CN118109397A - Preparation method and application of cartilage micro-tissue derived from human pluripotent stem cell differentiation - Google Patents

Abstract

Provides a preparation method and application for preparing cartilage micro-tissue from human pluripotent stem cell differentiation source. The chondrocyte micro-tissue described herein comprises chondrocytes derived from human pluripotent stem cell differentiation and extracellular matrix proteins secreted therefrom, and has higher expression levels of extracellular matrix proteins COL2A1, ACAN, COL9A1, SPARC, COL11A1 and SOX9 and similar expression levels of HAPLN1, and reduced MFAP5 expression compared to adult primary chondrocytes. The chondrocyte microstructure described herein has higher expression levels of the chondrocyte extracellular matrix-related genes COL2A1, ACAN, SOX9, COL9A1, COL11A1, SPARC and hyaline chondrocyte marker gene HAPLN1, and lower expression levels of the fibrocartilage cell marker gene MFAP5 than the autologous chondrocyte products currently on the market.

Description

Preparation method and use of cartilage microtissue derived from differentiation of human pluripotent stem cells

Technical Field

The present invention relates to the field of cell culture, and in particular to a preparation method and application of cartilage microtissue derived from differentiation of human pluripotent stem cells.

Background Art

Human pluripotent stem cells include human embryonic stem cells (ESC) and human induced pluripotent stem cells (iPSC, iPS cells). The latter is induced by introducing specific transcription factors into adult cells to change the fate of cells, becoming pluripotent stem cells with the ability of self-replication, expansion and multi-lineage directed differentiation. This technology avoids the immune rejection and ethical issues in the field of regenerative medicine research and is a great revolution in the field of life sciences. Like embryonic stem cells (ESC), iPS cells can self-renew and maintain an undifferentiated state. They have the same cell morphology, karyotype, telomerase activity, and in vitro differentiation potential as ESCs, and express surface marker molecules specific to pluripotent stem cells. iPS cells are of great application value in theoretical research and clinical application. iPS cells can be directed and induced to differentiate into a variety of mature tissue cells in vitro, and play an important role in the treatment of various diseases such as blood system diseases, nervous system diseases, and degenerative diseases. They are regarded as important cell materials for future regenerative medicine.

Human pluripotent stem cells have been shown to be able to differentiate into multiple different tissue types, including ectoderm, mesoderm, and endoderm. Previous studies have shown that human pluripotent stem cells can successfully differentiate into chondrocytes in vitro.

At present, the main methods for preparing chondrocyte microtissues are (1) spontaneous aggregation and culture of chondrocytes, (2) directed induction and differentiation of mesenchymal stem cells (MSC), and (3) co-culture of MSC or chondrocytes combined with biomaterials. However, the above methods for preparing chondrocyte microtissues have their own defects and shortcomings: (1) For example, the method of spontaneous aggregation of autologous chondrocytes to form chondrocyte microtissues represented by the German Spherox product, all of which are derived from autologous non-weight-bearing articular cartilage. After enzymatic digestion, the cartilage tissue needs to be cultured in vitro for a long time (5 to 10 weeks) to form sufficient chondrocyte microtissues for patients (Huang B J, Hu J C, Athanasiou KA. Cell-based tissue engineering strategies used in the clinical repair of articular cartilage [J]. Biomaterials, 2016, 98: 1-22). In addition, due to individual differences and the poor proliferation ability and dedifferentiation of chondrocytes in vitro, the efficacy of chondrocyte microtissues prepared by this method is limited; at the same time, secondary surgery is likely to cause donor site lesions, which greatly reduces clinical patient acceptance. (2) The directional induction of MSC into chondrocyte microtissues is a multi-factor controlled process that requires the optimization of the proportions of multiple components. The existing induction technology has a relatively low induction differentiation rate and a long time period, requiring 14-28 days to form cartilage, and cannot effectively differentiate all MSCs into uniform and stable chondrocyte microtissues, resulting in low density, insufficient density, and poor quality of cartilage after induction, which is not conducive to use in tissue engineering. In addition, due to the large differences between MSC batches, it is difficult to achieve large-scale, stable, and uniform preparation. (3) In addition to being limited by the above-mentioned seed cell source, the chondrocyte microtissues produced by co-culturing MSC or chondrocytes combined with biomaterials will also be accompanied by the characteristics of the biomaterials themselves, such as biocompatibility, in vivo degradation rate, residues, and safety issues.

Therefore, in order to further transform the application of chondrocytes derived from the directed differentiation of human pluripotent stem cells, it is necessary to establish a high-throughput, large-scale culture method for preparing cartilage microtissues that can be used in clinical cartilage repair transplantation.

Summary of the invention

The present invention aims to develop a method for large-scale preparation of stable and uniform chondrocyte microtissues with therapeutic functions from chondrocytes derived from human pluripotent stem cells, for the treatment of diseases related to articular cartilage damage. Represented by human iPS cells, the inventors have completed the determination of the directional differentiation scheme of human pluripotent stem cells into chondrocytes, pharmacodynamic research, quality research and process research.

Compared with traditional two-dimensional plane adherent differentiation culture of chondrocytes, three-dimensional cultured chondrocyte microtissues can express higher chondrocyte functional maturation-related genes (such as COL2A1, ACAN, SOX9, etc.), while secreting a large amount of natural hyaline cartilage extracellular matrix such as type II collagen and aggrecan. In animal experiments on the repair of articular cartilage defects in vivo, it was also confirmed that three-dimensional cultured chondrocyte microtissues have a stronger ability to repair hyaline cartilage damage. However, due to technical and cost limitations, previous studies were mostly isolated primary chondrocytes that were cultured in vitro for a short period of time or irregular microtissues were generated on a small scale in the laboratory.

The main purpose of the present invention is to use human pluripotent stem cells to differentiate into chondrocyte microtissues and their identification, and to prepare stable, uniform, and therapeutic chondrocyte microtissues on a large scale. By optimizing the culture conditions, mature chondrocytes are induced to spontaneously aggregate and self-assemble in a microplate to generate chondrocyte microtissues.

The inventors have successfully developed a new method for large-scale preparation of human chondrocyte three-dimensional microtissues, which can significantly improve the yield and quality of chondrocyte microtissues after induction of human pluripotent stem cells. The three-dimensional chondrocyte microtissues differentiated by iPS cells have higher expression levels of chondrocyte extracellular matrix-related genes COL2A1, ACAN, SOX9, COL9A1, COL11A1, SPARC and hyaline chondrocyte marker gene HAPLN1, and lower expression levels of fibrochondrocyte marker gene MFAP5 than the autologous chondrocyte products currently on the market (2D autologous chondrocyte products represented by MACI and 3D autologous chondrocyte microtissue products represented by German Spherox).

In one aspect, provided herein is a method for preparing chondrocyte microtissue, comprising:

(A1) differentiating iPS cells into chondrogenic precursor cells, wherein the chondrogenic precursor cells are positive for CD73, CD90, CD105, and CD44, and negative for CD11b, CD19, CD31, CD34, CD45, and HLADR;

(B1) statically culturing cartilage precursor cells in a 3D cell culture manner to aggregate and form individual spheres or spheroid-like bodies in a cartilage precursor cell culture medium, wherein the cartilage precursor cell culture medium comprises DMEM high glucose medium as a basal medium, and contains 1%-10% Knockout serum replacement, 10-40 ng/ml EGF, and 10-40 ng/ml FGF2;

(C1) replacing the cartilage precursor cell culture medium with a chondrocyte microtissue culture medium and statically culturing individual spheres or spheroids for 1-28 days, wherein the chondrocyte microtissue culture medium comprises DMEM high glucose medium as a basal medium, and contains 0.5%-10% Knockout serum replacement, 0.5%-10% insulin-transferrin-selenium supplement, 0.5-10 mM sodium pyruvate, 50-200 U/mL penicillin, 50-200 μg/mL streptomycin, 0.05-1 mM sodium ascorbate, 0.05-1 uM dexamethasone, 10-100 μg/mL proline, 10-100 ng/mL TGF-β3, and 10-100 ng/mL BMP2; and

(D1) Individual spheres or spheroids suspended in chondrocyte microtissue culture medium are cultured with shaking until chondrocyte microtissues are obtained.

In one embodiment, the diameter of the chondrocyte microtissue ranges from 100 μm to 2000 μm. In one embodiment, the shaking culture is a shaking culture at 60 rpm to 80 rpm.

In one embodiment, step (A1) comprises:

(1) culturing iPS cells for 20-28 hours in a medium for inducing iPS cell differentiation, wherein the medium comprises DMEM/F12 medium and contains 0.5-5% penicillin, 0.5-5% streptomycin, 0.5-5% ITS-A, 1-10% B27, 0.5-5% NEAA, and 50-200 μM β-mercaptoethanol;

(2) Induction in the presence of 10-50 ng/mL WNT3a and 50-100 ng/mL Activin A for 20-28 hours;

(3) induced for 20-28 hours in the presence of 10-50 ng/mL WNT3a, 10-50 ng/mL Activin A, and 10-50 ng/mL FGF2;

(4) induced for 20-28 hours in the presence of 10-50 ng/mL WNT3a, 5-50 ng/mL Activin A, 10-50 ng/mL FGF2, and 20-80 ng/mL BMP4;

(5) subculturing the cells in the presence of 10-50 ng/mL FGF2, 20-80 ng/mL BMP4, 1-10 ng/mL NT4, and 100 ng/mL Follistatin, and culturing for 3-5 days;

(6) induced for 20-28 hours in the presence of 10-50 ng/mL FGF2, 20-80 ng/mL BMP4, and 1-10 ng/mL NT4;

(7) passage the cells in the presence of 10-50 ng/mL FGF2, 10-50 ng/mL BMP4, 10-50 ng/mL GDF5, 1-10 ng/mL NT4, and 5-50 ng/mL TGFβ3, and culture for 1-3 days; and

(8) The cells were cultured for 3-6 days in the presence of 10-50 ng/mL FGF2, 20-80 ng/mL GDF5, 1-10 ng/mL NT4, and 5-50 ng/mL TGFβ3; the cells were then passaged for more than 15 generations to obtain cartilage precursor cells.

The "%" in the above steps refers to volume percentage.

In one embodiment, cartilage precursor cells are cultured for 2-5 days in step (B1) to aggregate to form spheres or spheroids. In one embodiment, spheres or spheroids are cultured in a chondrocyte microtissue culture medium with shaking for 1-16 days in step (D1). In one embodiment, the shaking culture is a shaking culture at 60rpm-80rpm. In one embodiment, steps (B1) and (C1) are performed in a multiwell plate selected from a 24-well plate, a 96-well plate, a 384-well plate or a 948-well plate. The multiwell plate can be an EB-disk948.

In another aspect, the present invention provides a method for preparing chondrocyte microtissue, comprising:

(A2) differentiating induced pluripotent stem (iPS) cells into chondrogenic precursor cells, wherein the chondrogenic precursor cells are positive for CD73, CD90, CD105, and CD44, and negative for CD11b, CD19, CD31, CD34, CD45, and HLADR;

(B2) statically culturing cartilage precursor cells in a 3D cell culture manner to aggregate and form individual spheres or spheroid-like bodies in a cartilage precursor cell culture medium, wherein the cartilage precursor cell culture medium comprises DMEM high glucose medium as a basal medium, and contains 1%-10% Knockout serum replacement, 10-40 ng/ml EGF, and 10-40 ng/ml FGF2;

(C2) replacing the cartilage precursor cell culture medium with a chondrocyte microtissue culture medium and culturing the individual spheres or spheroids statically for 1-4 days, wherein the chondrocyte microtissue culture medium comprises DMEM high glucose medium as a basal medium, and contains 0.5%-10% Knockout serum replacement, 0.5%-10% insulin-transferrin-selenium supplement, 0.5-10 mM sodium pyruvate, 50-200 U/mL penicillin, 50-200 μg/mL streptomycin, 0.05-1 mM sodium ascorbate, 0.05-1 uM dexamethasone, 10-100 μg/mL proline, 10-100 ng/mL TGF-β3 and 10-100 ng/mL BMP2; and

(D2) shaking and culturing the suspended plurality of spheres or spheroids in a chondrocyte microtissue culture medium until a chondrocyte microtissue fusion body is obtained, which comprises a plurality of adherent spheres or spheroids and has a diameter of 0.8 mm to 3 mm. Preferably, the shaking culture is carried out at 60 rpm to 80 rpm.

The "%" in the above steps refers to volume percentage.

In one embodiment, the cartilage precursor cells are cultured for 2-5 days in step (B2) to aggregate to form spheres or spheroids. In one embodiment, steps (B2) and (C2) are performed in a multi-well plate selected from a 24-well plate, a 96-well plate, a 384-well plate or a 948-well plate. In one embodiment, step (A2) comprises:

(1) culturing iPS cells for 20-28 hours in a medium for inducing iPS cell differentiation, wherein the medium comprises DMEM/F12 medium and contains 0.5-5% penicillin, 0.5-5% streptomycin, 0.5-5% ITS-A, 1-10% B27, 0.5-5% NEAA, and 50-200 μM β-mercaptoethanol;

(2) Induction in the presence of 10-50 ng/mL WNT3a and 50-100 ng/mL Activin A for 20-28 hours;

(3) induced for 20-28 hours in the presence of 10-50 ng/mL WNT3a, 10-50 ng/mL Activin A, and 10-50 ng/mL FGF2;

(4) induced for 20-28 hours in the presence of 10-50 ng/mL WNT3a, 5-50 ng/mL Activin A, 10-50 ng/mL FGF2, and 20-80 ng/mL BMP4;

(5) subculturing the cells in the presence of 10-50 ng/mL FGF2, 20-80 ng/mL BMP4, 1-10 ng/mL NT4, and 100 ng/mL Follistatin, and culturing for 3-5 days;

(6) induced for 20-28 hours in the presence of 10-50 ng/mL FGF2, 20-80 ng/mL BMP4, and 1-10 ng/mL NT4;

(7) passage the cells in the presence of 10-50 ng/mL FGF2, 10-50 ng/mL BMP4, 10-50 ng/mL GDF5, 1-10 ng/mL NT4, and 5-50 ng/mL TGFβ3, and culture for 1-3 days; and

(8) The cells were cultured for 3-6 days in the presence of 10-50 ng/mL FGF2, 20-80 ng/mL GDF5, 1-10 ng/mL NT4, and 5-50 ng/mL TGFβ3; the cells were then passaged for more than 15 generations to obtain cartilage precursor cells.

In another aspect, the present invention provides a chondrocyte microtissue, which comprises chondrocytes and extracellular matrix proteins secreted therefrom, and has higher expression levels of the extracellular matrix proteins COL2A1, ACAN, COL9A1, SPARC, COL11A1 and SOX9 and similar expression levels of HAPLN1 as compared to adult primary chondrocytes, and reduced expression of MFAP5.

In one embodiment, the chondrocyte microtissues express COL2A1, ACAN, COL9A1, SPARC, COL11A1 and SOX9 proteins at least 2-fold increased compared to adult primary chondrocytes.

In one embodiment, the chondrocyte microtissue expresses HAPLN1 at an amount that is at least 80% of the amount of HAPLN1 expressed by adult primary chondrocytes.

In one embodiment, the MFAP5 expression level of the chondrocyte microtissue is at most 80% of the MFAP5 expression level of adult primary chondrocytes;

In one embodiment, the diameter of the chondrocyte microtissue ranges from 100 μm to 2000 μm.

In one embodiment, the chondrocyte microtissue has the ability to develop into mature cartilage tissue in vivo.

In one embodiment, the chondrocyte microtissue further expresses COLII and Lubricin proteins.

In one embodiment, the survival rate of chondrocytes in the chondrocyte microtissue is above 90%.

In one embodiment, chondrocyte microtissues are prepared by the methods described herein.

In another aspect, the present invention provides a kit comprising the chondrocyte microtissue described herein.

In another aspect, the present invention provides cartilage tissue formed from the chondrocyte microtissues described herein.

In another aspect, the present invention provides the use of the chondrocyte microtissue described herein in the preparation of a medicament or a kit for treating cartilage defects, cartilage degeneration, bone degeneration, osteoarthritis, and/or for cartilage regeneration, bone regeneration or cartilage transplantation.

The beneficial effects of the present invention include:

1. A 3D culture method for preparing chondrocyte microtissues from human pluripotent stem cells is provided, which has extremely high differentiation efficiency and cell viability.

2. For the 3D culture method of this article, the inventors have discovered the optimal culture conditions, such as culture medium and culture time.

3. The inventors found that chondrocyte precursor cells differentiated from iPS cells reached a stable optimal level of chondrocyte markers (COL2A1, ACAN, COL9A1, SPARC, COL11A1 and SOX9 expression, HAPLN1 expression and MFAP5 expression) on the 8th to 16th day (for example, 9th, 10th, 11th, 12th, 13th, 14th or 15th day) when induced into chondrocyte microspheres.

4. The present invention provides a method for large-scale production of chondrocyte microtissues, which is carried out using a multi-well plate selected from a 24-well plate, a 96-well plate, a 384-well plate or a 948-well plate, such as EB-disk948.

5. The chondrocyte microtissue described in this article has high consistency and purity, and the tissue regenerated in vivo has the same mechanical biomechanical properties as the reported human natural hyaline cartilage, with an elastic modulus of about 27 MPa.

6. The chondrocyte microtissue described in this article will not cause immune rejection reaction in the body and has the potential to develop into osteochondral tissue in vivo.

7. The chondrocyte microtissue described in this article has a good filling and repairing effect on knee joint cartilage defects.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the morphology of P0 and P15 chondrogenic precursor cells (ipCDC) after directed differentiation of iPS cells (the left picture is P0, and the right picture is P15).

Figure 2 shows the expression of surface antigens of P10 ipCDC after directed differentiation of iPS cells. Red (left) and blue (right) represent the isotype antibody control histogram and the corresponding surface marker antibody histogram, respectively.

FIG. 3 shows the HE staining results of the samples obtained 6 weeks after the P15 ipCDCs were transplanted into the subcutaneous tissues of NOD-SCID mice after directed differentiation of iPS cells.

Figure 4 shows the formation process of chondrocyte microtissue derived from iPS cell differentiation obtained by 3D induction method. Figure A shows the morphological changes of chondrocyte microtissue; Figure B shows the changes in the diameter of chondrocyte microtissue.

Figure 5 shows chondrocyte microtissues of different sizes derived from iPS cell differentiation obtained by 3D induction method (from left to right, 1×10 ⁴ /well, 5×10 ^{4 /} well, 1×10 ^{5 /} well cartilage spheres, with diameters of 0.5 mm, 0.8 mm, 1.0 mm, respectively).

Figure 6 shows the large-scale preparation of chondrocyte microtissue derived from iPS cell differentiation obtained by 3D induction method. ipCDC was inoculated into EB-Disk948 (middle left), and the induced cartilage microtissue was transferred to a 10 cm culture dish (middle right) and cultured on a shaker at 60 rpm-80 rpm (the upper left picture shows 2×10 ⁴ cells/microwell, the upper right picture shows 5×10 ⁴ cells/microwell, the lower left picture has a magnification of 40×, and the lower right picture is a photo taken with a mobile phone).

Figures A-B of Figure 7 are cell viability detection of chondrocyte microtissues. Figure 7 A shows the dead/live cell staining of chondrocyte microtissues derived from iPS cell differentiation and chondrocyte microtissues derived from adult tissues obtained by 3D induction method (3D7d, 3D14d, CDC 3D7d, green indicates live cells, red indicates dead cells). Figure 7 B shows the cell viability results of chondrocyte microtissues (3D20d) after digestion into single cells by flow cytometry.

FIG8 shows the special staining results of chondrocyte microtissue derived from iPS cell differentiation obtained by 3D induction method (3D10d, from left to right, HE staining, Safranin O fast green staining, and toluidine blue staining).

FIG. 9 shows immunofluorescence staining of chondrocyte microtissues derived from iPS cell differentiation obtained by the 3D induction method, which are chondrocyte microtissues at 5d, 10d, and 15d of induction, respectively.

Figures 10A-10C show the differences in gene expression levels between chondrocyte microtissues derived from human iPS cell differentiation and autologous articular chondrocytes and autologous chondrocyte microtissues obtained by 2D and 3D induction methods (CDC P0 is human primary articular chondrocytes, CDC P2 3D8d is cartilage microspheres formed by human P2 articular chondrocytes, and EB948 3D8d is chondrocyte microtissues induced by EB948 disk method for 8 days). Figure 10A shows the gene expression of chondrocyte microtissues induced by 2D method; Figure 10B shows the gene expression of chondrocyte microtissues induced by 3D method; Figure 10C shows the gene expression of CRTAC1 and EBF3 genes in chondrocyte microtissues induced by 3D method.

FIG11 shows the results of gross observation and pathological staining of chondrocyte microtissues derived from iPS cell differentiation obtained by 3D induction method and subcutaneously transplanted in NOD-SCID mice and C57BL/6 mice for 6 weeks, (A) gross observation, (B) pathological staining.

FIG12 shows the results of mechanical properties testing of chondrocyte microtissues derived from differentiation of human iPS cells obtained by 3D induction method and removed from NOD-SCID mice 8 weeks after subcutaneous transplantation.

Figure 13 shows the repair of cartilage defects in the weight-bearing area of the rabbit knee joint by filling chondrocyte microtissue derived from iPS cell differentiation obtained by human 3D induction method.

(A) In vitro preliminary test

Graft: chondrocyte microtissue 3D8d, number of grafts: 20 to 30 per injured area

(B) In vivo animal testing

Graft: Chondrocyte microtissue 3D8d, number of grafts: 20 per injured area

(C) Animal experimental results of chondrocyte microtissues obtained by 3D induction method to repair cartilage defects in rabbit weight-bearing area one month after transplantation (the yellow dotted line indicates the cartilage defect, from top to bottom are gross observation, HE, safranin O fast green, Alcian blue, toluidine blue staining, and Masson staining).

(D) Calculation of the cartilage regeneration and repair ratio of chondrocyte microtissues obtained by 3D induction method to repair cartilage defects in the weight-bearing area of rabbits one month after transplantation.

FIG. 14 shows the microtissue morphology of chondrocytes differentiated from iPS-derived chondrocyte precursor cells (2D induction method).

Figure 15A shows that chondrocyte microtissues (3D2d, 3D3d, 3D4d) with different induction times adhere to each other after concentrated shaking culture to form a larger volume of chondrocyte microtissue fusion; Figure 15B shows the mutual adhesion and fusion process of chondrocyte microtissues of different diameters; Figure 15C is a local enlarged view of the mutual adhesion and fusion of chondrocyte microtissues (3D2d+3D3d) with a diameter of 2 mm, and the white arrows indicate the mutual adhesion and fusion phenomenon of cells on the surface of adjacent chondrocyte microtissues.

FIG. 16 shows a schematic diagram of the process of differentiation of iPS cells into chondrogenic precursor cells.

FIG. 17 shows a schematic diagram of the differentiation process of iPS-derived chondrogenic precursor cells into chondrogenic microtissues (2D induction method).

FIG. 18 shows a schematic diagram of the differentiation process of iPS-derived chondrogenic precursor cells into chondrocyte microtissues (3D induction method).

Figure 19 shows the single-cell sequencing results of chondrocyte microtissues (3D8d) derived from directed differentiation of human iPS cells. Figure 19A shows the nonlinear dimensionality reduction tSNE cell clustering analysis; Figure 19B shows the correspondence between Cluster and cell type; Figure 19C shows the number of cells of different cell types; Figure 19D shows the cell type identification results; Figure 19E shows the scores of different cell types.

DETAILED DESCRIPTION

As used herein, the term "comprising" is synonymous with "including", "containing", and is inclusive or open-ended, and does not exclude additional unrecited elements or method steps. "Comprising" is a technical term used in the claim language, meaning that the elements are present, but other elements may also be added and still form a structure or method within the scope of the claim.

As used herein, "chondrocyte microtissue" is also referred to as cartilage microtissue, which is formed by 3D culture of cartilage precursor cells. Chondrocyte microtissue may include chondrocytes, and optionally may include extracellular matrix proteins secreted by chondrocytes. Extracellular matrix proteins secreted by chondrocytes include, but are not limited to, COL2A1 (collagen type IIalpha 1chain), ACAN (aggrecan), COL9A1 (collagen type IX alpha 1chain), SPARC (secreted protein acidic and cysteine rich), COL11A1 (collagen type XI alpha1chain), SOX9 (SRY-box transcription factor 9), HAPLN1 (hyaluronan andproteoglycan link protein 1) and/or MFAP5 (Microfibril Associated Protein 5). Chondrocyte microtissues described herein have a higher expression of COL2A1, ACAN, COL9A1, SPARC, COL11A1 and/or SOX9 compared to adult primary chondrocytes. The chondrocyte microtissues described herein may have similar HAPLN1 expression levels compared to adult primary chondrocytes. The chondrocyte microtissues described herein may have reduced MFAP5 expression compared to adult primary chondrocytes. For example, the expression of COL2A1, ACAN, COL9A1, SPARC, COL11A1 and SOX9 proteins in the chondrocyte microtissues is increased by at least 2 times, for example 3-20 times, such as 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 times compared to adult primary chondrocytes. The HAPLN1 expression level of the chondrocyte microtissue can be at least 80% of the expression level of HAPLN1 of adult primary chondrocytes, for example 85%-300%, such as 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, or 290%. The HAPLN1 expression level of the chondrocyte microtissue can be up to 80% of the expression level of MFAP5 of adult primary chondrocytes, for example 20%-75%, for example 30%, 40%, 50%, 60% or 70%. The chondrocyte microtissue described herein can also express COLII (Collagen II) and Lubricin (also known as PRG4, proteoglycan 4) proteins.

The chondrocyte microtissue can be in the form of a sphere or a spheroid, and can have a diameter range of 500-1200um. The chondrocyte microtissue comprises a dense structure formed by the chondrocyte microtissue. The chondrocyte survival rate in the chondrocyte microtissue is above 90%, for example, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%. Importantly, the chondrocyte microtissue described herein has the ability to develop into mature cartilage tissue in vivo. The chondrocyte microtissue can also be a chondrocyte microtissue fusion with a diameter of 0.8mm-3mm formed by the mutual adhesion of multiple spheres or spheroids.

As used herein, "chondrocyte precursor cells" refer to chondrocytes induced by induced pluripotent stem cells (iPS cells) by induction means (such as induction means already available in the prior art). Although there are already methods of inducing iPS cells into chondrocytes in the prior art, this does not mean that the method for preparing chondrocyte precursor cells of the present invention is known or completely known. Chondrocyte precursor cells can be aggregated into chondrocyte microtissues by the methods herein. The chondrocyte precursor cells described herein may be positive for CD73, CD90, CD105, and CD44, and may be negative for CD11b, CD19, CD31, CD34, CD45, and HLADR.

Method for preparing cartilage precursor cells

In this article, cartilage precursor cells can be prepared by a specific method. For example, the method can include one or more of the following steps:

(1) culturing iPS cells for 20-28 hours in a medium for inducing iPS cell differentiation, wherein the medium comprises DMEM/F12 medium and contains 0.5-5% penicillin, 0.5-5% streptomycin, 0.5-5% ITS-A, 1-10% B27, 0.5-5% NEAA, and 50-200 μM β-mercaptoethanol;

(2) Induction in the presence of 10-50 ng/mL WNT3a and 50-100 ng/mL Activin A for 20-28 hours;

(3) induced for 20-28 hours in the presence of 10-50 ng/mL WNT3a, 10-50 ng/mL Activin A, and 10-50 ng/mL FGF2;

(4) induced for 20-28 hours in the presence of 10-50 ng/mL WNT3a, 5-50 ng/mL Activin A, 10-50 ng/mL FGF2, and 20-80 ng/mL BMP4;

(5) subculturing the cells in the presence of 10-50 ng/mL FGF2, 20-80 ng/mL BMP4, 1-10 ng/mL NT4, and 100 ng/mL Follistatin, and culturing for 3-5 days;

(6) induced for 20-28 hours in the presence of 10-50 ng/mL FGF2, 20-80 ng/mL BMP4, and 1-10 ng/mL NT4;

(7) passage the cells in the presence of 10-50 ng/mL FGF2, 10-50 ng/mL BMP4, 10-50 ng/mL GDF5, 1-10 ng/mL NT4, and 5-50 ng/mL TGFβ3, and culture for 1-3 days; and

(8) The cells were cultured for 3-6 days in the presence of 10-50 ng/mL FGF2, 20-80 ng/mL GDF5, 1-10 ng/mL NT4, and 5-50 ng/mL TGFβ3; the cells were then passaged for more than 15 generations to obtain cartilage precursor cells.

In step (1), iPS cells can be cultured in a culture medium that induces iPS cell differentiation for 20-28 hours, such as 21, 22, 23, 24, 25, 26, 27 hours. As a basal culture medium, the culture medium may include DMEM/F12 culture medium. The culture medium may further include 0.5-5% penicillin, such as 1%, 2%, 3% or 4% penicillin. The culture medium may include 0.5-5% streptomycin, such as 1%, 2%, 3% or 4% streptomycin. The culture medium may include 0.5-5% ITS-A, such as 1%, 2%, 3% or 4% ITS-A. The culture medium may include 1-10% B27, such as 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8% or 9% B27. The culture medium may include 0.5-5% NEAA, such as 1%, 2%, 3% or 4% NEAA. The culture medium may contain 50-200 μM β-mercaptoethanol, for example 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 110 μM, 120 μM, 130 μM, 140 μM, 150 μM, 160 μM, 170 μM, 180 μM or 190 μM.

In step (2), the induction is carried out for 20-28 hours, for example, 21, 22, 23, 24, 25, 26, 27 hours, in the presence of 10-50 ng/mL WNT3a and 50-100 ng/mL Activin A. The concentration of WNT3a is 10-50 ng/mL, for example, 20, 30, 40 ng/mL. The concentration of Activin A is 50-100 ng/mL, for example, 60, 70, 80 or 90 ng/mL.

In step (3), the induction may be carried out for 20-28 hours, for example, 21, 22, 23, 24, 25, 26, 27 hours, in the presence of 10-50 ng/mL WNT3a, 10-50 ng/mL Activin A and 10-50 ng/mL FGF2. The concentration of WNT3a may be 10-50 ng/mL, for example, 20, 30 or 40 ng/mL. The concentration of Activin A may be 10-50 ng/mL, for example, 20, 30 or 40 ng/mL. The concentration of FGF2 may be 10-50 ng/mL, for example, 20, 30 or 40 ng/mL.

In step (4), the induction may be carried out for 20-28 hours, for example, 21, 22, 23, 24, 25, 26, 27 hours, in the presence of 10-50 ng/mL WNT3a, 5-50 ng/mL Activin A, 10-50 ng/mL FGF2, and 20-80 ng/mL BMP4. The concentration of WNT3a may be 10-50 ng/mL, for example, 20, 30, or 40 ng/mL. The concentration of Activin A may be 10-50 ng/mL, for example, 20, 30, or 40 ng/mL. The concentration of BMP4 may be 20-80 ng/mL, for example, 30, 40, 50, 60, or 70 ng/mL.

In step (5), the cells may be passaged in the presence of 10-50 ng/mL FGF2, 20-80 ng/mL BMP4, 1-10 ng/mL NT4 and 50-500 ng/mL Follistatin, and cultured for 3-5 days, such as 4 days. The concentration of FGF2 may be 10-50 ng/mL, such as 20, 30 or 40 ng/mL. The concentration of BMP4 may be 20-80 ng/mL, such as 30, 40, 50, 60 or 70 ng/mL. The concentration of Follistatin may be 100, 200, 300 or 400 ng/mL. The concentration of NT4 may be 2, 3, 4, 5, 6, 7, 8, 9 ng/mL NT4.

In step (6), the induction may be carried out for 20-28 hours, for example, 21, 22, 23, 24, 25, 26, 27 hours, in the presence of 10-50 ng/mL FGF2, 20-80 ng/mL BMP4 and 1-10 ng/mL NT4. The concentration of FGF2 may be 10-50 ng/mL, for example, 20, 30 or 40 ng/mL. The concentration of BMP4 may be 20-80 ng/mL, for example, 30, 40, 50, 60 or 70 ng/mL. The concentration of NT4 may be 2, 3, 4, 5, 6, 7, 8, 9 ng/mL NT4.

In step (7), the cells may be passaged in the presence of 10-50 ng/mL FGF2, 10-50 ng/mL BMP4, 10-50 ng/mL GDF5, 1-10 ng/mL NT4, and 5-50 ng/mL TGFβ3, and cultured for 1-3 days, such as 2 days. The concentration of FGF2 may be 10-50 ng/mL, such as 20, 30, or 40 ng/mL. The concentration of BMP4 may be 10-50 ng/mL, such as 20, 30, or 40 ng/mL. The concentration of GDF5 may be 10-50 ng/mL, such as 20, 30, or 40 ng/mL. The concentration of NT4 may be 2, 3, 4, 5, 6, 7, 8, 9 ng/mL NT4. The concentration of TGFβ3 may be 10, 15, 20, 25, 30, 35, or 40 ng/mL

In step (8), the cells may be cultured for 3-6 days, such as 4 or 5 days, in the presence of 10-50 ng/mL FGF2, 20-80 ng/mL GDF5, 1-10 ng/mL NT4, and 5-50 ng/mL TGFβ3. The concentration of FGF2 may be 10-50 ng/mL, such as 20, 30, or 40 ng/mL. The concentration of NT4 may be 2, 3, 4, 5, 6, 7, 8, 9 ng/mL NT4. The concentration of TGFβ3 may be 10, 15, 20, 25, 30, 35, or 40 ng/mL. The concentration of GDF5 may be 30, 40, 50, 60, or 70 ng/mL.

Before step (1), iPS cells can be added to a culture dish coated with Vitronectin (Nuwacell, RP01002) and cultured for 1-3 days, such as 24 hours or 2 days. The culture medium used is mTESR1 (Stem Cell Technologies, 85850). The culture medium may contain 1-5% double antibody (penicillin and streptomycin). The culture medium may contain 1-50um (e.g., 10, 20, 30, 40μM) Y27632 (Stem Cell Technologies, 72308). The added cell concentration can be more than 5,000 cells per square centimeter, such as a cell concentration of 10,000 cells per square centimeter. In steps (1)-(8), the culture medium for inducing iPS differentiation contains DMEM/F12, 1-5% (e.g., 2%, 3%, 4%) bispecific antibody, 0.5-10% (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%) ITS-A, 1-10% (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%) B27, 0.5-10% (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%) NEAA and 10-200 μM (e.g., 50, 90, 100, 150, 160, 170 μM) β-mercaptoethanol as the basal culture medium.

The cartilage precursor cells described herein may be positive for CD73, CD90, CD105, and CD44, and may be negative for CD11b, CD19, CD31, CD34, CD45, and HLADR.

Method for preparing chondrocyte microtissue

The present invention provides a method for preparing chondrocyte microtissue, which comprises one or more of the following:

(A1) differentiating induced pluripotent stem (iPS) cells into chondrogenic precursor cells, wherein the chondrogenic precursor cells are positive for CD73, CD90, CD105, and CD44, and negative for CD11b, CD19, CD31, CD34, CD45, and HLADR;

(B1) statically culturing cartilage precursor cells in a 3D cell culture manner in a cartilage precursor cell culture medium to aggregate and form spheres or spheroids, wherein the cartilage precursor cell culture medium comprises DMEM high glucose medium as a basal medium, and contains 1%-10% Knockout serum replacement, 10-40 ng/ml EGF, and 10-40 ng/ml FGF2;

(C1) replacing the chondrogenic precursor cell culture medium with a chondrogenic microtissue culture medium and culturing the spheres or spheroids statically for 1-28 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 days), preferably 5-9 days, wherein the chondrogenic microtissue culture medium comprises DMEM high glucose medium as the basal medium and comprises 0.5%-10% Knockout serum replacement, 0.5%-10% insulin-transferrin-selenium supplement, 0.5-10 mM sodium pyruvate, 50-200 U/mL penicillin, 50-200 μg/mL streptomycin, 0.05-1 mM sodium ascorbate, 0.05-1 uM dexamethasone, 10-100 μg/mL proline, 10-100 ng/mL TGF-β3, and 10-100 ng/mL BMP2; and

(D1) The suspended spheres or spheroids are cultured with shaking in a culture dish containing chondrocyte microtissue culture medium until chondrocyte microtissues are obtained.

The diameter of the chondrocyte microtissue can range from 100 μm to 2000 μm, for example, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800 or 1900 μm.

In step (A1), chondrogenic precursor cells derived from human iPS cell directed differentiation and meeting the quality inspection requirements can be inoculated into a T175 culture flask at a density of 50,000/cm ² and cultured in a 37°C, 5% CO ₂ incubator. When the confluence of chondrogenic precursor cells reaches 80% to 90%, the cell morphology is round, oval, polygonal or short spindle (Figure 1), and the cell surface markers meet the standards of chondrogenic precursor cell surface markers (Figure 2).

In step (B1), the cartilage precursor cells may be cultured for 2-5 days (e.g., 2.5, 3, 3.5, 4 or 4.5 days) to aggregate and form spheres or spheroids. The inoculation amount of the cartilage precursor cells is 1×10 ⁴ /well to 9×10 ^{5 /} well, such as 1×10 ⁴ /well, 5×10 ^{4 /} well or 1×10 ^{5 /} well.

In step (D1), the spheroids or spheroid-like cells are cultured with shaking in a culture dish containing chondrocyte microtissue culture medium for 1-16 days (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 days).

In this article, steps (A1)-(D1) can be carried out at room temperature and _CO2 . Shaking culture can be shaking culture at 60rpm-80rpm (e.g., 65, 70 or 75rpm). Steps (B1) and (C1) can be carried out in a multi-well plate selected from a 24-well plate, a 96-well plate, a 384-well plate or a 948-well plate. Preferably, the multi-well plate is a low-attachment or ultra-low-attachment multi-well plate. The multi-well plate can achieve 3D cell culture of cells.

Method for preparing chondrocyte microtissue fusion formed by mutual adhesion

The present invention also provides a method for preparing a chondrocyte microtissue fusion body formed by mutual adhesion, which comprises:

(A2) differentiating induced pluripotent stem (iPS) cells into chondrogenic precursor cells, wherein the chondrogenic precursor cells are positive for CD73, CD90, CD105, and CD44, and negative for CD11b, CD19, CD31, CD34, CD45, and HLADR;

(B2) statically culturing cartilage precursor cells in a 3D cell culture manner in a cartilage precursor cell culture medium to aggregate and form individual spheres or spheroid-like bodies, wherein the cartilage precursor cell culture medium comprises DMEM high glucose medium as a basal medium, and contains 1%-10% Knockout serum replacement, 10-40 ng/ml EGF, and 10-40 ng/ml FGF2;

(C2) replacing the cartilage precursor cell culture medium with a chondrocyte microtissue culture medium and culturing the individual spheres or spheroids statically for 1-4 days, wherein the chondrocyte microtissue culture medium comprises DMEM high glucose medium as a basal medium, and contains 0.5%-10% Knockout serum replacement, 0.5%-10% insulin-transferrin-selenium supplement, 0.5-10 mM sodium pyruvate, 50-200 U/mL penicillin, 50-200 μg/mL streptomycin, 0.05-1 mM sodium ascorbate, 0.05-1 uM dexamethasone, 10-100 μg/mL proline, 10-100 ng/mL TGF-β3 and 10-100 ng/mL BMP2; and

(D2) shaking and culturing the suspended multiple spheres or spheroids in a chondrocyte microtissue culture medium until a larger chondrocyte microtissue fusion body is obtained, which contains multiple adherent spheres or spheroids and has a diameter of 0.8 mm-3 mm. Preferably, the shaking culture is carried out at 60 rpm-80 rpm.

Herein, the chondrogenic precursor cell culture medium may comprise DMEM high glucose medium as the basal medium, and comprise 1%-10% (e.g. 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%) Knockout serum replacement, 10-40 ng/ml (e.g. 20, 25, 30, 35 ng/ml) EGF and 10-40 ng/ml (e.g. 20, 25, 30, 35 ng/ml) FGF2.

The chondrocyte microtissue culture medium may comprise DMEM high glucose medium as a basal medium, and 0.5%-10% (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%) Knockout serum replacement, 0.5%-10% (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%) insulin-transferrin-selenium supplement, 0.5-10 mM sodium pyruvate, 50-200 U/mL penicillin, 50-200 μg/mL streptomycin, 0.05-1 mM (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 10.1% pyruvate, 50-200 U/mL penicillin, 50-200 μg/mL streptomycin, 0.05-1 mM (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8, 0.9, 10.1% pyruvate, 0. .7, 0.8 or 0.9 mM) sodium ascorbate, 0.05-1 uM (e.g., 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9 uM) dexamethasone, 10-100 μg/mL (e.g., 20, 30, 40, 50, 60, 70, 80, 90 μg/mL) proline, 10-100 ng/mL (e.g., 20, 30, 40, 50, 60, 70, 80, 90 ng/mL) TGF-β3 and 10-100 ng/mL (e.g., 20, 30, 40, 50, 60, 70, 80, 90 ng/mL) BMP2.

After the suspension of cartilage precursor cells in the cartilage precursor cell culture medium is inoculated into the wells of the multi-well plate, centrifugation is performed to allow the cartilage precursor cells to settle to the bottom of the wells. The multi-well plate can be a 24-well plate, a 96-well plate, a 384-well plate or a 948-well plate. The multi-well plate can be an EB-disk948.

Drugs or test kits

The chondrocyte microtissue described herein can be prepared into a kit or medicine with other necessary ingredients, such as culture medium, etc. The kit may further include instruments or reagents for transplantation, such as buffer, etc. The chondrocyte microtissue described herein can be directly applied to the body in a certain amount, or it can also form cartilage tissue in vitro, and then the cartilage tissue is applied to the subject. The subject can be a subject in need of cartilage transplantation, such as a mammal, particularly a human. The subject can suffer from cartilage defects, cartilage degeneration, bone degeneration, osteoarthritis, and/or need cartilage regeneration, bone regeneration or cartilage transplantation.

Example

The embodiments of the present application will be described in detail below in conjunction with the examples, and those skilled in the art will appreciate that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. The specific conditions not specified in the examples are carried out according to the conditions recommended by normal conditions or manufacturers. The reagents used or the instruments not specified by the manufacturer are all conventional products that can be obtained commercially. The application should not be construed as being limited to the specific examples described.

Example 1: Preparation of chondrocyte microtissues from chondrocyte precursor cells derived from human iPS cells

Materials and methods

Material :

Human iPS cells: Saibei, CA4025106

GMP grade cell digestion solution TryplE: Thermo Fisher, 12563029.

Culture medium for chondrogenic precursor cells derived from human iPS cell differentiation: DMEM high glucose medium (gbico, 12430062), 5% Knockout serum replacement (Thermo Fisher, 12618013), 20 ng/ml EGF (Stemimmune, EME-EF-1000), 20 ng/ml FGF2 (Stemimmune, HST-F2-1000).

Chondrocyte microtissue culture medium: DMEM high glucose medium (gbicol, 12430062), 1% Knockout serum replacement (Thermo Fisher, 12618013), 1% insulin-transferrin-selenium supplement (gbicol, 41400-045), 1mM sodium pyruvate (gbicol, 11360070), 100U/mL penicillin 100μg/mL streptomycin (gbicol, 10378016), 0.1mM sodium ascorbate (MCE, HY-107837), 0.1uM dexamethasone (sigma, D4902), 40μg/mL proline (sigma, P5607), 20ng/mL TGF-β3 (Stemimmune, HST-TB3-1000), 20ng/mL BMP2 (peprotech, 120-02C).

method:

Directed differentiation of human iPS cells into chondrogenic precursor cells

Refer to the differentiation method of Griffith LA, Arnold KM, Sengers BG, et al. Ascaffold-free approach to cartilage tissue generation using human embryonic stem cells. [J]. Nature Publishing Group, 2021 (1). First, iPS cells were added to a Vitronectin-coated culture dish at a cell concentration of 10,000 cells per square centimeter and cultured for 24 hours. The culture medium was mTESR1, 1% double antibody and 10μM Y27632; all culture media for inducing iPS differentiation contained DMEM/F12, 1% double antibody, 1% ITS-A, 2% B27, 1% NEAA and 90μM β-mercatoethanol as basal culture medium 1. After culturing iPS for 24 hours, the original culture medium was discarded, and the first induction culture medium containing basal culture medium 1, 25ng/mL WNT3a and 50ng/mL Activin A was added to continue culturing for 24 hours. The first induction medium was discarded, and the second induction medium containing basal medium 1, 25 ng/mL WNT3a, 25 ng/mL Activin A and 20 ng/mL FGF2 was added and cultured for a further 24 h. The second induction medium was discarded, and the third induction medium containing basal medium 1, 25 ng/mL WNT3a, 10 ng/mL Activin A, 20 ng/mL FGF2 and 40 ng/mL BMP4 was added to continue culturing for 24 h; the third induction medium was discarded, the cells were washed once with PBS, 2 ml TrypLE ^TM Select was added to digest the cells at 37°C for 3 min, the cells were collected into a 50 ml centrifuge tube, centrifuged at 1200 rpm for 5 min, and the fourth induction medium containing basal medium 1, 20 ng/mL FGF2, 40 ng/mL BMP4, 2 ng/mL NT4 and 100 ng/mL Follistatin was added to resuspend the cells, and the cells were passaged at a ratio of 1:2 and continued to be cultured for 4 days; the fourth induction medium was discarded, and the fifth induction medium containing basal medium 1, 20 ng/mL FGF2, 40 ng/mL BMP4 and 2 ng/mL NT4 was added to continue culturing for 24 h; the fifth induction medium was discarded, the cells were washed once with PBS, and 2 ml TrypLE ^TM Select was added. Digest the cells at 37°C for 3 minutes, collect the cells into a 50ml centrifuge tube, centrifuge at 1200rpm for 5 minutes, add the sixth induction medium containing basal medium 1, 20ng/mL FGF2, 20ng/mL BMP4, 20ng/mL GDF5, 2ng/mL NT4 and 10ng/mL TGFβ3 to resuspend the cells, subculture at a ratio of 1:2, and continue to culture for 2 days; discard the sixth induction medium, add the seventh induction medium containing basal medium 1, 20ng/mL FGF2, 40ng/mL GDF5, 2ng/mL NT4 and 10ng/mL TGFβ3 to continue culturing for 4 days, and cartilage precursor cells can be obtained. During this induction differentiation period, it is necessary to replace the seventh induction medium every day. The above culture is carried out in a 37°C, 5% CO ₂ incubator. Then, the cartilage precursor cells can be subcultured and expanded in the cartilage precursor cell culture medium derived from human iPS cell differentiation at a ratio of 1:2-1:3 for at least 15 generations (Figure 16).

Cell morphology assay

After the chondrogenic precursor cells derived from iPS cells were inoculated into a culture dish containing the culture medium for chondrogenic precursor cells derived from human iPS cells, the cell morphology was observed and photographed every day using an inverted microscope (Zeiss, Primovert). The chondrogenic precursor cells derived from iPS cells were small in size and round, oval, polygonal or short spindle-shaped (Figure 1).

Flow cytometry detection of cell surface markers

After culturing in the seventh induction medium for 4 days, the original medium was discarded, the cells were washed once with PBS, 2 ml of TrypLE ^TM Select was added to the culture dish to digest the cells at 37°C for 3 min, the cells were collected into a 50 ml centrifuge tube, centrifuged at 1200 rpm for 5 min, and PBS was added to resuspend the cells. 20 ul of the cell suspension was taken and counted using a cell counter (Countstar, IC1000), and the cell density was adjusted to 1×10 ⁷ /ml. Take 14 flow cytometry tubes, add 0.1 ml of the adjusted cell suspension to each tube, and then add the following corresponding antibodies to each tube according to the antibody instructions: IgG1 ISO-PE (Thermo Fisher, 12-4714-82), CD73-PE (Thermo Fisher, 12-0739-42), CD105-PE (Thermo Fisher, 12-1057-42), CD11b-PE (Thermo Fisher, 12-0118-42), IgG1 ISO-FITC (Thermo Fisher, 11-4714-81), CD90-FITC (Thermo Fisher, 11-0909-42), CD19-FITC (Thermo Fisher, 11-0199-42), CD31-FITC (Thermo Fisher, 11-0319-42), CD34-FITC (Thermo Fisher, 11-0319-42), CD47-PE (Thermo Fisher, 11-0118-42), CD47-PE (Thermo Fisher, 11-0118-42), CD47-PE (Thermo Fisher, 11-0118-42), CD47-PE (Thermo Fisher, 11-0118-42), CD47-PE (Thermo Fisher, 11-0118-42), CD47-PE (Thermo Fisher, 11-0 Fisher, 11-0349-42), CD45-FITC (Thermo Fisher, 11-0459-42), IgG2bISO-FITC (Thermo Fisher, 11-4031-82), CD44-FITC (Thermo Fisher, 11-0441-82), IgG2b ISO-FITC (Thermo Fisher, 11-4732-81), HLA-DR-FITC (Thermo Fisher, 11-9956-42), incubate at 4°C in the dark for 30 min. Add 2 ml PBS, gently mix the cells, centrifuge at 400 g for 5 minutes, and remove the supernatant. Add 2 ml PBS, gently mix the cells, centrifuge at 400 g for 5 minutes, and remove the supernatant. Add 0.5 ml PBS, gently mix the cells, and use Lyric flow cytometer to detect cell surface markers. Use the cells in the blank control tube to circle the target cell population on the FS/SS scatter plot, and use the cells in the FITC/PE isotype control antibody tube to adjust the fluorescence threshold and define the range of positive cells. Then collect the fluorescence signal of the sample tube, collect 10,000 single cells in each tube, and record and save the data. The surface markers of cartilage precursor cells should be CD73, CD90, CD105, and CD44 with a positive rate of more than 95% and CD11b, CD19, CD31, CD34, CD45, and HLADR with a positive rate of less than 2.0% (Figure 2). HE staining of transplanted cartilage precursor cells and new tissues in NOD-SCID mice

In order to study the ability of chondrocytes derived from human iPS cells to differentiate into cartilage tissue in vivo, we transplanted P15 chondrocytes derived from iPS cells into the inner thigh muscles of NOD-SCID immunodeficient mice (8-10 weeks). First, the chondrocytes were collected, resuspended in matrigel (Corning, 354277), and the cell suspension was extracted with a 1 ml syringe and transported to the animal room on ice. 1×10 ⁷ chondrocytes were injected into the inner thigh muscles of 8-10 week old NOD-SCID immunodeficient mice with a syringe. The bulge at the injection site was observed, and the mice were euthanized after 6 weeks. The new tissue on the recipient mice was stripped and cut, and the new tissue blocks were fixed with 4% paraformaldehyde at 4°C overnight, dehydrated, paraffin-embedded, and 5 μm sections were cut, and special staining was performed using a hematoxylin and eosin (HE) staining kit (Solebol, G1120).

Results: Six weeks after the P15 chondrocytes derived from iPS cells were transplanted into NOD-SCID immunodeficient mice, HE staining revealed that the new tissue was composed of a large number of "paving stone"-like chondrocytes and extracellular matrix-like cartilage tissue (Figure 3). This shows that the chondrocytes derived from iPS cells have the potential to differentiate into cartilage tissue in vivo.

Preparation of chondrocyte microtissues by 2D induction method

First, the chondrocyte precursor cells derived from human iPS cell directed differentiation that meet the quality inspection requirements are inoculated into a T175 culture flask at a density of 50,000/cm ² and cultured in a 37°C, 5% CO ₂ incubator. When the confluence of the chondrocyte precursor cells reaches 80% to 90%, the cell morphology is round, oval, polygonal or short spindle (Figure 1), and the cell surface markers meet the standards of chondrocyte precursor cell surface markers (Figure 2), then 5 mL of GMP-grade cell digestion solution TryplE is added to the culture flask, gently rotated horizontally to evenly distribute the digestion solution, and placed flat in a 37°C incubator for digestion for 5 minutes. Lift the culture flask and observe the cell detachment with the naked eye under the light, and observe the cells digested into bright single cells under the microscope (if the digestion process is slow, you can gently tap the side wall of the culture flask to accelerate cell detachment through vibration). After all the cells have fallen off, immediately add 5mL of cartilage precursor cell culture medium derived from human iPS cell differentiation, rotate and mix to terminate digestion, add 10mL PBS to the culture flask to wash the cells, and then transfer the cells to a 50mL centrifuge tube. Add 10mL PBS again, wash and collect the remaining cells, place them in a centrifuge tube, balance, centrifuge at 1200rpm for 5min, and gently remove the supernatant. Add 50mL PBS to resuspend the cells, centrifuge at 1200rpm for 5min again, and carefully aspirate the supernatant. Use 20mL of cartilage precursor cell culture medium to resuspend the cell pellet, aspirate 20uL of cell suspension and add it to the Countstar counting plate for counting and calculating the number of cells. Use cartilage precursor cell culture medium to adjust the cell concentration to 1.66×10 ⁵ /mL, inoculate in a TC-96 well plate (Corning, 3599), add 100μL of cell suspension to each well, that is, each well contains 1.66×10 ⁴ cells, and place in a 37°C, 5% CO ₂ incubator for culture. After 3 days of culture, discard the culture medium in the wells, slowly add 100 μL of 37°C preheated PBS to wash the cells once, discard the PBS, and then slowly add 100 μL of 37°C preheated chondrocyte microtissue culture medium to the well plate to start differentiation into chondrocytes, which is recorded as 2D0d. On the 7th day of chondrocyte differentiation (2D7d), the chondrocyte microtissue was transferred to a 10 cm culture dish, placed on a shaker, set the speed to 60-80 rpm, and continued to culture until the 22nd day (2D22d), during which the medium was changed every 2 to 3 days (Figure 17). Results:

As shown in Figure 14, after the chondrocytes differentiated from human iPS cells were inoculated into the TC96 well plate and cultured for 4 hours, the chondrocytes began to adhere to the wall and gradually spread and extend. After 3 days of continuous culture, the cell confluence reached about 85%-90%, and the cell morphology was round, oval, polygonal or short spindle. At this time, the cells were switched to the chondrocyte microtissue culture medium and began to differentiate into chondrocytes, which was recorded as 2D0d. The cell morphology was mostly round. When induced differentiation culture was carried out on 2D5d, the cell density increased significantly and showed a multilayered cell state. The cells aggregated to form multiple cartilage nodules and rolled up spontaneously from the edge of the well plate to the center. When induced differentiation culture was carried out on 2D7d, the cells gradually aggregated into irregular microtissues and fell off from the bottom of the well plate. At this time, the chondrocyte microtissue was transferred to a 15 cm culture dish, and the chondrocyte microtissue culture medium was added. The cells were placed on a horizontal shaker and cultured at 60 rpm-80 rpm. When cultured to 2D12-2D15d, the chondrocyte microtissue was milky white, with a smooth and bright surface and a dense texture. The cells were continued to be cultured to 2D22d, and the changes in cell morphology and culture medium color, the internal cell state of the chondrocyte microtissue, and the adhesion and fusion between the chondrocyte microtissues during the induction and differentiation of chondrocyte precursor cells derived from human iPS cells into chondrocyte microtissues were continuously observed.

Preparation of chondrocyte microtissues using 3D induction method

First, the chondrocyte precursor cells derived from human iPS cell directed differentiation that meet the quality inspection requirements are inoculated into a T175 culture flask at a density of 50,000/cm ² and cultured in a 37°C, 5% CO ₂ incubator. When the confluence of the chondrocyte precursor cells reaches 80% to 90%, the cell morphology is round, oval, polygonal or short spindle (Figure 1), and the cell surface markers meet the standards of chondrocyte precursor cell surface markers (Figure 2), then 5 mL of GMP-grade cell digestion solution TryplE is added to the culture flask, gently rotated horizontally to evenly distribute the digestion solution, and placed flat in a 37°C incubator for digestion for 5 minutes. Lift the culture flask and observe the cell detachment with the naked eye under the light, and observe the cells digested into bright single cells under the microscope (if the digestion process is slow, you can gently tap the side wall of the culture flask to accelerate cell detachment through vibration). After all the cells have fallen off, immediately add 5mL of chondrogenic precursor cell culture medium derived from human iPS cell differentiation, rotate and mix to terminate digestion, add 10mL PBS to the culture flask to wash the cells, and then transfer the cells to a 50mL centrifuge tube. Add 10mL PBS again, wash and collect the remaining cells, place them in a centrifuge tube, balance, centrifuge at 1200rpm for 5min, and gently remove the supernatant. Add 50mL PBS to resuspend the cells, centrifuge at 1200rpm for 5min again, and carefully aspirate the supernatant. Use 20mL of chondrogenic precursor cell culture medium to resuspend the cell pellet, aspirate 20uL of cell suspension and add it to the Countstar counting plate to count and calculate the number of cells. The cell concentration was adjusted to 5×10 ⁵ /mL using chondrogenic precursor cell culture medium, and the cells were inoculated in a ULA-96 well plate (Corning, 7007), with 100uL of cell suspension added to each well, i.e., 5×10 ⁴ cells per well (different cell inoculation amounts were also set up to induce the formation of chondrogenic microtissues, i.e., 1×10 ⁴ cells were inoculated per well, and 1×10 ⁵ cells were inoculated per well), and the cells were cultured in a 37°C, 5% CO ₂ incubator. After 24 hours of inoculation and culture, the chondrogenic precursor cells derived from human iPS cell differentiation began to aggregate and gradually took on an irregular spherical shape. Change the medium on the second day. On the third day of culture, discard the old medium in the well, slowly add 100uL 37°C preheated PBS to wash the cells once, discard the PBS, and then slowly add 100uL 37°C preheated chondrocyte microtissue culture medium to the well plate to start differentiation into chondrocytes. This time is recorded as 3D0d. On the 3D7d of cartilage differentiation, transfer the chondrocyte microtissue to a 15cm culture dish, place it on a shaker, set the speed to 60-80rpm, and continue to culture until 3D22d. During this period, change the medium every 2 to 3 days. Figure 18 shows a schematic diagram of the differentiation process of chondrocyte precursor cells derived from iPS differentiation into chondrocyte microtissues (3D induction method).

result

As shown in Fig. 4 A, after chondrocyte precursor cells were inoculated into ULA-96 well plates for 2h, the edge of the cell mass gradually gathered spontaneously, and after 1 day, the cells gathered to form a regular sphere, and after 2-3 days of cultivation, the sphere became milky white, with a smooth and bright surface and a dense texture, and then switched to chondrocyte microtissue culture medium. Between 3D0d-3D6d thereafter, cells formed dense chondrocyte microtissues, and the volume gradually slightly shrank, and then the volume remained essentially unchanged, with a diameter of about 700um, a milky white regular sphere, bright luster, smooth surface, and a certain hardness (Fig. 4 B). At 3D7d, this chondrocyte microtissue was transferred to a 15cm culture dish, chondrocyte microtissue culture medium was added, and it was placed on a horizontal shaker and cultured to 3D22d at 60rpm-80rpm, and the adhesion and fusion between the culture medium color change, the internal cell state of the chondrocyte microtissue, and the cartilage microspheres were continuously observed.

We also found that different chondrocyte microtissues of different sizes can be prepared by inoculating different amounts of chondrocyte precursor cells. As shown in Figure 5, 1×10 ⁴ cells, 5×10 ⁴ cells, and 1×10 ⁵ cells per well can form chondrocyte microtissues with diameters of approximately 0.5 mm, 0.8 mm, and 1.0 mm, respectively.

Generally, 3×10 ⁷ cells can be harvested from one T175 bottle, which can be used for 6 ULA-96 well plates, and finally 6×96=576 chondrocyte microtissues can be obtained. The demand for each patient is calculated based on 40-70 pellets, and the amount of cells in one T175 bottle can be used clinically for 8-14 patients. Assuming that each cell culture box can accommodate 300 ULA-96 well plates, each cell culture box can produce 96×300=28,800 chondrocyte microtissues, which can be used for 411-720 clinical patients.

Multiple chondrocyte microtissues adhere to each other and fuse to form a chondrocyte microtissue fusion

As shown in Figure 15A, chondrocyte microtissues differentiated from iPS cells at different induction times were transferred to BeaverNano ^™ 6-well suspension cell culture plates (Beaver Biotechnology, 40406), 8 chondrocyte microtissues were placed in each well, 4 mL of chondrocyte microtissue culture medium was added, and the plates were cultured on a horizontal shaker with a rotation speed set at 60 rpm-80 rpm so that all cartilage spheres gathered in the center of the well plate, recorded as 3DXd+3DYd.

Results: After multiple chondrocyte microtissues (3D2d, 3D3d, and 3D4d) were transferred and concentrated together for shaking culture (3D induction culture for a total of 8 days), contact adhesion occurred between the chondrocyte microtissues and gradually fused into a larger sphere (chondrocyte microtissue fusion).

As shown in FIG. 15B and FIG. 15C, chondrocyte microtissues (3D2d) differentiated from iPS cells of different diameters were transferred to BeaverNano ^TM 6-well suspension cell culture plates (Beaver Biotechnology, 40406), 8 chondrocyte microtissues were placed in each well, 4 mL chondrocyte microtissue culture medium was added, and the plates were placed on a horizontal shaker for culture, and the speed was set to 60rpm-80rpm, so that all cartilage spheres gathered in the center of the plate, which was recorded as 3D2d+3D0d. When culturing 3D2d+3D3d, chondrocyte microtissues began to adhere to each other, and cells on the surface of adjacent chondrocyte microtissues adhered and fused; when culturing 3D2d+3D5d, the cell adhesion and fusion at the edge of the chondrocyte microtissue was higher, and it was difficult to distinguish a single chondrocyte microtissue; when culturing 3D2d+3D6d, multiple chondrocyte microtissues were closely connected to form a larger milky white cartilage-like tissue with bright luster, smooth surface and certain hardness. The diameter of chondrocyte microtissues increased from 0.45mm, 0.75mm, and 1.2mm to 1.0mm (about 2.2 times), 1.8mm (about 2.4 times), and 2.6mm (2.2 times). On this basis, the diameter, number, or shape of the mold of a single chondrocyte microtissue can be adjusted to obtain chondrocyte microtissue fusions of different shapes and sizes. In this way, the larger chondrocyte microtissue formed by the mutual adhesion of multiple chondrocyte microtissues is easier to adhere to and fix to the cartilage defect site than a single chondrocyte microsphere during transplantation, and is less likely to fall off, thereby improving the surgical treatment effect; at the same time, it can meet the clinical needs of patients with different cartilage defect areas.

Example 2: Large-scale preparation of cartilage microtissues

Although the method of forming cartilage microtissue based on a 96-well plate in Example 1 above can be used to prepare a quantity of cartilage cell microtissues that can meet early clinical needs, this method requires relatively more manpower and time costs and is not suitable for later larger-scale industrial production. To further solve the above problems, we have further improved this process and discovered a method for preparing cartilage microtissues with a larger production scale and easier operation. The specific description is as follows:

When the confluence of chondrocyte precursor cells reaches 80% to 90%, add clinical-grade digestion solution TryplE to the bottle and cover the bottom of the bottle, and place it in a 37°C incubator for digestion for 3 to 5 minutes. Add preheated chondrocyte precursor cell culture medium to terminate digestion, blow evenly, collect cells in a 50mL centrifuge tube, centrifuge at 1200rpm for 5 minutes, and discard the supernatant. Use preheated chondrocyte precursor cell culture medium to adjust the cell concentration to 2.37×10 ⁸ /mL, use a 1mL pipette tip to gently blow 3 times to mix the cells. Pipette 2mL of the above cell suspension and gently add it to each EB-disk948 (about 6cm in diameter) so that the cell suspension covers the surface of EB-disk948. Let it stand for 2 minutes to allow the cells to be evenly distributed on the surface of EB-Disk948. Then centrifuge at 300g for 1 minute to allow the cells to settle to the bottom of each microwell. Return to the biosafety cabinet and gently add 6 mL of chondrocyte precursor cell culture medium from the side wall of EB-Disk948 to avoid damaging the seeded cells. EB-Disk948 was cultured in a 37°C, 5% CO ₂ incubator. After 3 days, the old culture medium was gently discarded, and chondrocyte microtissue culture medium was added to EB-Disk948 to begin inducing differentiation into chondrocyte microtissues (recorded as 3D0d). After 7 days (3D7d), the formed chondrocyte microtissue was transferred to a 10 cm culture dish (or culture bag, bioreactor), and cultured for 15 days (3D22d) on a 60rpm-80rpm shaker, during which the medium was changed every 2 to 3 days. During 3D0d to 3D22d, chondrocyte microtissues were collected every 2 days, and total RNA was isolated from chondrocyte microtissues (EB948 3D8d) derived from iPS cell differentiation using RNeasy Mini Kit (Qiagen, 74004) according to the product instructions, and DNase I digestion was performed on the column. 1 μg of total RNA was used as a template, and cDNA was synthesized using the iScript cDNA synthesis kit (BioRad, 1708891), which was stored at -20°C for later use. After being induced into chondrocyte microtissues, each EB-Disk948 can prepare 948 chondrocyte microtissues. The demand for each patient is calculated based on 40-70 pellets, which can be used for 13 to 23 patients (Figure 6). Assuming that each incubator can accommodate 300 EB-Disk948, each incubator can produce 300×948=284,400 chondrocyte microtissues, which can be used for 4062-7110 clinical patients. Therefore, this method can be used to achieve large-scale preparation and production of stable and uniform chondrocyte microtissues to meet the needs of clinical patients.

Example 3: Live and dead cell staining and flow cytometry detection of cell viability of chondrocyte microtissues derived from human iPS cell directed differentiation

The live/dead cell viability assay kit (abcam, ab287858, Lot: GR3451473-4) provides a dual-color fluorescence method based on the simultaneous determination of live and dead cells using two different dyes. Live cell dyes easily and completely penetrate the cell membrane, and live cells and intracellular esterases hydrolyze the dyes to produce hydrophilic, highly fluorescent compounds that remain in the cell cytoplasm and can be measured at Ex/Em=485/530nm. Dead cell dyes enter damaged cell membranes, and after binding to nucleic acids, the fluorescence is enhanced 40 times, producing bright red fluorescence in dead cells (Ex/Em=495/635nm).

The chondrocyte microtissues (3D7d, 3D14d) formed by chondrocyte precursor cells derived from iPS cells on the 7th and 14th days of induction differentiation and the chondrocyte microtissues derived from adult tissues (CDC 3D7d) were placed in TC96 well plates, and 200ulPBS was added to wash the chondrocyte microtissues twice. The PBS was discarded, and 200ul assay buffer containing live and dead cell staining solution was added to immerse the chondrocyte microtissues. The control group only added the assay buffer without live and dead cell staining solution, incubated at 37°C for 15min, and then observed and collected images using a fluorescence microscope (Keyence, BZ-X800).

Take 12 chondrocyte microtissues (3D20d) formed by chondrocyte precursor cells differentiated from iPS cells after 20 days of induction differentiation in a 15ml centrifuge tube, wash the chondrocyte microtissue twice with PBS, discard the PBS, add 2ml0.2% type II collagenase to the tube, digest at 37℃ for 60min, add 2ml chondrocyte microtissue culture medium to terminate the reaction, and obtain a chondrocyte microtissue single cell suspension. Take 20μl cell suspension for counting, resuspend the cells with PBS and divide them into 4 1.5ml centrifuge tubes, each containing 1×10 ⁶ cells, centrifuge at 500g for 5min, remove the supernatant, add 1ml assay buffer containing dead and live cell staining solution (see the table below) to the tube, and incubate at 37℃ in the dark for 15min. Wash the cells once with PBS, discard the supernatant, and finally resuspend them in 0.5ml assay buffer, and use Lyric flow cytometer to detect cell viability. FITC channel represents live cells, and APC channel represents dead cells.

Table 1: Flow cytometry experimental configuration for each group

The staining results showed that on the 7th and 14th days of induced differentiation, there were a large number of cells inside the chondrocyte microtissues, the cells were in good condition, round or oval, most of them were living cells, and there were very few dead cells. The morphology of the cells inside the chondrocyte microtissues (CDC 3D7d) derived from adult tissues was mostly spindle-shaped and fibrous, the internal cells were unevenly distributed, and there were many dead cells (Figure 7A). In addition, after the chondrocyte microtissues were digested into single cells, the cell viability was detected using a flow cytometer according to the product instructions. The results showed that the cell viability inside the chondrocyte microtissues derived from iPS cell directed differentiation was 99.7% (Figure 7B).

Example 4: Staining of micro-tissue pathological sections of chondrocytes derived from human iPS cells

On the 5th, 10th and 15th days of in vitro differentiation induction, chondrocyte microtissues were fixed with 4% paraformaldehyde (PFA), dehydrated, paraffin-embedded, and 5 μm sliced, and specially stained with hematoxylin and eosin (HE) staining kit (Solebau, G1120), toluidine blue staining kit (Solebau, G3668), and modified safranin O-fast green staining kit (Solebau, G1371). For immunofluorescence staining, the white slides were immersed in xylene for 10 min 3 times, 100% ethanol for 5 min 2 times, 95% ethanol for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and 50% ethanol for 5 min, and then washed with pure water. Wash with PBS on a shaker 3 times, 5 min each time; permeabilize with 0.4% Triton X-100 at room temperature for 1 h; wash with PBS on a shaker 3 times, 5 min each time; add 5% donkey serum in a wet box at room temperature for 30 min to block nonspecific binding sites. Knock off the excess donkey serum, add the primary antibodies diluted with 5% donkey serum in the wet box: COLII (abcam, ab185430), ACAN (abnova, PAB27837), Lubricin (abcam, ab28484), SOX9 (santa cruz biotech, sc-166505), and place the wet box at 4°C overnight. Wash with PBS on a shaker for 3 times, 10 min each time; add fluorescein-labeled secondary antibody in a wet box in a light-proof environment and react at room temperature for 70 min; wash with PBST on a shaker for 3 times, 5 min each time, and then rinse with pure water; add DAPI (Thermo fisher, 62247) in a wet box and react at room temperature for 10 min; wash with PBST on a shaker for 3 times, 5 min each time, and then rinse with pure water; seal the slides with anti-fluorescence attenuation sealing agent, and observe and collect images with a fluorescence microscope (Keyence, BZ-X800).

Toluidine Blue O dye is an alkaline dye that can bind to cations in cells, staining the nucleus purple or dark blue and the cytoplasm light blue. The principle of the modified Safranin O/Fastgreen cartilage staining method is that basophilic cartilage combines with the alkaline dye Safranin O to show red. Safranin O is a cationic dye that binds to polyanions. Its display of cartilage tissue is based on the combination of cationic dyes with anionic groups (chondroitin sulfate or keratan sulfate) in polysaccharides. Safranin O coloring is approximately proportional to the concentration of anions, which indirectly reflects the content and distribution of proteoglycans in the matrix. The special staining results showed that after 10 days of induction of cartilage precursor cells derived from iPS cells to differentiate into chondrocyte microtissues, the chondrocyte microtissue contained a large number of cells, and the cells were surrounded by rich type II collagen and proteoglycans (Figure 8).

The protein encoded by the Lubricin (also known as PRG4) gene is a large proteoglycan synthesized by chondrocytes and some synoviocytes located on the surface of articular cartilage. This protein contains chondroitin sulfate and keratin sulfate glycosaminoglycans. It acts as a boundary lubricant on the surface of cartilage, contributing to the elastic absorption and energy dissipation of synovial fluid. Immunofluorescence staining results showed that on the 5th, 10th and 15th days of induction of differentiation, chondrocyte microtissues expressed COLII, ACAN, Lubricin and SOX9, and gradually increased with the extension of induction time (Figure 9).

Example 5: Single-cell sequencing of chondrocyte microtissues derived from directed differentiation of human iPS cells

Take an appropriate amount of chondrocyte microtissue (3D8d) formed by chondrocyte precursor cells differentiated from iPS cells after 8 days of induction differentiation in a 15ml centrifuge tube, wash the chondrocyte microtissue twice with PBS, discard the PBS, add 2ml0.2% type II collagenase to the tube, digest at 37℃ for 60min, add 2ml chondrocyte microtissue culture medium to terminate the reaction, and obtain a chondrocyte microtissue single cell suspension. Entrust a scientific research service testing agency (Beijing Geek Gene Technology Co., Ltd.) to perform 10× single-cell transcriptome sequencing.

The results of 10× single-cell transcriptome sequencing showed that the vast majority of cell types in the chondrocyte microtissue (3D8d) formed by chondrocyte precursor cells differentiated from iPS cells after 8 days of induction differentiation were chondrocytes, and the other small number of cells were only mesenchymal stem cells (MSC). There were no iPS cells remaining, and the purity of chondrocytes was 95.22% (A-E in Figure 19).

In this method, the differentiation efficiency of chondrogenic progenitor cells derived from human iPS cells into mature chondrocytes was extremely high (A-E in Figures 8, 9, and 19).

Example 6: Gene expression in chondrocyte microtissues derived from human iPS cell directed differentiation

Material

CDC P0 refers to human articular chondrocytes of generation P0 (Sciencell, #4650).

The culture medium for human articular chondrocytes was DMEM high glucose (gbicol, 12430062), 10% FBS (gbicol, 10099141), 20 ng/ml TGFb3 (Stemimmune, HST-TB3-1000), 100 U/mL penicillin and 100 μg/mL streptomycin (gbicol, 10378016).

CDC P2 3D8d is a chondrocyte microtissue formed by 3D culture of human P2 articular chondrocytes for 8 days. The preparation method is as follows: take out the P0 generation human articular chondrocytes from liquid nitrogen, immediately put them into a 37°C water bath to thaw, add 1ml of thawed P0 generation human articular chondrocytes to 9ml pre-cooled α-MEM (Hyclone, SH30265.01), and centrifuge at 1200rpm for 5min; remove the supernatant, resuspend the cells in human articular chondrocyte culture medium, inoculate them into T75 culture bottles at a density of 10000/ ^cm2 , and place them in a 37°C, 5% CO2 incubator for 3-5 days. When the cell confluence reaches 80%-90%, add 3mL of GMP-grade cell digestion solution TryplE to the culture bottle, gently rotate horizontally to evenly distribute the digestion solution, and place it flat in a 37°C incubator for 5min of digestion. Lift the culture bottle and observe the cell detachment under the light with the naked eye, and observe the cells digested into bright single cells under the microscope (if the digestion process is slow, you can gently tap the side wall of the culture bottle to accelerate the cell detachment through vibration). After all the cells are detached, immediately add 3mL of human articular chondrocyte culture medium, rotate and mix to terminate the digestion, collect the cells into a 15mL centrifuge tube, and then use 8mlPBS to wash the culture bottle and collect the cells into a 15ml centrifuge tube. Centrifuge at 1200rpm for 5min, gently remove the supernatant, and obtain P1 generation human articular chondrocytes. Obtain P2 generation human articular chondrocytes by the same passaging method, adjust the cell concentration to 5×10 ⁵ /mL using human chondrocyte culture medium, inoculate in ULA-96 well plates (Corning, 7007), add 100uL of cell suspension to each well, that is, each well contains 5×10 ⁴ cells, and place in a 37°C, 5% CO ₂ incubator for culture. After 24 hours of culture, human articular chondrocytes began to gather and gradually became irregular spheres. The culture was continued for 8 days, during which the medium was changed every 2 to 3 days. On the 8th day of human articular chondrocyte microtissue culture, total RNA was isolated from human articular chondrocyte microtissue (CDC P2 3D8d) using RNeasy Mini Kit (Qiagen, 74004) according to the product instructions, and DNase I digestion was performed on the column. 1 μg of total RNA was taken as a template, and cDNA was synthesized using iScript cDNA synthesis kit (BioRad, 1708891) and stored at -20°C for future use.

EB948 3D8d: In the large-scale preparation of cartilage microtissues in Example 2, chondrocyte microtissues cultured for 8 days were prepared using the EB948 disk method and recorded as EB948 3D8d.

method

During the in vitro differentiation into chondrocyte microtissues, total RNA was isolated from chondrocyte microtissues every 2 days using RNeasy Mini Kit (Qiagen, 74004) according to the product instructions, and DNase I digestion was performed on the column. 1 μg of total RNA was used as a template, and cDNA was synthesized using iScript cDNA synthesis kit (BioRad, 1708891). Real-time PCR was performed using a two-step PCR amplification program using the TB green Premix Ex Taq (TAKARA, RR820A) qPCR kit on the CFX96 Real Time PCR Detection System (Bio Rad). The formula 2 ^-ΔΔCt was used to calculate the relative expression of genes, and ACTB or B2M was used for gene normalization. The primer sequences are shown in the table below. Reference Yamashita (Yamashita A, Morioka M, Yahara Y, Okada M, Kobayashi T, Kuriyama S, Matsuda S, Tsumaki N. Generation of scaffoldless hyaline cartilaginous tissue from humaniPSCs. Stem Cell Reports. 2015Mar 10;4(3):404-18) and Kawata (Kawata M, Mori D, KankeK, Hojo H, Ohba S, Ch. ung UI, Yano F, Masaki H, Otsu M, Nakauchi H, Tanaka S, Saito T. Simple and Robust Differentiation of Human Pluripotent Stem Cells toward Chondrocytes by Two Small-Molecule Compounds. Stem Cell Reports. 2019Sep 10; 13(3):530-544) and G. Roll (method for preparing transplantable cartilage tissue, authorization publication number CN110944684B) and others published articles or patents.

Table 2: Primers for detecting markers

Human hyaline cartilage extracellular matrix encoding genes mainly include COL2A1 (type II collagen), ACAN (proteoglycan), COL9A1 (type IX collagen), and COL11A1 (type XI collagen). The expression of ACAN gene in chondrocyte microtissue is positively correlated with the cartilage regeneration and repair ability after transplantation. Therefore, it can be potentially used as a drug release test and is generally considered to be a standard for chondrocyte microtissue quality and product release (reference Vonk, LA, G., Hernigou, J., Kaps, C., & Hernigou, P. (2021). Role of Matrix-Associated Autologous Chondrocyte Implantation with Spheroids in the Treatment of Large Chondral Defects in the Knee: A Systematic Review. International Journal of Molecular Sciences, 22(13), 7149.). SOX9 (SRY-Box transcription factor 9) is a protein-coding gene that determines the synthesis and secretion of type II collagen during chondrocyte differentiation and plays an important role in chondrocyte differentiation and bone development. The SPARC (secreted acidic and cysteine-rich protein) gene encodes a cysteine-rich acidic matrix-associated protein. This encoded protein is required for collagen calcification in bones, but it is also involved in the synthesis of extracellular matrix and promotes changes in cell shape. HAPLN1 (hyaluronan and proteoglycan binding protein 1) is a protein-coding gene and a biomarker specific for articular chondrocytes, and MFAP5 (microfibril-associated protein 5) gene encodes a 25-kD microfibril-associated glycoprotein that is a component of extracellular matrix microfibrils and is considered a fibroblast-specific biomarker.

In patent CN110944684B (G. Roll et al., method for preparing transplantable cartilage tissue, authorization announcement number CN110944684B), when the expression levels of the chondrogenesis consistency marker gene CRTAC1 (Homo sapiens cartilage acidic protein 1, NM_018058) and the chondrogenesis purity marker gene EBF3 (Homo sapiens early B-cell factor 3, NM_001005463) of the chondrocyte microtissue meet the following conditions: CRTAC1/R≥0.012, EBF3/R≤0.1, it indicates that the chondrocyte microtissue has high consistency and purity.

result

As shown in Figure 10A and Table 3, compared with adult primary chondrocytes (CDC P0) and adult chondrocyte microtissues (CDCP2 3D8d), chondrocyte microtissues derived from iPS cell differentiation (2D induction method) highly expressed chondrocyte extracellular matrix-related genes COL2A1, COL9A1, COL11A1, SPARC and SOX9 transcription factors, and their expression levels increased first and then decreased with the extension of induction differentiation time; while the expression levels of ACAN and HAPLN genes were lower than those of adult primary chondrocytes (CDC P0) except for 3D8d; in addition, the expression level of MFAP5, a marker gene representing fibrochondrocytes, increased rapidly in the early stage of induction differentiation into chondrocytes, which was much higher than that of adult primary chondrocytes (CDC P0) and adult chondrocyte microtissues (CDC P23D8d). This indicates that the chondrocyte microtissues obtained by the 2D induction method may be more inclined to form fibrocartilage rather than hyaline cartilage tissue required clinically.

Table 3: qPCR raw CT values of chondrocyte microspheres induced by 2D method

Table 3: qPCR raw CT values of chondrocyte microspheres induced by 2D method

Table 3: qPCR raw CT values of chondrocyte microspheres induced by 2D method

As shown in Figure 10B and Table 4, compared with adult primary chondrocytes (CDC P0) and adult chondrocyte microtissues (CDCP2 3D8d), iPS cell-derived chondrocyte microtissues (3D induction method) highly expressed cartilage extracellular matrix-related genes COL2A1, ACAN, COL9A1, COL11A1, HAPLN1, SPARC, and SOX9 transcription factors, and their expression levels basically showed a gradual increase trend with the extension of induction differentiation time, and basically maintained a stable high expression at 3D8d (Figure 10B). On the other hand, iPS cell-derived chondrocyte microtissues had similar or higher MFAP5 expression levels than adult primary chondrocytes (Figure 10B), indicating that they contained very few fibroblasts or mesenchymal stem cells, which was consistent with the results of 10× single-cell sequencing (Figure 19). Judging from the differences in gene expression levels of chondrocyte microtissues obtained by 2D and 3D induction methods, the 3D induction method can upregulate the expression of hyaline chondrocyte-related genes, such as COL2A1, ACAN, COL9A1, COL11A1, SPARC, HAPLN1, and SOX9, and downregulate the expression of fibrochondrocyte marker gene MFAP5.

Table 4: qPCR raw CT values of chondrocyte microspheres induced by 3D method

Table 4: qPCR raw CT values of chondrocyte microspheres induced by 3D method

Table 4: qPCR raw CT values of chondrocyte microspheres induced by 3D method

As shown in FIG. 10C and Table 5, the chondrocyte microtissue (3D8d) obtained by the directional induction and differentiation of iPS obtained by the 3D induction method in this method has CRTAC1/R=60≥0.012 and EBF3/R=0≤0.1, indicating that it has high consistency and purity, which is consistent with the 10× single-cell sequencing results (FIG. 19). The patent of G. Roll et al. (Method for preparing transplantable cartilage tissue, authorization announcement number CN110944684B) requires CRTAC1/R≥0.012 and EBF3/R≤0.1, which can indicate that the chondrocyte microtissue has good consistency and purity.

Table 5: qPCR raw CT values of CRTAC1 and EBF3 in chondrocyte microspheres induced by 3D method:

The above data fully demonstrate that the chondrocyte microtissue derived from iPS cell differentiation obtained by the 3D induction method in this method is mainly hyaline cartilage rather than fibrous cartilage; it also shows that the chondrocyte precursor cells derived from iPS cell differentiation reach a stable optimal level on the 8th day (3D8d) of induction into chondrocyte microspheres.

Example 7: Determination of the ability of chondrocyte microtissues derived from directed differentiation of human iPS cells to develop into cartilage tissue in vivo.

method

In vivo implantation

In order to study the ability of chondrocyte microtissues derived from human iPS cells (3D8d chondrocyte microtissues) to form teratomas and mature into hyaline cartilage tissue in vivo, we transplanted chondrocyte microtissues derived from iPS cells into NOD-SCID immunodeficient mice (8-10 weeks) and C57BL/6N mice (7-8 weeks) subcutaneously. First, the mice were restrained, the hair on both sides of the ventral and dorsal skin was shaved, and the mice were anesthetized with 2.5% tribromoethanol. After the mice had no obvious pain reflex, the skin was disinfected with iodine tincture and alcohol, a 0.5 cm wound was opened on the skin, and a 1.5 cm deep channel was bluntly separated along the skin opening with tweezers. The chondrocyte microtissue was removed from the culture medium, washed once with PBS, and then the chondrocyte microtissue was placed at the bottom of the subcutaneous channel. One chondrocyte microtissue was transplanted at each site, and the opening was sutured with a skin suturer. The bulge of the ventral and dorsal skin was observed. The animals were killed after 6 weeks, and the regenerated tissue was grossly observed, pathologically stained, and biomechanically tested.

Gross observation of regenerated tissue

As shown in Figure 11A, after 6 weeks of subcutaneous transplantation of chondrocyte microtissues in NOD-SCID mice and C57BL/6 mice, large tissue protrusions were visible to the naked eye, no teratoma was formed at the transplantation site, and no immune rejection or other adverse reactions were observed. After 6 weeks of transplantation, the subcutaneous transplanted chondrocyte microtissues of NOD-SCID mice and C57BL/6 mice were surgically removed, and it was found that the volume of chondrocyte microtissues was larger than that before transplantation, the regenerated tissue was milky white, smooth in surface, and had a certain hardness, and the chondrocyte microtissues grew well under the skin.

Biomechanical testing of regenerated tissue

Methods: According to the method of Yang et al. (Yang Y, Wang X, Zha K, Tian Z, Liu S, Sui X, Wang Z, Zheng J, Wang J, Tian X, Guo Q, Zhao J. Porcine fibrin sealant combined with autologous chondrocytes successfully promotes full-thickness cartilage regeneration in a rabbit model. J Tissue Eng Regen Med. 2021 Sep; 15 (9): 776-787), the surface of the cartilage-like tissue regenerated subcutaneously in NOD-SCID immunodeficient mice was first placed perpendicular to the indentation axis, and a cylindrical flat-end indenter with a diameter of 1 mm (BOSE3220 EM USA) was used to continuously record the force and displacement applied to the regenerated cartilage-like tissue at a speed of 0.005 mm/s for 300 s. The elastic modulus of the repaired cartilage was calculated based on the stress-strain curve.

Results: After 8 weeks of subcutaneous transplantation of human iPSC-derived chondrocyte microtissues into mice, the regenerated tissue had the same mechanical biomechanical properties as the reported human natural hyaline cartilage, with an elastic modulus of about 27 MPa (Figure 12 and Table 6).

Table 6: The chondrocyte microtissues derived from human iPS cells were removed from NOD-SCID mice 8 weeks after subcutaneous transplantation and the results of mechanical properties testing were performed.

Pathological staining

The regenerated cartilage-like tissues were obtained after subcutaneous transplantation of chondrocyte microtissues in NOD-SCID immunodeficient mice and C57BL/6 mice for 6 weeks, fixed with 4% paraformaldehyde (PFA) for 2 days, dehydrated, paraffin-embedded, and 5 μm sliced, and specially stained with hematoxylin and eosin (HE) staining kit (Solebol, G1120), toluidine blue staining kit (Solebol, G3668), modified safranin O-fast green staining kit (Solebol, G1371) and Alcian blue staining kit (Solebol, G1562). Alcian blue (Alcian blue) is a copper-titanium cyanine conjugate dye. This cationic dye is combined with an acidic group, that is, Alcian blue forms an insoluble complex with anionic groups such as carboxyl and sulfate groups contained in the tissue. Alcian blue is composed of a central copper-containing phthalocyanine ring connected to four isothiourea groups through thioether bonds. The isothiourea group is moderately alkaline, making Alcin blue a cationic dye. The different pH values of the dye solution can be used to distinguish the types of mucus substances.

Results: As shown in Figure 11A, the subcutaneous regenerated cartilage-like tissues of NOD-SCID immunodeficient mice and C57BL/6 mice were composed of a large number of hyaline chondrocytes wrapped in extracellular matrix proteoglycans, and the internal cells were arranged in a typical "paving stone" shape.

result

The results of gross observation, pathological staining and biomechanical testing of the regenerated cartilage-like tissue 6 weeks after subcutaneous transplantation of chondrocyte microtissues in NOD-SCID immunodeficient mice and C57BL/6 mice showed that no teratoma was formed 6 weeks after subcutaneous transplantation of chondrocyte microtissues, and the chondrocyte microtissues grew and developed well under the skin (Figure 11, Panel A). The results of histopathological sections showed that the regenerated tissue was composed of a large number of hyaline chondrocytes wrapped in extracellular matrix proteoglycans (Figure 11, Panel B), and human osteochondral tissue with the same mechanical and biomechanical properties as natural hyaline cartilage (Figure 12), which shows that the chondrocyte microtissue derived from iPS cell differentiation will not cause immune rejection in the body, and has the potential to develop into osteochondral tissue in vivo.

Example 8: Repair of cartilage defects in the weight-bearing area of the rabbit knee joint by filling chondrocyte microtissue derived from directed differentiation of human iPS cells

Construction of cartilage defect model in weight-bearing area of rabbit knee joint

The New Zealand rabbits that passed the quarantine were weighed and anesthetized by injecting 2.5% sodium amobarbital solution (1mL/kg-3mL/kg) through the ear vein. The hair of the hind legs and knees were cut and the skin was prepared. The limbs were fixed in the supine position and disinfected twice with iodine tincture after draping. A 3.0-4.0 cm incision was made on the medial side of the knee joint of the New Zealand rabbit's hind limb (or a longitudinal incision on the lateral side of the patella) to expose the joint surface. After checking whether there was effusion or adhesion in the knee joint cavity and the articular cartilage was flat and normal in color, a 4mm corneal trephine was used to drill holes on the medial condyle of the femur, and the residual cartilage fragments were removed with a curette and the bottom was repaired and leveled (Figure 13, Figure A). The patella was repositioned and the joint capsule and skin incision were sutured layer by layer. Penicillin was injected intramuscularly immediately after surgery to prevent infection, and the injection was continued for 7 days. A full-thickness articular cartilage defect animal model was established. The defect site was 4 mm in diameter and 1-2 mm in depth. The food intake, defecation, mental state, and wound infection of each group of animals were observed and recorded every day.

Repair of cartilage defects in the weight-bearing area of the rabbit knee joint by filling chondrocyte microtissue derived from directed differentiation of human iPS cells Transplantation procedure

Refer to the method of Boehm et al. (Boehm E, Minkus M, Scheibel M. Autologous chondrocyte implantation for treatment of focal articular cartilage defects of the humeral head. J Shoulder Elbow Surg. 2020 Jan; 29 (1): 2-11). After successfully establishing the cartilage defect model in the weight-bearing area of the rabbit knee joint, use a curette to remove the residual cartilage fragments at the injured site and repair the bottom to make it flat. Then, the chondrocyte microtissue derived from iPS cell differentiation is evenly spread and filled in the articular cartilage defect. It is recommended to transplant 10 to 70 chondrocyte microtissues per square centimeter, keep the blood dry, and let it stand for 15 minutes to allow the cartilage spheres to fully adhere to the joint tissue (Figure 13 A and B), and then suture the wound layer by layer. Samples were collected at 1 month and 3 months after surgery. The sampled areas included the cartilage in the repaired area, surrounding normal cartilage, subchondral bone and cancellous bone. All samples were fixed in 4% paraformaldehyde, decalcified and dehydrated in 10% EDTA.

result

The results of gross observation and histopathological staining showed that there was basically no new tissue regeneration at the cartilage defect site in the model group. Chondrocyte microtissues derived from iPS cell differentiation were transplanted into the cartilage defect site of rabbits. One month later, the defect site was filled and covered with regenerated tissue (Figure C in Figure 13). The results of HE, toluidine blue, safranin O fast green, alcian blue and Masson staining showed that the regenerated cartilage tissue was rich in a large number of chondrocytes and cartilage extracellular matrix, such as proteoglycans, collagen fibers, etc., and had a high degree of fusion with the surrounding natural cartilage. After Image J software analysis, the proportion of regenerated tissue area in the model control group and the iPS cell differentiation-derived chondrocyte microtissue treatment group to the defect area was 30.05% and 63.30%, respectively (Figure D in Figure 13 and Table 7). This shows that the chondrocyte microtissue derived from iPS cell differentiation has a good filling and repair effect on rabbit knee cartilage defects.

Table 7: Calculation of cartilage regeneration and repair ratio

The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims (10)

1. A method of preparing a chondrocyte microstructure comprising:

(A1) Differentiating iPS cells into cartilage precursor cells, wherein the cartilage precursor cells are positive for CD73, CD90, CD105 and CD44 and negative for CD11b, CD19, CD31, CD34, CD45 and HLADR;

(B1) Stationary culturing cartilage precursor cells in a 3D cell culture manner in a cartilage precursor cell culture medium to aggregate to form individual spheres or spheroids, wherein the cartilage precursor cell culture medium comprises DMEM high sugar medium as a basal medium and comprises 1% -10% knockout serum replacement, 10-40ng/ml EGF and 10-40ng/ml FGF2;

(C1) Replacing the chondrocyte precursor cell medium with chondrocyte micro-tissue medium and culturing the individual spheres or spheroids for 1-28 days, preferably 5-9 days, wherein the chondrocyte micro-tissue medium comprises DMEM high sugar medium as basal medium and comprises 0.5% -10% knockout serum replacement, 0.5% -10% insulin-transferrin-selenium additive, 0.5-10mM sodium pyruvate, 50-200U/mL penicillin, 50-200 μg/mL streptomycin, 0.05-1mM sodium ascorbate, 0.05-1uM dexamethasone, 10-100 μg/mL proline, 10-100ng/mL TGF- β3 and 10-100ng/mL BMP2; and

(D1) The individual spheres or spheroids in suspension are shake-cultured in the chondrocyte micro-tissue culture medium until chondrocyte micro-tissue is obtained, preferably in the diameter range of 100 μm to 2000 μm, preferably shake-cultured at 60rpm to 80 rpm.

2. The method of claim 1, wherein step (A1) comprises:

(1) Culturing iPS cells in se:Sup>A medium that induces iPS cell differentiation for 20-28 hours, the medium comprising DMEM/F12 medium and comprising 0.5-5% penicillin, 0.5-5% streptomycin, 0.5-5% its-se:Sup>A, 1-10% b27, 0.5-5% nease:Sup>A, and 50-200 μΜβ -mercaptoethanol;

(2) Inducing in the presence of 10-50ng/mL WNT3a and 50-100ng/ML ACTIVIN A for 20-28 hr;

(3) Inducing in the presence of 10-50ng/mL WNT3a, 10-50ng/ML ACTIVIN A and 10-50ng/mL FGF2 for 20-28 hours;

(4) Inducing in the presence of 10-50ng/mL WNT3a, 5-50ng/ML ACTIVIN A, 10-50ng/mL FGF2 and 20-80ng/mL BMP4 for 20-28 hr;

(5) Passaging the cells in the presence of 10-50ng/mL FGF2, 20-80ng/mL BMP4, 1-10ng/mL NT4, and 100ng/mL Follistatin, and culturing for 3-5 days;

(6) Inducing in the presence of 10-50ng/mL FGF2, 20-80ng/mL BMP4 and 1-10ng/mL NT4 for 20-28 hr;

(7) Passaging the cells in the presence of 10-50ng/mL FGF2, 10-50ng/mL BMP4, 10-50ng/mL GDF5, 1-10ng/mL NT4, and 5-50ng/mL TGF beta 3, and culturing for 1-3 days; and

(8) Culturing the cells in the presence of 10-50ng/mL FGF2, 20-80ng/mL GDF5, 1-10ng/mL NT4, and 5-50ng/mL TGF beta 3 for 3-6 days; the cells were then passaged for more than 15 passages to obtain cartilage precursor cells.

3. The method according to any one of claims 1 or 2, wherein the cartilage precursor cells are cultured in step (B1) for 2-5 days to aggregate to form spheres or spheroids; and/or shaking culturing the spheroids or spheroids in chondrocyte microtissue medium for 1-28 days in step (D1); and/or wherein steps (B1) and (C1) are performed in a multi-well plate selected from the group consisting of a 24-well plate, a 96-well plate, a 384-well plate, or a 948-well plate, such as EB-disk 948.

4. A method of preparing a chondrocyte microstructure comprising:

(A2) Differentiating the induced pluripotent stem cell iPS cells into cartilage precursor cells, wherein the cartilage precursor cells are positive for CD73, CD90, CD105 and CD44 and negative for CD11b, CD19, CD31, CD34, CD45 and HLADR;

(B2) Stationary culturing cartilage precursor cells in a 3D cell culture manner in a cartilage precursor cell culture medium to aggregate to form individual spheres or spheroids, wherein the cartilage precursor cell culture medium comprises DMEM high sugar medium as a basal medium and comprises 1% -10% knockout serum replacement, 10-40ng/ml EGF and 10-40ng/ml FGF2;

(C2) Replacing the chondrocyte precursor cell culture medium with chondrocyte micro-tissue culture medium and standing the individual spheres or spheroids for 1-28 days, wherein the chondrocyte micro-tissue culture medium comprises DMEM high sugar culture medium as a basal medium and comprises 0.5% -10% knockout serum replacement, 0.5% -10% insulin-transferrin-selenium additive, 0.5-10mM sodium pyruvate, 50-200U/mL penicillin, 50-200 μg/mL streptomycin, 0.05-1mM sodium ascorbate, 0.05-1uM dexamethasone, 10-100 μg/mL proline, 10-100ng/mL TGF- β3 and 10-100ng/mL BMP2; and

(D2) The plurality of spheres or spheroids in suspension are shake-cultured in the chondrocyte micro-tissue culture medium until a larger volume of chondrocyte micro-tissue fusion is obtained, comprising a plurality of adherent spheres or spheroids, preferably having a diameter of 0.8mm-3mm, preferably shake-cultured at 60rpm-80 rpm.

5. The method of claim 4, wherein the cartilage precursor cells are cultured in step (B2) for 2-5 days to aggregate to form spheres or spheroids; and/or wherein steps (B2) and (C2) are performed in a multi-well plate selected from a 24-well plate, a 96-well plate, a 384-well plate, or a 948-well plate, such as EB-disk 948; and/or step (A2) comprises step (A1) of claim 1.

6. A chondrocyte micro-tissue comprising chondrocytes derived from human pluripotent stem cell differentiation and extracellular matrix proteins secreted therefrom, and having higher expression levels of extracellular matrix proteins COL2A1, ACAN, COL9A1, SPARC, COL11A1 and SOX9 and similar expression levels of HAPLN1, and reduced MFAP5 expression, as compared to adult primary chondrocytes;

Preferably, the amount of expression of COL2A1, ACAN, COL9A1, SPARC, COL11A1 and SOX9 proteins of the chondrocyte microstructure is increased by at least 2-fold compared to an adult primary chondrocyte;

Preferably, the amount of HAPLN1 expression of the chondrocyte microstructure is at least 80% of the amount of HAPLN1 expression of the adult primary chondrocyte;

Preferably, the MFAP5 expression level of the chondrocyte microstructure is at most 80% of the MFAP5 expression level of the adult primary chondrocyte;

preferably, the chondrocyte microstructure has a diameter in the range of 100 μm to 2000 μm;

preferably, the chondrocyte microtissue has the ability to develop into mature cartilage tissue in vivo;

preferably, the chondrocyte microtissue also expresses COLII and Lubricin proteins;

preferably, the chondrocyte survival rate in the chondrocyte microstructure is above 90%.

7. The chondrocyte micro-tissue according to claim 6, prepared by the method according to any one of claims 1-5.

8. A kit comprising the chondrocyte micro-tissue according to claim 6 or 7.

9. Cartilage tissue formed from the chondrocyte micro-tissue according to claim 6 or 7.

10. Use of a chondrocyte micro-tissue according to claim 6 or 7 in the manufacture of a medicament or a kit for the treatment of cartilage defects, cartilage degeneration, bone degeneration, osteoarthritis, and/or for cartilage regeneration, bone regeneration or cartilage transplantation.