive growth in medium E in the presence of FGF-2 by performing comparative genomic hybridization (CGH) array analysis on 10 donor samples cultured for passages 3 to 11.

[0177] Consistent with previous studies, Y chromosome loss is a common acquired mutation in normal aging in men and was observed in a proportion (43%) of older donors (Thompson, DJ et al. 2019. Nature 575, 652-657; Stumm, M. et al. 2012. Osteoarthritis and cartilage 20, 1039-1045).

[0178] In an in vivo study, we evaluated the potential tumorigenic effect of human Cartibeads implanted in SCID mice. Cartibeads did not grow and were barely detectable 6 months after implantation in SCID mice, confirming the absence of potential tumorigenicity over a 6-month follow-up (Figure 9A-C, Table 5).

[0179] In vivo preclinical efficacy testing To move towards clinical applications, we performed preclinical efficacy studies in adult Gottingen minipigs, selected for their cartilage maturity and similar knee tissue to 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 this study. A first surgical procedure was performed to harvest cartilage biopsies (approximately 30 mg) from the knee to generate autologous minipig Cartibeads. A second surgical procedure was performed to induce 4-5 lesions per animal / right knee and implant the autologous Cartibeads. The minipigs were followed up for 3-6 months (Figure 10A; Figure 11). Prior to implantation, the amount of GAG in minipig Cartibeads was quantified, which ranged on average from 40 to 50 μg / bead (Figure 10B). Three months after implantation, we confirmed the hyaline nature of the lesions grafted with minipig Cartibeads by Safranin-O staining (Figure 10C). Similar results were obtained from the grafted lesions 6 months after implantation (Figure 10D). Complete fusion of the assembled Cartibeads and their integration into the surrounding native cartilage and subchondral bone was observed in all minipigs at the 3 and 6 month follow-up periods (Figures 10C-D). In summary, autologous Cartibeads proved their efficacy in terms of lesion survival while preserving hyaline quality. They also proved to be safe, as neither joint degeneration (Figure 11, middle panel) nor ectopic or hypertrophic tissue formation was noted on macroscopic examination by two independent operators (data not shown).

[0180] Discussion and conclusion The main outcome of this study is a method that can reverse the loss of chondrocyte phenotype during proliferation (chondrocyte dedifferentiation). This solves an important problem facing cell therapies that use chondrocytes as starting material. The data show that the novel three-step method can result in high-quality cartilage with hyaline characteristics, regardless of the patient's age and the arthritic state of the joint.

[0181] The Cartibead method allows cell expansion from harvesting very small cartilage samples (approximately 30 mg in our preclinical minipig studies) as opposed to an average of 260 mg in conventional human chondrocyte-based cell therapy (Brittberg, M. 2008. Injury 39 Suppl 1, S40-49), reducing donor site morbidity. The method demonstrated a 20-fold higher GAG / Cartibead ratio than previously reported cartilage microtissues (Bartz, C. et al. 2016. J Transl Med 14, 317), which were suggested to contain less hyaline and more fibrocartilage. Consistent with these results, we obtained GAG / DNA ratios that were on average at least 3-fold higher than other published methods (Mumme, M. et al. 2016. Lancet 388, 1985-1994; Dang, PN, Solorio, LD & Alsberg, E. 2014. Tissue engineering. Part A 20, 3163-3175) (Figure 2B).

[0182] Redifferentiation was due to the removal of FGF-2. However, this removal in 3D culture was not sufficient to induce the synthesis of hyaline matrix in a two-step manner. Chondrocyte redifferentiation most likely requires cell attachment to matrix-coated flasks in 2D culture and the induction of specific cell signaling pathways. Starvation of FGF-2 shuts down genes involved in pluripotency and stemness, including several genes in the WNT signaling pathway. WNT signaling is involved in both the inhibition and stimulation of chondrogenic differentiation of adult precursor 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 and 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).

[0183] In this study, we identified WNT5A, WNT5B, and WNT7B as WNT isoforms likely involved in this mechanism. In parallel with the downregulation of the WNT pathway, we observed an increase in the expression of genes involved in inflammatory pathways (interleukins, cytokines). In wound healing and tissue repair, the dynamic balance between pro- and anti-inflammatory factors is important for effective healing (Gerhard T. Laschober et al. 2011. Rejuvenation Research 14, 119-131; Bosurgi, L. et al. 2017. Science 356, 1072-1076). Thus, the data suggest that inflammation is a prerequisite for tissue regeneration associated with the biosynthesis of hyaline matrix (Karin, M. & Clevers, H.2016. Nature 529, 307-315).

[0184] Human Cartibeads did not grow when implanted in SCID mice and even disappeared after 6 months. This is consistent with a similar study (Zscharnack, M. et al. 2015. J Transl Med 13, 160). Furthermore, autologous implantation of Cartibeads in minipigs showed stable integration into the lesion, maintained high levels of GAG, and successfully repaired the cartilage damage 6 months after implantation. In summary, we have shown the feasibility of implanting Cartibeads with high levels of GAG in a large animal model, supporting the possibility of this long-term cartilage repair. As a result, Cartibeads represent a breakthrough in the field of cartilage repair and can now enter Phase I clinical trials (FIH: First-in-Human) for autologous cartilage implantation in patients with cartilage damage.

Claims

1. A method for the in vitro production of hyaline cartilage tissue, comprising: i) culturing chondrocytes in an adherent culture system in a dedifferentiation culture medium that activates the Wnt signaling pathway to obtain chondrocytes with a fibroblast-like cell morphology; ii) culturing said fibroblast-like chondrocytes in an adherent culture system in a redifferentiation culture medium that inactivates the Wnt signaling pathway to obtain chondrocytes with the complete ability to resynthesize hyaline matrix; iii) culturing the chondrocytes obtained in step ii) in a three-dimensional culture system in an induction / maturation culture medium that maintains the inactivation of the Wnt signaling pathway; A method comprising:

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

3. The method according to claim 1 or 2, wherein the dedifferentiation culture medium comprises FGF-2.

4. The method according to any one of claims 1 to 3, wherein the de-differentiation culture medium contains FGF-2, and the re-differentiation culture medium and the induction / maturation culture medium are FGF-2-free medium.

5. The method according to any one of claims 1 to 4, wherein the redifferentiation culture medium and the induction / maturation culture medium contain TGF-β.

6. The method according to any one of claims 1 to 5, wherein the redifferentiation culture medium and the induction / maturation culture medium contain TGF-β3.

7. The method according to any one of claims 1 to 6, wherein the redifferentiation culture medium and the induction / maturation culture medium comprise TGF-β3 and FGF7.

8. The method according to any one of claims 1 to 7, wherein the regeneration culture medium comprises a platelet lysate.

9. The method according to any one of claims 1 to 8, wherein the de-differentiation, re-differentiation and / or induction / maturation culture medium comprises serum.

10. The method of any one of claims 1 to 9, wherein the induction / maturation culture medium comprises at least one component selected from the group consisting of insulin, IGF-1, selenium, transferrin, and ethanolamine.

11. In step iii), the chondrocytes are treated with an aqueous solution of less than 10% (v / v) O ２ The method according to any one of claims 1 to 10, wherein the cells are cultured in a low-oxygen atmosphere comprising:

12. The method according to any one of claims 1 to 11, wherein the chondrocytes are cultured in step i) for 10 to 15 days, in step ii) for 4 to 8 days, and / or in step iii) for 10 to 15 days.

13. The method according to any one of claims 1 to 12, wherein the chondrocytes of step i) are isolated from a subject.

14. The method according to any one of claims 1 to 13, wherein the chondrocytes of step i) are isolated from cartilage tissue of a human or equine subject.

15. Engineered cartilage tissue in the form of spheroids presenting a glycosaminoglycan (GAG) content per spheroid of 10-100 μg / spheroid, characterized in that said engineered cartilage tissue is obtained by the method according to any one of claims 1 to 14.

16. 16. A pharmaceutical composition for use in treating cartilage defects and cartilage degenerative diseases in a subject in need thereof, comprising the engineered cartilage tissue of claim 15.

17. 17. The pharmaceutical composition according to claim 16 for use in autologous transplantation.

18. 17. The pharmaceutical composition of claim 16 for use in allogeneic transplantation.

19. 1. A method for screening for molecules that inhibit a cartilage degeneration process, comprising: i) contacting the engineered cartilage tissue of claim 15 with one or more candidate molecules; ii) selecting a molecule that inhibits the cartilage degeneration process; A method comprising:

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