CN118109397A

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

Info

Publication number: CN118109397A
Application number: CN202211522543.0A
Authority: CN
Inventors: 请求不公布姓名
Original assignee: Beisai Hongsheng Beijing Biotechnology Co ltd
Current assignee: Beisai Hongsheng Beijing Biotechnology Co ltd
Priority date: 2022-11-30
Filing date: 2022-11-30
Publication date: 2024-05-31
Also published as: WO2024114336A1

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 application of cartilage micro-tissue derived from human pluripotent stem cell differentiation

Technical Field

The invention relates to the field of cell culture, in particular to a preparation method and application of cartilage micro-tissues based on human pluripotent stem cell differentiation sources.

Background

Human pluripotent stem cells include human embryonic stem cells (embryonic stem cell, ESC) and human induced pluripotent stem cells (human induced pluripotent stem cell, iPSC, iPS cells), which are pluripotent stem cells with the ability to self-replicate, expand and multi-lineage direct differentiation by inducing cell fate changes through the introduction of specific transcription factors into adult cells. The technology avoids the problems of immune rejection and ethical issues in the research field of regenerative medicine, and is a great revolution in the field of life science. Like Embryonic Stem Cells (ESCs), iPS cells are capable of self-renewal and maintenance of an undifferentiated state, have the same cell morphology, karyotype, telomerase activity, in vitro differentiation potential as ESCs, and express surface marker molecules specific to pluripotent stem cells. The iPS cells have great application value in theoretical research, clinical application and other aspects, can directionally induce and differentiate various mature tissue cells in vitro, play an important role in treating various diseases such as blood system diseases, nervous system diseases, degenerative diseases and the like, and are regarded as important cell materials of future regenerative medicine.

Human pluripotent stem cells have been demonstrated to be capable of directed differentiation into a number of different tissue types including ectodermal, mesodermal and endodermal. Previous studies have shown that human pluripotent stem cells can successfully differentiate into chondrocytes in vitro with a directed differentiation.

At present, the method for preparing the chondrocyte microstructure mainly comprises (1) spontaneous aggregation culture formation of chondrocytes, (2) directional induced differentiation of mesenchymal stem cells (MESENCHYMAL STEM CELL, MSC), and (3) co-culture of MSC or chondrocyte combined biological materials. However, the preparation of chondrocyte microstructures using the above method has respective drawbacks and disadvantages: (1) The method for forming the chondrocyte micro-tissue by spontaneous aggregation of autologous chondrocytes represented by German Spherox products is exemplified, the chondrocytes are all derived from articular cartilage in an autologous non-load bearing region, the chondrocyte micro-tissue (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). which is enough for patients to use can be formed by long-time culture in vitro (5-10 weeks) after enzyme digestion of the chondrocyte, and in addition, the chondrocyte micro-tissue prepared by the method has limited curative effect due to individual difference and poor in-vitro culture proliferation capacity and dedifferentiation phenomenon of the chondrocyte; meanwhile, the secondary operation is easy to cause lesions of the donor area, and the acceptance of clinical patients is greatly reduced. (2) The directional induction and differentiation of MSC into chondrocyte micro-tissue are a multi-factor control process, the proportion of various components is required to be optimized, the induction and differentiation rate of the existing induction technology is relatively low, the time period is long, cartilage can be formed only by 14-28 days, and all MSC cannot be effectively differentiated into uniform and stable chondrocyte micro-tissue, so that the induced cartilage has low density, is not compact enough, has poor cartilage quality and is unfavorable for use in tissue engineering; in addition, since the MSC has large batch-to-batch variation, large-scale, stable and uniform preparation is difficult to achieve. (3) The cartilage cell micro-tissue prepared by the co-culture of MSC or cartilage cells and biological materials is limited by the sources of the seed cells, and the characteristics of the biological materials such as biocompatibility, in vivo degradation rate, residue and safety are accompanied.

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

Disclosure of Invention

The invention aims to develop a method for preparing stable and uniform chondrocyte micro-tissues with curative effect on diseases related to articular cartilage injury on a large scale by directionally differentiating chondrocytes from human pluripotent stem cells. The inventor completes the determination of the directional differentiation scheme of the human pluripotent stem cells to the chondrocytes, pharmacodynamics research, quality research and process research by taking the human iPS cells as the representative.

Compared with the traditional two-dimensional planar adherent differentiation culture chondrocyte, the three-dimensional culture chondrocyte microstructure can express higher cartilage cell function maturation related genes (such as COL2A1, ACAN, SOX9 and the like) and simultaneously secrete a large amount of natural hyaline cartilage extracellular matrixes such as type II collagen, aggrecan and the like. In the animal experimental study of in vivo repair of articular cartilage defects, it is also demonstrated that the three-dimensional cultured chondrocyte micro-tissue has a more powerful hyaline cartilage damage repair capacity. However, due to technical and cost reasons, previous studies have mostly been to culture isolated primary chondrocytes in vitro for a short period of time or to generate irregular micro-tissues on a small scale in a laboratory.

The invention mainly aims to directionally differentiate human pluripotent stem cells into cartilage cell micro-tissues and identify the cartilage cell micro-tissues, and prepare the cartilage cell micro-tissues with stability, uniformity and curative effect on a large scale, and induce mature cartilage cells to spontaneously aggregate in a micropore plate and self-assemble by optimizing culture conditions to generate the cartilage cell micro-tissues.

The inventor successfully develops a brand-new method for preparing the three-dimensional microstructure of the human chondrocyte on a large scale, and the method can remarkably improve the yield and quality of the microstructure of the chondrocyte after the induction of the human pluripotent stem cells. The three-dimensional chondrocyte microstructure induced to differentiate by iPS cells 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 fibrochondrocyte marker gene MFAP5 than the autologous chondrocyte products (2D autologous chondrocyte products represented by MACI, 3D autologous chondrocyte microstructure products represented by germany Spherox) currently on the market.

In one aspect, provided herein is a method of preparing a chondrocyte micro-tissue, 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 by standing, wherein the chondrocyte micro-tissue medium comprises DMEM high sugar 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

(D1) The individual spheres or spheroids in suspension are shake-cultured in chondrocyte microtissue medium until chondrocyte microtissue is obtained.

In one embodiment, the chondrocyte microstructure has a diameter in the range of 100 μm to 2000 μm. In one embodiment, the shaking culture is a shaking culture at 60rpm-80 rpm.

In one embodiment, step (A1) comprises:

(1) Culturing iPS cells in se:Sup>A medium that induces differentiation of iPS cells 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.

The "%" in the above steps is volume percent.

In one embodiment, the cartilage precursor cells are cultured in step (B1) for 2-5 days to aggregate to form spheres or spheroids. In one embodiment, the spheres or spheroids are shake-cultured in chondrocyte microtissue medium in step (D1) for 1-16 days. In one embodiment, the shaking culture is a shaking culture at 60rpm-80 rpm. In one embodiment, 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. The porous plate may be an EB-disk948.

In another aspect, the invention provides a method of preparing a chondrocyte microtissue 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 1-4 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 chondrocyte micro-tissue fusion is obtained, which comprises a plurality of adherent spheres or spheroids and has a diameter of 0.8mm-3mm, preferably shake-culture is at 60rpm-80 rpm.

The "%" in the above steps is volume percent.

In one embodiment, the cartilage precursor cells are cultured in step (B2) for 2-5 days 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:

In another aspect, the present invention provides a chondrocyte microstructure comprising chondrocytes and extracellular matrix proteins secreted therefrom, and having a higher expression level of extracellular matrix proteins COL2A1, ACAN, COL9A1, SPARC, COL11A1 and SOX9 and a similar expression level of HAPLN1, and reduced MFAP5 expression, as compared to an adult primary chondrocyte.

In one embodiment, 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.

In one embodiment, the amount of HAPLN1 expression by the chondrocyte microstructure is at least 80% of the amount of HAPLN1 expression by the adult primary chondrocyte.

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

in one embodiment, the chondrocyte microstructure has a diameter in the range of 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 also expresses COLII and Lubricin proteins.

In one embodiment, the chondrocyte viability in the chondrocyte microstructure is greater than 90%.

In one embodiment, the chondrocyte microtissue is prepared by the methods described herein.

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

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

In another aspect, the invention provides the use of a chondrocyte micro-tissue as described herein in the manufacture of a medicament or kit for the treatment of cartilage defects, cartilage degeneration, bone degeneration, osteoarthritis, and/or for cartilage regeneration, bone regeneration or cartilage grafting.

The beneficial effects of the invention include:

1. A3D culture method for preparing chondrocyte micro-tissue from human pluripotent stem cells is provided, which has extremely high differentiation efficiency and cell viability.

2. For the 3D culture methods herein, the inventors found optimal culture conditions, such as medium and culture time.

3. The inventors found that chondrocyte precursor cells derived from iPS cell differentiation reached stable optimal levels of chondrocyte markers (COL 2A1, ACAN, COL9A1, SPARC, COL11A1 and SOX9 expression levels, HAPLN1 expression levels, and MFAP5 expression) on days 8-16 (e.g., 9, 10, 11, 12, 13, 14, or 15 days) of induction into cartilage microspheres.

4. The present invention provides a method for mass production of chondrocyte micro-tissue 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-disk 948.

5. The chondrocyte micro-tissue has higher consistency and purity, and the tissue regenerated in vivo has the same mechanical biomechanical property as reported natural transparent cartilage of human, and the elastic modulus is about 27 MPa.

6. The chondrocyte micro-tissue described herein does not cause the body to produce immune rejection reactions, and has the potential to develop into osteochondral tissue in vivo.

7. The chondrocyte microstructure described herein has good filling and repairing effects on knee cartilage defects.

Drawings

Fig. 1 shows the morphology of P0 and P15 generation cartilage precursor cells (ipCDC) after iPS cell directed induction differentiation (left panel is P0, right panel is P15).

Fig. 2 shows the expression of the surface antigen of P10 generation ipCDC after the directional induction and differentiation of iPS cells. Red (left) and blue (right) represent isotype control antibody (Isotype antibody control) histograms and corresponding surface marker antibody histograms, respectively.

FIG. 3 shows HE staining results obtained after transplanting P15 generation ipCDC after iPS cell directed induction differentiation into NOD-SCID mice subcutaneously for 6 weeks.

Fig. 4 is a process of forming chondrocyte micro-tissue from which iPS cells are differentiated by a 3D induction method, and a graph is a morphological change of the chondrocyte micro-tissue; panel B shows the change in diameter of the chondrocyte microtissue.

Fig. 5 shows cartilage cell microstructures (from left to right, cartilage pellets of 1×10 ⁴/hole, 5×10 ^4/ hole, and 1×10 ^5/ hole, respectively, with diameters of 0.5mm, 0.8mm, and 1.0 mm) of different specifications from which iPS cells were differentiated were obtained by a 3D induction method.

FIG. 6 shows a 3D induction method for large-scale preparation of chondrocyte micro-tissue derived from iPS cell differentiation, ipCDC was inoculated into EB-Disk948 (middle left), induced cartilage micro-tissue was transferred into 10cm petri dishes (middle right) and shake cultured at 60rpm-80rpm (upper panel left is 2X10 ⁴ cells/microwell, upper panel right is 5X 10 ⁴ cells/microwell, lower panel left magnification is 40X, lower panel right is cell phone photograph).

FIG. 7A-B is a graph showing the cell viability assay of chondrocyte microtissue. Fig. 7 a shows the 3D induction method to obtain dead/living cell staining (3D 7D, 3D14D, CDC 3D7D, green for living cells and red for dead cells) of chondrocyte micro-tissue derived from iPS cell differentiation and chondrocyte micro-tissue derived from adult tissue. Panel B of FIG. 7 shows the results of flow cytometry for cell viability following digestion of chondrocyte microtissue (3D 20D) into single cells.

Fig. 8 shows the results of specific staining of chondrocyte microstructures derived from iPS cell differentiation obtained by 3D induction (3D 10D, left to right HE staining, safranin O fast green staining, toluidine blue staining, respectively).

Fig. 9 shows immunofluorescent staining of chondrocyte micro-tissues derived from iPS cell differentiation obtained by 3D induction method, which were induced for 5D, 10D, and 15D respectively.

Fig. 10A to 10C show differences in gene expression levels between chondrocyte microstructures derived from differentiation of human iPS cells and autologous articular chondrocytes and autologous chondrocyte microstructures obtained by 2D and 3D induction methods (CDC P0 is human primary articular chondrocytes, CDC P2 3D8D is cartilage microsphere formed by human P2-generation articular chondrocytes, EB948 3D8D is chondrocyte microstructure induced by EB948 disk method for 8 days). FIG. 10A shows the expression of cartilage cell micro-tissue genes induced and differentiated by the 2D method; FIG. 10B is a diagram showing the expression of cartilage cell micro-tissue genes formed by the 3D method induced differentiation; FIG. 10C is a diagram showing the expression of CRTAC1 and EBF3 genes in chondrocyte micro-tissues formed by the induction and differentiation of the 3D method.

FIG. 11 shows the results of general observation and pathological staining after 6 weeks of subcutaneous transplantation of chondrocyte micro-tissue derived from differentiation of iPS cells obtained by 3D induction method in NOD-SCID mice and C57BL/6 mice, (A) general observation, (B) pathological staining.

FIG. 12 shows the results of mechanical property tests performed on chondrocyte microstructures derived from human iPS cell differentiation obtained by the 3D induction method taken out after 8 weeks of subcutaneous transplantation in NOD-SCID mice.

Fig. 13 shows that cartilage cells derived from iPS cell differentiation obtained by the human 3D induction method are used for repairing cartilage defects in the load zone of the knee joint of rabbits through micro-tissue filling.

(A) In vitro Pre-test

And (3) transplanting: chondrocyte microtissue 3D8D, number of transplants: 20 to 30 damaged areas

(B) In vivo animal test

And (3) transplanting: chondrocyte microtissue 3D8D, number of transplants: 20 per lesion area

(C) Animal experiment results (yellow dotted line represents cartilage defect, and general observation, HE, safranine O solid green, alisxin blue, toluidine blue dyeing and Pinus masson dyeing are respectively carried out from top to bottom) after cartilage cell micro-tissue repair rabbit load area cartilage defect transplantation obtained by the 3D induction method is carried out for 1 month.

(D) And calculating the cartilage regeneration repair proportion of the cartilage cell microstructure obtained by the 3D induction method after the cartilage defect in the loading area of the rabbit is transplanted for 1 month.

Fig. 14 shows the microstructure morphology of chondrocyte in which iPS differentiation-derived chondrocyte precursor cells were directionally differentiated (2D induction method).

FIG. 15A shows the formation of larger-volume chondrocyte micro-tissue fusion by mutual adhesion of chondrocyte micro-tissues (3D 2D, 3D3D, 3D 4D) after concentrated shake culture at different induction times; FIG. 15B is a graph showing the process of adhesion fusion of chondrocyte microtissue of different diameters to each other; FIG. 15C is a partial enlarged view showing the adhesion fusion of cartilage cell micro-tissues (3D2d+3D3d) having a diameter of 2mm, and white arrows indicate the adhesion fusion of cells on the surfaces of adjacent cartilage cell micro-tissues.

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

Fig. 17 shows a schematic diagram of the differentiation process (2D induction method) of iPS differentiation-derived chondrocyte precursor cells into chondrocyte micro-tissues.

Fig. 18 shows a schematic diagram of the differentiation process (3D induction method) of iPS differentiation-derived chondrocyte precursor cells into chondrocyte micro-tissues.

Fig. 19 shows the results of single cell sequencing of chondrocyte micro-tissue (3D 8D) derived from directional differentiation of human iPS cells. FIG. 19A is a graph of a nonlinear dimension reduction tSNE cell Cluster Cluster analysis; FIG. 19B is a graph showing Cluster versus cell type correspondence; FIG. 19C is a graph showing cell numbers of different cell types; panel D of FIG. 19 shows cell type identification; panel E of FIG. 19 shows scoring values for 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 claim language to mean that the recited element is present, but other elements may be added and still form a construct or method within the scope of the recited claims.

As used herein, a "chondrocyte micro-tissue," also known as a cartilage micro-tissue, is formed from cartilage precursor cells by 3D culture. The chondrocyte microtissue may comprise chondrocytes, and optionally may comprise extracellular matrix proteins secreted by chondrocytes. Extracellular matrix proteins secreted by chondrocytes include, but are not limited to COL2A1(collagen type II alpha 1chain)、ACAN(aggrecan)、COL9A1(collagen type IX alpha 1chain)、SPARC(secreted protein acidic and cysteine rich)、COL11A1(collagen type XI alpha 1chain)、SOX9(SRY-box transcription factor 9)、HAPLN1(hyaluronan and proteoglycan link protein 1) and/or MFAP5 (Microfibril Associated Protein 5). The chondrocyte micro-tissue described herein has a higher expression level of COL2A1, ACAN, COL9A1, SPARC, COL11A1 and/or SOX9 than adult primary chondrocytes. The chondrocyte microtissue described herein may have similar amounts of HAPLN1 expression compared to adult primary chondrocytes. The chondrocyte microtissue 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 of the chondrocyte micro-tissue is increased by at least 2-fold, e.g. 3-20-fold, such as4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19-fold compared to adult primary chondrocytes. The HAPLN1 expression level of the chondrocyte microstructure may be at least 80%, 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% of the HAPLN1 expression level of the adult primary chondrocyte. The HAPLN1 expression level of the chondrocyte microstructure may be up to 80%, such as 20% -75%, such as 30%, 40%, 50%, 60% or 70% of the MFAP5 expression level of the adult primary chondrocyte. The chondrocyte microtissue described herein may also express COLII (Collagen II) and Lubricin (also known as PRG4, proteoglycan 4) proteins.

Chondrocyte microstructures can take the form of spheres or spheroids and can have a diameter in the range of 500-1200 um. The chondrocyte microstructure comprises a dense structure formed by the chondrocyte microstructure. The chondrocyte survival rate in the chondrocyte microstructure is 90% or more, 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 microstructure can also be a chondrocyte microstructure fusion body with the diameter of 0.8mm-3mm formed by mutually adhering a plurality of spheres or spheroids.

As used herein, "chondrocyte precursor cells" refers to chondrocytes induced by induction means such as those known in the art to induce induced pluripotent stem cells (iPS cells). Although there has been induction of iPS cells into chondrocytes in the prior art, this is not meant to be an admission that the preparation method of the cartilage precursor cells of the present invention is known or completely known. The chondrocyte precursor cells may be aggregated into chondrocyte micro-tissue by the methods herein. The cartilage precursor cells described herein can be positive for CD73, CD90, CD105 and CD44, and can be negative for CD11b, CD19, CD31, CD34, CD45 and HLADR.

Preparation method of cartilage precursor cells

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

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

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

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

In step (4), induction may be performed 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 hours, e.g., 21, 22, 23, 24, 25, 26, 27 hours. The concentration of WNT3a may be from 10 to 50ng/mL, for example, 20, 30 or 40ng/mL. The concentration of Activin A may be 10-50ng/mL, for example 20, 30 or 40ng/mL. The concentration of BMP4 may be 20-80ng/mL, e.g., 30, 40, 50, 60 or 70ng/mL.

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

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

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

In step (8), the cells may be cultured 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, e.g., 4 or 5 days. The concentration of FGF2 may be 10-50ng/mL, e.g., 20, 30, or 40ng/mL. The concentration of NT4 may be 2, 3, 4, 5, 6, 7, 8, 9ng/mL NT4. The concentration of TGF-beta 3 may be 10, 15, 20, 25, 30, 35 or 40ng/mL. The concentration of GDF5 may be 30, 40, 50, 60 or 70ng/mL.

Before step (1), iPS may be added to a Vitronectin (Nuwacell, RP 01002) -coated petri dish for 1-3 days, e.g., 24h or 2 days. The culture medium used for the culture was mTESR1 (Stem Cell Technologies, 85850). The medium may contain 1-5% of diabodies (penicillin and streptomycin). The medium may comprise 1-50um (e.g., 10, 20, 30, 40. Mu.M) Y27632 (Stem Cell Technologies, 72308). The cell concentration added may be 5000 cells per square centimeter or more, for example 10000 cells per square centimeter. In steps (1) - (8), the culture medium inducing iPS differentiation contains DMEM/F12,1-5% (e.g., 2%, 3%, 4%) diabodies, 0.5-10% (e.g., 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%) ITS-se:Sup>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%) nease:Sup>A, and 10-200 μm (e.g., 50, 90, 100, 150, 160, 170 μm) β -mercaptoethanol as se:Sup>A basal medium.

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

Preparation method of chondrocyte microstructure

The present invention provides a method of preparing chondrocyte microtissue comprising one or more of:

(A1) 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;

(B1) Stationary culturing cartilage precursor cells in a 3D cell culture manner in a cartilage precursor cell culture medium to aggregate to form 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 microtissue medium and culturing the spheres or spheroids by standing 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 chondrocyte microtissue 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, 10-100 ng- β 3, and 10-100ng/mL BMP2; and

(D1) The suspended spheres or spheroids are shake-cultured in a petri dish containing chondrocyte micro-tissue culture medium until chondrocyte micro-tissue is obtained.

The chondrocyte microstructure may have a diameter in the range of 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 the step (A1), cartilage precursor cells meeting quality inspection requirements of human iPS cell directional differentiation sources can be inoculated into 1T 175 culture flask, the inoculation density is 50000/cm ², and the culture flask is placed into a 37 ℃ and 5% CO ₂ incubator for culture. When the confluence of the cartilage precursor cells reaches 80% -90%, the cell morphology is round, oval, polygonal or short shuttle (figure 1), and the cell surface markers meet the standards of the cartilage 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 to form spheres or spheroids. The chondrocyte precursors are seeded in an amount of 1X 10 ⁴/well to 9X 10 ^5/, e.g.1X 10 ⁴/well, 5X 10 ^4/ or 1X 10 ^5/.

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

Herein, the steps (A1) - (D1) may be performed under the conditions of room temperature and CO ₂. The shaking culture may be a shaking culture at 60rpm-80rpm (e.g., 65, 70 or 75 rpm). Steps (B1) and (C1) may be 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. Preferably, the porous plate is a low adhesion or ultra low adhesion porous plate. The multi-well plate can realize 3D cell culture of cells.

Method for preparing cartilage cell micro-tissue fusion formed by mutual adhesion

Also provided herein is a method of preparing a chondrocyte microtissue fusion formed by mutual adhesion, comprising:

(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 and having a diameter of 0.8mm-3mm, preferably shake culture is at 60rpm-80 rpm.

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

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

Centrifugation may be performed after seeding the chondrocyte suspension in the chondrocyte precursor cell medium into the wells of the multiwell plate to settle the chondrocyte precursor cells to the bottom of the wells. The multi-well plate may be a 24-well plate, 96-well plate, 384-well plate, or 948-well plate. The porous plate may be an EB-disk948.

Medicaments or kits

The chondrocyte microtissue described herein may be prepared as a kit or medicament with other necessary ingredients, such as culture medium, and the like. The kit may further comprise an instrument or reagent for implantation, such as a buffer or the like. The chondrocyte microtissue described herein may be applied directly to the body in an amount or may be formed in vitro and then applied to the subject. The subject may be a subject in need of cartilage transplantation, such as a mammal, particularly a human. The subject may have cartilage defects, cartilage degeneration, bone degeneration, osteoarthritis, and/or require cartilage regeneration, bone regeneration, or cartilage grafting.

Examples

Embodiments of the present application will be described in detail below with reference to examples, which will be understood by those skilled in the art, for illustrating the present application only and should not be construed as limiting the scope of the present application. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention. The application should not be construed as being limited to the particular embodiments described.

Example 1: preparation of chondrocyte microstructure from cartilage precursor cells derived from human iPS cell differentiation

Materials and methods

Materials:

Human iPS cells: saibei, CA4025106

GMP-grade cell digest TryplE: thermo Fisher,12563029.

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

Chondrocyte micro-tissue culture medium: DMEM high sugar 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 100 μg/mL streptomycin (gbicol, 10378016), 0.1mM sodium ascorbate (MCE, HY-107837), 0.1uM dexamethasone (sigma, D4902), 40 μg/mL proline (sigma, P5607), 20ng/mL TGF- β3 (Stemimmune, HST-TB 3-1000), 20ng/mL BMP2 (peprotech, 120-02C).

The method comprises the following steps:

directional differentiation of human iPS cells into cartilage precursor cells

Reference is made to the differentiation method of Griffith L A,Arnold K M,Sengers B G,et al.Ascaffold-free approach to cartilage tissue generation using human embryonic stem cells.[J].Nature Publishing Group,2021(1). Firstly, adding the iPS into a culture dish coated with the Vitronectin at a cell concentration of 10000 cells per square centimeter for culturing for 24 hours, wherein the culture medium is mTESR < 1 >, 1% of double antibody and 10 mu M Y27632; all medise:Sup>A inducing iPS differentiation then contained DMEM/F12,1% diabody, 1% ITS-A, 2% B27,1% NEAA, and 90. Mu.M. Betse:Sup>A. -mercatoethanol as basal medium 1. After the iPS was cultured for 24 hours, the original medium was discarded, and the culture was continued for 24 hours by adding the first induction medium containing basic medium 1, 25ng/mL WNT3a and 50ng/ML ACTIVIN A. The first induction medium was discarded and the culture was continued for 24 hours with the addition of a second induction medium containing basal medium 1, 25ng/mL WNT3a, 25ng/ML ACTIVIN A and 20ng/mL FGF 2. Discarding the second induction culture medium, adding a third induction culture medium containing WNT3a of 25ng/mL, WNT3a of 10ng/ML ACTIVIN A, FGF2 of 20ng/mL and BMP4 of 40ng/mL, and continuously culturing for 24h; discarding the third induction medium, washing the cells once by PBS, adding 2mL of TrypLE ^TM Select cells for 3min at 37 ℃, centrifuging at 1200rpm for 5min after the cells are collected in a 50mL centrifuge tube, adding a fourth induction medium resuspended cells containing basic medium 1, 20ng/mL FGF2, 40ng/mL BMP4, 2ng/mL NT4 and 100ng/mL Follistatin, and carrying out passage according to the proportion of 1:2, and continuously culturing for 4 days; discarding the fourth induction medium, adding a fifth induction medium containing basic medium 1, 20ng/mL FGF2, 40ng/mL BMP4 and 2ng/mL NT4, and continuously culturing for 24h; discarding the fifth induction medium, washing the cells once by PBS, adding 2mL of TrypLE ^TM Select cells for 3min at 37 ℃, centrifuging at 1200rpm for 5min after the cells are collected in a 50mL centrifuge tube, adding a sixth induction medium containing basic medium 1, 20ng/mL FGF2, 20ng/mL BMP4, 20ng/mL GDF5, 2ng/mL NT4 and 10ng/mL TGFbeta 3, and carrying out passage according to the proportion of 1:2, and continuously culturing for 2 days; the sixth induction medium is discarded, and a seventh induction medium containing basic medium 1, 20ng/mL FGF2, 40ng/mL GDF5, 2ng/mL NT4 and 10ng/mL TGF beta 3 is added for continuous culture for 4 days, so that the cartilage precursor cells can be obtained. During this induced differentiation, a new seventh induction medium needs to be changed daily. The above culture was carried out at 37℃in a 5% CO ₂ incubator. The chondrocyte precursors may then be passaged in a human iPS cell differentiation derived chondrocyte precursor cell culture medium at a ratio of 1:2-1:3 for at least 15 more passages (fig. 16).

Cell morphometry

After the chondrocyte precursor cells derived from the differentiation of iPS cells were inoculated into a culture dish containing a culture medium for chondrocyte precursor cells derived from the differentiation of human iPS cells, the cell morphology change was observed daily by an inverted microscope (Zeiss, primovert), and recorded by photographing. The chondrocyte precursor cells derived from iPS cell differentiation were small in volume and were round, oval, polygonal or short-shuttle in shape (fig. 1).

Flow cytometer for detecting cell surface markers

After 4 days of incubation in seventh induction medium, the original medium was discarded, the cells were washed once with PBS, 2ml TrypLE ^TM Select 37℃were added to the petri dish to digest the cells for 3min, the cells were collected into a 50ml centrifuge tube, centrifuged at 1200rpm for 5min, and the cells were resuspended in PBS. 20ul of the cell suspension was counted using a cell counter (Countstar, IC 1000) and the cell density was adjusted to 1X 10 ⁷/ml. The 14 tributary tubes were taken, 0.1ml of the cell suspension after concentration adjustment was added to each tube, and then the following corresponding antibodies ：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,11-0349-42)、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),4℃ were added to each tube according to the antibody instructions and incubated for 30min in the dark. 2ml PBS was added, the cells were gently mixed, centrifuged at 400g for 5 minutes, and the supernatant was removed. 2ml PBS was added, gently mixed, centrifuged at 400g for 5 minutes, and the supernatant was removed. 0.5ml PBS was added, the cells were gently mixed, and cell surface markers were detected using Lyric flow cytometer. The target cell population was collected by circling blank control tube cells on the FS/SS scatter plot, and fluorescence threshold was adjusted with FITC/PE isotype control antibody tube cells to define the positive cell range. Fluorescence signals of the sample tubes were then collected, 10000 single cells were collected per tube, and data were recorded for storage. The cartilage precursor cell surface markers should be above 95% positive for CD73, CD90, CD105, CD44 and below 2.0% positive for CD11b, CD19, CD31, CD34, CD45, HLADR (fig. 2). HE staining of NOD-SCID mice transplanted with cartilage precursor cells and new tissue

To investigate the ability of human iPS committed differentiation-derived chondrocyte precursor cells to differentiate into cartilage tissue in vivo, we transplanted iPS cell differentiation-derived P15 generation chondrocyte precursor cells into thigh inner muscle of NOD-SCID immunodeficient mice (8-10 weeks). Cartilage precursor cells were first collected, resuspended using matrigel (Corning, 354277), the cell suspension was withdrawn with a 1ml syringe and transferred onto the animal house on ice. 1X 10 ⁷ cartilage precursor cells were injected into the medial thigh muscle of 8-10 week old NOD-SCID immunodeficient mice using a syringe. The injection site was observed to be convex, the mice were euthanized after 6 weeks, the neotissues on the recipient mice were dissected and the neotissue mass was fixed overnight at 4℃with 4% paraformaldehyde, dehydrated, paraffin embedded, sectioned at 5 μm and specially stained using Hematoxylin Eosin (HE) staining kit (Solebao, G1120).

Results: after transplanting P15 generation cartilage precursor cells from iPS directed differentiation into NOD-SCID immunodeficient mice for 6 weeks, HE staining results revealed that the new tissue was cartilage-like tissue composed of a large number of "paving stone" like chondrocytes and extracellular matrix (FIG. 3). This suggests that chondrocyte precursor cells from which iPS cells are directionally differentiated have the potential to differentiate into cartilage tissue in vivo.

Preparation of cartilage cell micro-tissue by 2D induction method

Firstly, inoculating cartilage precursor cells which meet the quality inspection requirement and are derived from directional differentiation of human iPS cells into 1T 175 culture flasks, and culturing the cells in a 5% CO ₂ incubator at 37 ℃ at the inoculation density of 50000/cm ². After the confluence of the cartilage precursor cells reaches 80% -90%, the cell morphology is round, oval, polygonal or short shuttle (figure 1), the cell surface markers meet the standards of the cartilage precursor cell surface markers (figure 2), 5mL of GMP (good manufacturing practice) level cell digestive juice TryplE is added into a culture flask, and the digestive juice is evenly distributed by slightly rotating horizontally, and is placed in a culture box at the temperature of 37 ℃ for digestion for 5min. The flask was lifted and visually inspected under a lamp to see the cells detached and visually inspected under a mirror to see if the cells were digested into bright single cells (e.g., the digestion process was slow, the flask side wall could be gently tapped, and the cell detachment was accelerated by shaking). After the cells were completely shed, 5mL of a cartilage precursor cell culture medium derived from differentiation of human iPS cells was immediately added, the digestion was stopped by spin mixing, 10mL of PBS was added to the flask to wash the cells, and then the cells were transferred to a 50mL centrifuge tube. 10mL of PBS was added again, the remaining cells were collected by washing, placed in a centrifuge tube, trimmed, centrifuged at 1200rpm for 5min, and the supernatant was gently removed. 50mL of PBS was added to resuspend the cells, again centrifuged at 1200rpm for 5min, and the supernatant was carefully aspirated away. Cell pellet was resuspended using 20mL of chondrocyte precursor cell medium, 20uL of cell suspension was aspirated and counted in Countstar counting plate, and cell number was calculated. The cell concentration was adjusted to 1.66×10 ⁵/mL using cartilage precursor cell culture medium, inoculated into TC-96 well plates (Corning, 3599), 100. Mu.L of cell suspension was added per well, i.e., 1.66×10 ⁴ cells per well, and cultured in a 5% CO ₂ incubator at 37 ℃. After 3 days of incubation, the medium in the wells was discarded, the cells were washed 1 time by slowly adding 100. Mu.L of 37℃pre-warmed PBS, PBS was discarded, and then differentiation to chondrocytes was initiated by slowly adding 100. Mu.L of 37℃pre-warmed chondrocyte microtexture medium to the well plate, which was designated 2D0D. On day 7 of differentiation into chondrocytes (2D 7D), chondrocyte microtissue was transferred to a 10cm dish, placed on a shaking table, and continuously cultured at a rotation speed of 60-80rpm until day 22 (2D 22D), with liquid changes every 2-3 days (FIG. 17). Results:

As shown in fig. 14, the cartilage precursor cells derived from the differentiation of the human iPS cells began to adhere to the wall and spread and extended gradually after being inoculated into a TC96 well plate for 4 hours. After 3 days of continuous culture, the cell confluence reaches about 85% -90%, and the cell morphology is round, oval, polygonal or short shuttle. At this time, the micro-tissue culture medium of the chondrocytes is switched to start differentiation into the chondrocytes, and the differentiation is marked as 2D0D, and the cell morphology is round; when induced to differentiate for 2D5D, the cell density is significantly increased and the cell assumes a multi-layered cell state, and the cells aggregate to form a plurality of cartilage nodules and spontaneously roll up from the edge of the well plate toward the center; when induced differentiation culture is carried out for 2D7D, cells gradually aggregate into irregular micro-tissues and fall off from the bottom of an orifice plate, at the moment, the cartilage cell micro-tissues are transferred into a 15cm culture dish, a cartilage cell micro-tissue culture medium is added, and the culture is carried out on the cartilage cell micro-tissues on a horizontal shaking table at 60rpm-80 rpm; and continuously observing the cell morphology and the color change of a culture medium, the internal cell state of the chondrocyte micro-tissues and the adhesion fusion condition among the chondrocyte micro-tissues in the process of inducing and differentiating the chondrocyte precursor cells derived from the human iPS cells into the chondrocyte micro-tissues in the culture process until the 2D12-2D15D chondrocyte micro-tissues are in milky color, smooth and bright in surface and compact in texture, and continuously culturing until the 2D 22D.

Preparation of cartilage cell micro-tissue by 3D induction method

Firstly, inoculating cartilage precursor cells which meet the quality inspection requirement and are derived from directional differentiation of human iPS cells into 1T 175 culture flasks, and culturing the cells in a 5% CO ₂ incubator at 37 ℃ at the inoculation density of 50000/cm ². After the confluence of the cartilage precursor cells reaches 80% -90%, the cell morphology is round, oval, polygonal or short shuttle (figure 1), the cell surface markers meet the standards of the cartilage precursor cell surface markers (figure 2), 5mL of GMP (good manufacturing practice) level cell digestive juice TryplE is added into a culture flask, and the digestive juice is evenly distributed by slightly rotating horizontally, and is placed in a culture box at the temperature of 37 ℃ for digestion for 5min. The flask was lifted and visually inspected under a lamp to see the cells detached and visually inspected under a mirror to see if the cells were digested into bright single cells (e.g., the digestion process was slow, the flask side wall could be gently tapped, and the cell detachment was accelerated by shaking). After the cells were completely shed, 5mL of a cartilage precursor cell culture medium derived from differentiation of human iPS cells was immediately added, the digestion was stopped by spin mixing, 10mL of PBS was added to the flask to wash the cells, and then the cells were transferred to a 50mL centrifuge tube. 10mL of PBS was added again, the remaining cells were collected by washing, placed in a centrifuge tube, trimmed, centrifuged at 1200rpm for 5min, and the supernatant was gently removed. 50mL of PBS was added to resuspend the cells, again centrifuged at 1200rpm for 5min, and the supernatant was carefully aspirated away. Cell pellet was resuspended using 20mL of chondrocyte precursor cell medium, 20uL of cell suspension was aspirated and counted in Countstar counting plate, and cell number was calculated. Cell concentration was adjusted to 5×10 ⁵/mL using cartilage precursor cell culture medium, inoculated into ULA-96 well plates (Corning, 7007), 100uL of cell suspension was added per well, i.e. 5×10 ⁴ cells per well (different cell inoculum sizes were also set to induce formation of chondrocyte microtissue experiments, i.e. 1×10 ⁴ cells per well and 1×10 ⁵ cells per well) and incubated in a 5% CO ₂ incubator at 37 ℃. After 24 hours of inoculation culture, the cartilage precursor cells from the differentiation of the human iPS cells begin to aggregate and gradually take on irregular spheres. Changing liquid on the 2 nd day, on the 3 rd day of culture, discarding old culture medium in the hole, slowly adding 100uL of PBS preheated at 37 ℃ to wash 1 time of cells, discarding PBS, then slowly adding 100uL of cartilage cell micro-tissue culture medium preheated at 37 ℃ into the hole plate to start differentiation to cartilage cells, namely 3D0D, on the 3 rd D7D of cartilage differentiation, transferring cartilage cell micro-tissue into a 15cm culture dish, placing the culture dish on a shaking table, setting the rotating speed to 60-80rpm, continuously culturing to 3D22D, and changing liquid once every 2-3 days. Fig. 18 shows a schematic diagram of the differentiation process (3D induction method) of iPS differentiation-derived chondrocyte precursor cells into chondrocyte micro-tissues.

Results

As shown in the A diagram of FIG. 4, after the cartilage precursor cells are inoculated into a ULA-96 well plate for 2 hours, the edges of the cell clusters gradually and spontaneously gather, the cells gather to form regular spheres after 1 day, the spheres become milky white after 2-3 days of culture, the surface is smooth and bright, the texture is compact, and the cartilage precursor cells are switched into a cartilage cell micro-tissue culture medium. Between the following 3D0D-3D6D, the cells formed dense chondrocyte micro-tissue, gradually decreasing in volume, then remaining substantially unchanged in volume, approximately 700um in diameter, in the form of milky regular spheres, bright in luster, smooth in surface, and having a certain hardness (panel B of fig. 4). At 3D7D, the chondrocyte micro-tissue is transferred to a15 cm culture dish, a chondrocyte micro-tissue culture medium is added, and the culture is carried out on the chondrocyte micro-tissue culture medium on a horizontal shaking table at 60rpm-80rpm until the culture is carried out to 3D22D, and the color change of the culture medium, the internal cell state of the chondrocyte micro-tissue and the adhesion and fusion condition among the chondrocyte micro-tissue are continuously observed.

We have also found that different amounts of chondrocyte precursor cell seeding can produce different sizes of chondrocyte microtissue. As shown in FIG. 5, 1X 10 ⁴ cells, 5X 10 ⁴ cells, and 1X 10 ⁵ cells were inoculated per well to form chondrocyte micro-tissues having diameters of about 0.5mm, 0.8mm, and 1.0mm, respectively.

Typically 1T 175 flask can harvest 3 x 10 ⁷ cells, can be used in 6 ULA-96 well plates, and finally obtain 6 x 96 = 576 chondrocyte micro-tissues, the required amount of each patient is calculated according to 40-70 pellets, and the cell amount of 1T 175 flask can be used for 8-14 patients clinically. 300 ULA-96 well plates can be accommodated per cell culture incubator, and 96×300=28800 chondrocyte micro-tissues can be produced per cell culture incubator for 411-720 clinical patients.

A plurality of cartilage cell micro-tissues are adhered and fused with each other to form a cartilage cell micro-tissue fusion body

As shown in panel A of FIG. 15, chondrocyte micro-tissues directionally differentiated by iPS cells with different induction times were transferred to BeaverNano ^TM well suspension cell culture plates (beaver organisms, 40406), 8 chondrocyte micro-tissues were placed in each well, 4mL of chondrocyte micro-tissue culture medium was added, and the culture was performed on a horizontal shaker, and the rotational speed was set at 60rpm-80rpm, so that all cartilage pellets were concentrated in the center of the well plate and denoted as 3DXD+3DYd.

Results: after a plurality of chondrocyte micro-tissues (3D 2D, 3D3D and 3D 4D) are respectively transferred and concentrated together and subjected to shake culture (8 days total of 3D induction culture), contact adhesion can occur among the chondrocyte micro-tissues, and the chondrocyte micro-tissues are gradually fused into a larger sphere (chondrocyte micro-tissue fusion body).

As shown in B of fig. 15 and C of fig. 15, chondrocyte micro-tissues (3D 2D) of different diameter sizes of iPS cells were transferred to BeaverNano ^TM well suspension cell culture plates (castoreum organism, 40406), 8 chondrocyte micro-tissues were placed into each well, 4mL of chondrocyte micro-tissue culture medium was added, and placed on a horizontal shaker for culture, and the rotation speed was set at 60rpm-80rpm, so that all of the chondrocyte pellets were concentrated in the center of the well plate, which was denoted as 3d2d+3d0d at this time. When the 3D2d+3D3d is cultured, the chondrocyte micro-tissues start to adhere to each other, and cells on the surfaces of adjacent chondrocyte micro-tissues are adhered and fused; when the 3D2d+3D5d is cultured, the cell adhesion fusion degree of the edge of the chondrocyte micro-tissue is higher, and the single chondrocyte micro-tissue is difficult to distinguish; when the 3D2d+3D6d is cultured, a plurality of cartilage cell micro-tissues are closely connected together to form a milk white cartilage tissue with larger volume, and the milk white cartilage tissue has bright luster, smooth surface and certain hardness. The micro-tissue diameters of the chondrocytes are increased from 0.45mm, 0.75mm, 1.2mm to 1.0mm (about 2.2 times), 1.8mm (about 2.4 times) and 2.6mm (2.2 times), respectively. On the basis, the diameter, the number or the shape of a mould of single chondrocyte micro-tissues can be adjusted to obtain chondrocyte micro-tissue fusion bodies with different shapes and sizes, so that larger chondrocyte micro-tissues formed by mutually adhering a plurality of chondrocyte micro-tissues are more easily adhered and fixed on cartilage defect parts than single chondrocyte microspheres during transplantation, are not easy to fall off, and improve the operation treatment effect; simultaneously can meet the clinical demands of patients with different cartilage defect areas.

Example 2: large-scale preparation of cartilage micro-tissue

Although the method of forming cartilage micro-tissue based on 96-well plate in example 1 above can prepare the number of cartilage cell micro-tissue that can meet the clinical requirement of early stage on in large scale, this method requires relatively much labor and time costs and is not suitable for industrial production of larger scale in later stage. To further solve the above problems, we have further improved the process and found a method for preparing cartilage micro-structures which has a larger production scale and is easier to handle. The specific description is as follows:

When the confluence of the cartilage precursor cells reaches 80-90%, adding clinical-grade digestive juice TryplE into the bottle to fill the bottle bottom, and putting the bottle into a 37 ℃ incubator to digest for 3-5 min. The digestion was stopped by adding pre-heated chondrocyte precursor cell culture medium, the blow was homogenized, the cells were collected in a 50mL centrifuge tube, centrifuged at 1200rpm for 5min, and the supernatant was discarded. The cell concentration was adjusted to 2.37X10 ⁸/mL using a pre-warmed cartilage precursor cell culture medium, gently blown 3 times using a 1mL pipette tip, and the cells were mixed. 2mL of the above cell suspension was gently added to each EB-disk948 (diameter: about 6 cm) to cover the surface of the EB-disk948 with the cell suspension. The mixture was allowed to stand for 2 minutes to uniformly distribute the cells on the surface of EB-Disk 948. Then 300g centrifugal 1 minutes, make the cell sedimentation to each microporous bottom. Returning to the biosafety cabinet, 6mL of cartilage precursor cell culture medium was gently added from the side wall of EB-Disk948 to avoid damaging the seeded cells. EB-Disk948 was incubated at 37℃in a 5% CO ₂ incubator. After 3 days, old medium was gently discarded, and the addition of chondrocyte microtexture medium to EB-Disk948 began to induce differentiation into chondrocyte microtexture (designated 3D 0D). After 7 days (3D 7D), the formed chondrocyte micro-tissue is transferred to a 10cm culture dish (or a culture bag or a bioreactor), and the culture is continued for 15 days (3D 22D) by a shaking table with the speed of 60rpm-80rpm, and liquid is changed every 2-3 days. During 3D0D to 3D22D, chondrocyte micro-tissues were collected every 2 days, total RNA was isolated from iPS cell differentiation-derived chondrocyte micro-tissues (EB 948 3D 8D) using RNEASY MINI KIT (Qiagen, 74004) according to the product instructions, and on-column DNase I cleavage was performed. 1 μg of total RNA was used as template, cDNA was synthesized using ISCRIPT CDNA SYNTHESIS KIT (BioRad, 1708891), and stored at-20℃for further use. After induction of chondrogenic micro-tissue, 948 chondrogenic micro-tissue can be prepared per EB-Disk948, with the demand for each patient calculated as 40-70 pellets for 13-23 patients (fig. 6). 300 EB-Disk948 can be accommodated per incubator, and 300×948= 284400 chondrocyte microstructures can be produced per incubator, and can be used for 4062-7110 clinical patients. Therefore, the method can realize large-scale preparation and production of stable and uniform chondrocyte micro-tissues, and meets the requirements of clinical patients.

Example 3: dead living cell staining and cell viability detection by flow cytometry are carried out on chondrocyte micro-tissues derived from directional differentiation of human iPS cells

A cell death and alive staining kit (live/dead cell viability assay kit, abcam, ab287858, lot: GR 3451473-4) provides a two-color fluorescence method based on the simultaneous determination of living and dead cells using two different dyes. The living cell dye readily penetrates the cell membrane intact, and the living cell and cytolactonase hydrolyzes the dye to produce a hydrophilic, strongly fluorescent compound that remains in the cytoplasm of the cell, as measured at Ex/em=485/530 nm. Dead cell dyes enter the damaged cell membrane, and after binding to nucleic acids, fluorescence is enhanced 40-fold, producing bright red fluorescence in dead cells (Ex/em=495/635 nm).

Chondrocyte micro-tissues (3D 7D, 3D 14D) formed by chondrocyte precursor cells from which iPS cells are differentiated on the 7 th day and 14 th day of induced differentiation and chondrocyte micro-tissues (CDC 3D 7D) from which adult tissues are derived are taken out in a TC 96-well plate, 200ul of PBS is added to wash the chondrocyte micro-tissues for 2 times, PBS is removed, 200ul of assay buffer containing dead living cell staining solution is added to soak the chondrocyte micro-tissues, only assay buffer containing no dead living cell staining solution is added to a control group, incubation is carried out for 15min at 37 ℃, and then a fluorescence microscope (Kirschner, BZ-X800) is used for image observation and acquisition.

Taking 12 chondrocyte micro-tissues (3D 20D) formed by chondrocyte precursor cells from iPS cell differentiation in a 15ml centrifuge tube after induced differentiation for 20 days, washing the chondrocyte micro-tissues for 2 times by using PBS, discarding the PBS, adding 2ml of 0.2% type II collagenase into the tube, digesting for 60min at 37 ℃, and adding 2ml of chondrocyte micro-tissue culture medium to terminate the reaction, thus obtaining the chondrocyte micro-tissue single-cell suspension. Mu.l of the cell suspension was counted, resuspended in PBS and then dispensed into 4 1.5ml centrifuge tubes containing 1X 10 ⁶ cells per tube, centrifuged at 500g for 5min, the supernatant removed, 1ml of assay buffer containing dead living cell stain (see Table below) added to the tube and incubated at 37℃for 15min in the absence of light. Cells were washed once with PBS, the supernatant was discarded, and finally resuspended in 0.5ml assay buffer and cell viability was measured using Lyric flow cytometry. FITC channel indicates living cells and APC channel indicates dead cells.

Table 1: flow cytometry assay configuration for each group

The staining results showed that the chondrocyte microtissue induced differentiation contained a large number of cells in the inside, had a good cell state, was round or oval, had very few dead cells, and had a morphology of cells in the chondrocyte microtissue derived from adult tissue (CDC 3D 7D) that was in the form of shuttle-formed fibers, had a non-uniform internal cell distribution, and had many dead cells (fig. 7 a). Further, the cell viability was measured by a flow cytometer according to the product instructions after the chondrocyte microstructure was digested into single cells, and the result showed that the cell viability inside the chondrocyte microstructure derived from the directional differentiation of iPS cells was 99.7% (fig. 7B).

Example 4: staining of human iPS directional differentiation-derived chondrocyte micro-tissue pathological sections

On days 5, 10 and 15 of in vitro induced differentiation, cartilage cell micro-tissues were fixed with 4% Paraformaldehyde (PFA), dehydrated, paraffin-embedded and sectioned at 5 μm, and special staining was performed using Hematoxylin Eosin (HE) staining kit (soribao, G1120), toluidine blue staining kit (soribao, G3668) and modified safranin O-fast green staining kit (soribao, G1371), respectively. For immunofluorescence staining, white flakes were sequentially immersed in xylene for 3 times for 10min, 100% ethanol for 5min for 2 times, 95% ethanol for 5min,85% ethanol for 5min,75% ethanol for 5min, and 50% ethanol for 5min, and then rinsed with pure water. Washing with PBS 3 times on a shaker for 5min each time; penetrating with 0.4% Triton X-100 at room temperature for 1 hr; washing with PBS 3 times on a shaker for 5min each time; 5% donkey serum was added dropwise to the wet box at room temperature for 30min, and the non-specific binding sites were blocked. Excess donkey serum was knocked off, a primary antibody ：COLII(abcam,ab185430)、ACAN(abnova,PAB27837)、Lubricin(abcam,ab28484)、SOX9(santa cruz biotech,sc-166505), diluted with 5% donkey serum was added dropwise to the wet box, and the wet box was placed at 4 ℃ overnight. Washing with PBS 3 times for 10min each time; dripping the secondary antibody marked by fluorescein in a wet box in a dark environment, and reacting for 70 minutes at room temperature; washing with PBST on a shaker for 3 times and 5min each time, and cleaning with pure water; DAPI (Thermo filter, 62247) was added dropwise to the wet box and reacted at room temperature for 10min; washing with PBST on a shaker for 3 times and 5min each time, and cleaning with pure water; the images were observed and collected using an anti-fluorescence decay capper and a fluorescence microscope (kenshi, BZ-X800).

Toluidine blue (Toluidine Blue O) dye liquor is a basic dye which can be combined with cations in cells, and the cell nucleus is dyed into purple or deep blue, and the cytoplasm is dyed into light blue. The staining principle of the modified Safranin O-fast green (Safranin O/FAST GREEN) cartilage staining method is that basophilic cartilage combined with the basic dye Safranin O appears red. Safranin O is a polyanionic-binding cationic dye showing that cartilage tissue is based on the binding of cationic dyes to anionic groups (chondroitin sulfate or keratan sulfate) in polysaccharides. Safranin O staining is approximately proportional to the concentration of anions, indirectly reflecting the proteoglycan content and distribution in the matrix. The special staining result shows that after the chondrocyte precursor cells from the differentiation of the iPS cells induce differentiation to the chondrocyte micro-tissues for 10 days, the chondrocyte micro-tissues contain a large number of cells, and the periphery of the cells is wrapped by the rich type II collagen and proteoglycan (figure 8).

The Lubricin (also called PRG 4) gene encodes a protein which is a large proteoglycan synthesized by chondrocytes and some synoviocytes located on the articular cartilage surface. The protein contains chondroitin sulfate and keratin sulfate glycosaminoglycan. It acts as a boundary lubricant on the cartilage surface, helping the elastic absorption and energy dissipation of synovial fluid. Immunofluorescent staining results showed that chondrocyte microtissue expressed COLII, ACAN, lubricin and SOX9 on days 5, 10 and 15 of induced differentiation and gradually increased with prolonged induction time (fig. 9).

Example 5: chondrocyte micro-tissue single cell sequencing of human iPS cell directional differentiation source

Taking a proper amount of cartilage cell micro-tissue (3D 8D) formed by chondrocyte precursor cells from iPS cell differentiation in a 15ml centrifuge tube after induced differentiation for 8 days, washing the cartilage cell micro-tissue with PBS for 2 times, discarding the PBS, adding 2ml of 0.2% type II collagenase into the tube, digesting for 60min at 37 ℃, and adding 2ml of cartilage cell micro-tissue culture medium to terminate the reaction, thus obtaining the cartilage cell micro-tissue single-cell suspension. The commission scientific service detection agency (beijing poda gene technologies limited) performed 10 x single cell transcriptome sequencing.

The 10×single cell transcriptome sequencing result shows that the most cell type inside the chondrocyte microstructure (3D 8D) formed by the chondrocyte precursor cells from iPS cell differentiation within 8 days of induced differentiation is chondrocyte (Chondrocytes), the other few cells are only Mesenchymal Stem Cells (MSCs), no iPS cell residue is present, and the chondrocyte purity is 95.22% (a-E of fig. 19).

In this method, the differentiation efficiency of the human iPS cell differentiation-derived chondrocyte precursor cells into mature chondrocytes was extremely high (fig. 8, 9, and 19 a-E).

Example 6: chondrocyte micro-tissue gene expression condition of human iPS cell directional differentiation source

Material

CDC P0 was P0 generation human articular chondrocytes (scientific, # 4650).

Human articular chondrocyte medium is DMEM high sugar (gbicol, 12430062), 10% FBS (gbicol, 10099141), 20ng/mL TGFb3 (Stemimmune, HST-TB 3-1000), 100U/mL penicillin 100 μg/mL streptomycin (gbicol, 10378016).

CDC P2 3D8D is a chondrocyte microstructure formed by 3D culture of human P2-generation articular chondrocytes for 8 days. The preparation method comprises the following steps: taking out the P0 generation human articular chondrocyte from liquid nitrogen, immediately placing the P0 generation human articular chondrocyte into a water bath kettle at 37 ℃ for thawing, adding 1ml of thawed P0 generation human articular chondrocyte into 9ml of precooled alpha-MEM (Hyclone, SH 30265.01), and centrifuging at 1200rpm for 5min; the supernatant was removed, and cells were resuspended using human articular chondrocyte medium, inoculated into T75 flasks at a density of 10000/cm ², and incubated at 37℃in a 5% CO2 incubator for 3-5 days. When the cell confluence reaches 80% -90%, 3mL GMP grade cell digestive juice TryplE is added into the culture flask, the digestive juice is evenly distributed by gentle horizontal rotation, and the culture flask is placed horizontally for digestion for 5min at 37 ℃. The flask was lifted and visually inspected under a lamp to see the cells detached and visually inspected under a mirror to see if the cells were digested into bright single cells (e.g., the digestion process was slow, the flask side wall could be gently tapped, and the cell detachment was accelerated by shaking). After all cells were shed, 3mL of human articular chondrocyte medium was immediately added, the mixing was stopped by spinning, the cells were collected to a 15mL centrifuge tube, the flask was then washed with 8mL PBS, and the cells were collected to a 15mL centrifuge tube. Centrifuging at 1200rpm for 5min, and gently removing supernatant to obtain P1 generation human articular cartilage cells. P2-generation human articular chondrocytes were obtained in the same passaging method, the cell concentration was adjusted to 5X 10 ⁵/mL using human chondrocyte medium, inoculated into ULA-96 well plates (Corning, 7007), 100uL of cell suspension was added per well, i.e., 5X 10 ⁴ cells per well, and placed in a 5% CO ₂ incubator at 37℃for culturing. After 24 hours of culture, the human articular chondrocytes begin to gather and gradually form an irregular sphere, and continue to culture for 8 days, and the liquid is changed every 2-3 days. On day 8 of human articular chondrocyte microtissue culture, total RNA was isolated from human articular chondrocyte microtissue (CDC P2 3D 8D) according to the product instructions using RNEASY MINI KIT (Qiagen, 74004) and subjected to on-column DNase I cleavage. 1 μg of total RNA was used as template, cDNA was synthesized using ISCRIPT CDNA SYNTHESIS KIT (BioRad, 1708891), and stored at-20℃for further use.

EB948 3D8D: in example 2, a cartilage micro-tissue was prepared on a large scale, and the cartilage micro-tissue was cultured for 8 days by the EB948 disk method, which was designated as EB948 3D8D.

Method of

In the process of inducing differentiation into chondrocyte micro-tissues in vitro, RNEASY MINI KIT (Qiagen, 74004) was used every 2 days to isolate total RNA from chondrocyte micro-tissues according to the product instructions, and DNase I cleavage on the column was performed. cDNA was synthesized using ISCRIPT CDNA SYNTHESIS KIT (BioRad, 1708891) using 1 μg of total RNA as template. Real-time PCR was performed using a TB green Premix Ex Taq (TAKARA, RR 820A) qPCR kit on a CFX96 REAL TIME PCR Detection System (Bio Rad) using a two-step PCR amplification procedure, using equation 2 ^-ΔΔCt to calculate the relative gene expression, ACTB or B2M gene normalization, primer sequences are shown in the table below. Refer to Yamashita(Yamashita A,Morioka M,Yahara Y,Okada M,Kobayashi T,Kuriyama S,Matsuda S,Tsumaki N.Generation of scaffoldless hyaline cartilaginous tissue from human iPSCs.Stem Cell Reports.2015Mar 10;4(3):404-18) and Kawata(Kawata M,Mori D,Kanke K,Hojo H,Ohba S,Chung 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.Rol (method for preparing transplantable cartilage tissue, bulletin No. CN 110944684B), et al.

Table 2: primers for determining markers

Human articular hyaline cartilage extracellular matrix coding genes mainly comprise COL2A1 (type II collagen), ACAN (proteoglycan), COL9A1 (type IX collagen) and COL11A1 (type XI collagen), wherein the expression level of ACAN gene in chondrocyte microstructure is in positive correlation with cartilage regeneration repair ability after transplantation, and thus can be potentially used as a drug effect release test, generally regarded as standard for quality and product release of chondrocyte microstructure (reference Vonk, L.A.,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, determines the synthesis and secretion of type II collagen in the process of chondrocyte differentiation, and plays an important role in the process of chondrocyte differentiation and skeletal development. The SPARC (secreted acid and cysteine-rich protein) gene encodes a cysteine-rich acid matrix-related protein that is required for collagen calcification in bone, but which is also involved in the synthesis of extracellular matrix and promotes changes in cell shape. HAPLN1 (hyaluronic acid and proteoglycan linker 1) is a protein encoding gene, a biomarker specific for articular chondrocytes, and MFAP5 (microfiber-related protein 5) gene encodes a 25-kD microfiber-related glycoprotein, which is a component of extracellular matrix microfibers, considered as a biomarker specific for fibroblasts.

In patent CN110944684B (g. Rol et al, method for preparing transplantable cartilage tissue, grant publication No. CN 110944684B), the expression levels of cartilage formation consistency marker gene CRTAC1 (homo sapiens chondroitins 1, nm_018058) and cartilage formation purity marker gene EBF3 (homo sapiens early B-cytokine 3, nm_001005463) representing cartilage cell micro-tissue satisfy the following conditions: CRTAC1/R is more than or equal to 0.012, and EBF3/R is less than or equal to 0.1, which shows that the cartilage cell microstructure has higher consistency and purity.

Results

As shown in fig. 10A and table 3, compared with the adult primary chondrocyte (cdcp 0) and adult chondrocyte microstructure (cdcp 2 3D 8D), the chondrocyte microstructure derived from iPS cell differentiation (2D induction method) highly expressed the chondrocyte extracellular matrix-related genes COL2A1, COL9A1, COL11A1, SPARC, and SOX9 transcription factors, and the expression amounts thereof tended to increase and decrease with the increase of the induced differentiation time; the expression level of ACAN and HAPLN genes is lower than that of adult primary chondrocytes (CDC P0) except 3D 8D; in addition, the expression level of MFAP5, a marker gene representing fibrochondrocytes, was rapidly increased in early stages of induction of differentiation into chondrocytes, which was far higher than that of adult primary chondrocytes (cdcp 0) and adult chondrocyte micro-tissues (cdcp 2 3D 8D). This suggests that chondrocyte microtissue resulting from the 2D induction method may be more prone to fibrocartilage formation than the clinically desirable hyaline cartilage tissue.

Table 3: chondrocyte microsphere qPCR original CT value formed by 2D method induced differentiation

Table 3 continues: chondrocyte microsphere qPCR original CT value formed by 2D method induced differentiation

As shown in fig. 10B and table 4, compared with the adult primary chondrocyte (cdcp 0) and adult chondrocyte microstructure (cdcp 2 3D 8D), the chondrocyte microstructure derived from iPS cell differentiation (3D induction method) highly expressed the chondrocyte extracellular matrix-related genes COL2A1, ACAN, COL9A1, COL11A1, HAPLN1, SPARC, and SOX9 transcription factors, and the expression amounts thereof were substantially gradually increased with the prolongation of the induced differentiation time, and maintained stable high expression at 3D8D (fig. 10B). On the other hand, chondrocyte microtissue derived from iPS cell differentiation has similar or higher MFAP5 expression level as that of adult primary chondrocytes (fig. 10B), indicating that very little fibroblasts or mesenchymal stem cells are contained therein, which is consistent with the 10×single cell sequencing result (fig. 19). From the difference in gene expression levels of chondrocyte micro-tissues obtained by the 2D and 3D induction methods, the 3D induction method can up-regulate the expression level of hyaline chondrocyte-related genes, such as COL2A1, ACAN, COL9A1, COL11A1, SPARC, HAPLN1, SOX9, and down-regulate the expression level of the fibrochondrocyte marker gene MFAP 5.

Table 4: chondrocyte microsphere qPCR original CT value formed by 3D method induced differentiation

Table 4 follow: chondrocyte microsphere qPCR original CT value formed by 3D method induced differentiation

As shown in fig. 10C and table 5, chondrocyte microstructure (3D 8D) CRTAC 1/r=60.gtoreq.0.012 and ebf 3/r=0.ltoreq.0.1 of iPS directionally induced differentiation obtained by the 3D induction method in the present method shows that it has higher consistency and purity, which is consistent with the 10×single cell sequencing result (fig. 19). G. The requirement of CRTAC1/R not less than 0.012 and EBF3/R not more than 0.1 in the Roer et al patent (method for preparing transplantable cartilage tissue, grant publication No. CN 110944684B) indicates that the chondrocyte microstructure has good consistency and purity.

Table 5: chondrocyte microspheres CRTAC1 and EBF3 qPCR original CT values formed by 3D method induced differentiation:

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The data fully demonstrate that chondrocyte microstructure derived from differentiation of iPS cells obtained by using a 3D induction method is mainly hyaline cartilage, but not fibrocartilage; meanwhile, it is demonstrated that chondrocyte precursor cells derived from differentiation of iPS cells reach a stable optimal level at day 8 (3D 8D) of induction into cartilage microspheres.

Example 7: the ability of human iPS cells to develop into cartilage tissue in vivo from directionally differentiated chondrocyte micro-tissues was determined.

Method of

In vivo implantation

To investigate the ability of human iPS directed differentiation-derived chondrocyte micro-tissue (3D 8D chondrocyte micro-tissue) to form teratomas and develop into hyaline cartilage tissue in vivo, we transplanted iPS cell differentiation-derived chondrocyte micro-tissue into NOD-SCID immunodeficient mice (8-10 weeks) and C57BL/6N mice (7-8 weeks) subcutaneously. Firstly, a mouse is kept, the fur on the two sides of the skin on the back side of the abdomen is shaved off, the mouse is anesthetized by 2.5% tribromoethanol, after the mouse has no obvious pain reflection, the skin is sterilized by iodine tincture and alcohol, the skin is opened with a wound of 0.5cm, and a channel with a depth of 1.5cm is passively separated along the opening of the skin by forceps. The chondrocyte micro-tissue is taken out from the culture solution, washed once by PBS, then the chondrocyte micro-tissue is put into the bottom of the subcutaneous tunnel, 1 chondrocyte micro-tissue is transplanted to each part, and the opening is sutured by a skin suture device. The condition of the bulge of the skin on the ventral back side was observed. Animals were sacrificed after 6 weeks for general observation, pathology staining, and biomechanical detection of regenerated tissue.

General observations of regenerated tissue

As shown in FIG. 11A, the cartilage cell micro-tissue was subcutaneously transplanted in NOD-SCID mice and C57BL/6 mice for 6 weeks, and large tissue projections were seen under the skin, and teratoma was not formed at the transplanted site, and immune rejection and other adverse reactions were not seen. After 6 weeks of transplantation, the NOD-SCID mice and the C57BL/6 mice were surgically removed for subcutaneous transplantation of chondrocyte micro-tissues, and the volume of the chondrocyte micro-tissues was found to be larger than that before transplantation, and the regenerated tissues were milky white, smooth in surface, and had a certain hardness, and the chondrocyte micro-tissues grew well under the skin.

Regenerated tissue biomechanical detection

The method comprises the following steps: specific implementation 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.2021Sep;15(9):776-787), the subcuticular tissue surface of NOD-SCID immunodeficient mice was first placed perpendicular to the axis of indentation and the force and displacement applied to the regenerated cartilage was continuously recorded at a rate of 0.005mm/s using a cylindrical flat-end indenter (BOSE 3220 EM USA) with a diameter of 1mm for 300 seconds. And calculating the elastic modulus of the repaired cartilage according to the stress-strain curve.

Results: the regenerated tissue of the chondrocyte micro-tissue derived from the human iPSC after being transplanted into the mouse body subcutaneously for 8 weeks has the same mechanical biomechanical property as the reported human natural hyaline cartilage, and the elastic modulus is about 27MPa (fig. 12 and table 6).

TABLE 6 results of mechanical test of chondrocyte microstructure derived from human iPS cell differentiation after 8 weeks of subcutaneous transplantation in NOD-SCID mice.

Pathological staining

The cartilage-like tissue regenerated after 6 weeks of subcutaneous transplantation of chondrocyte micro-tissue in NOD-SCID immunodeficient mice and C57BL/6 mice was dehydrated, paraffin-embedded and 5 μm sectioned with 4% Paraformaldehyde (PFA) for 2 days, and specially stained with Hematoxylin Eosin (HE) staining kit (Soilebao, G1120), toluidine blue staining kit (Soilebao, G3668), modified safranin O-fast green staining kit (Soilebao, G1371) and alisxin blue staining kit (Soilebao, G1562), respectively. Aripine blue (Alxin blue) is a copper-titanium-like cyanine conjugated dye, and this cationic dye is combined with an acidic group, i.e. the Alxin blue forms an insoluble complex with anionic groups contained in tissues, such as carboxyl groups and sulfate groups. The alisxin blue is formed by connecting a phthalocyanine ring with copper in the center and four isothiourea groups through thioether bonds. The isothiourea group is moderately alkaline, so that the alisxin blue band cations can distinguish the class of mucus substances by utilizing different pH values of dye liquor.

Results: as shown in panel A of FIG. 11, both NOD-SCID immunodeficient mice and C57BL/6 mice subcutaneous regenerated cartilage-like tissue consisted of a large number of extracellular matrix proteoglycan-encapsulated hyaline chondrocytes, with the internal cells arranged in a typical "paving stone" pattern.

Results

The results of general observation, pathology staining and biomechanical examination of the regenerated chondroid tissue after 6 weeks of subcutaneous transplantation of chondrocyte micro-tissue in NOD-SCID immunodeficient mice and C57BL/6 mice showed that no teratoma was formed after 6 weeks of subcutaneous transplantation of chondrocyte micro-tissue, and that the chondrocyte micro-tissue grew well under the skin (FIG. 11A). The result of histopathological section shows that the regenerated tissue is composed of a large number of transparent cartilage cells wrapped by extracellular matrix proteoglycan (B diagram of fig. 11) and has the same mechanical and biomechanical properties as the natural transparent cartilage (fig. 12), which shows that the cartilage cell micro-tissue derived from the differentiation of iPS cells does not cause the organism to generate immune rejection reaction, and has the potential of developing and forming the bone cartilage tissue in vivo.

Example 8: cartilage cell microstructure filling repair rabbit knee joint load area cartilage defect of human iPS cell directional differentiation

Construction of cartilage defect model of load zone of knee joint of rabbit

The quarantined New Zealand rabbits are weighed, and animals (1 mL/kg-3 mL/kg) are anesthetized by the injection of 2.5% sodium isopentobarbital solution through the ear margin vein. The back legs and knees are sheared to prepare skin, limbs are fixed in the supine position, and the towel is spread and then the iodine is sterilized for 2 times. Taking a 3.0-4.0 cm incision on the inner side of the knee joint (or a longitudinal incision on the outer side of the patella) of a New Zealand rabbit, and exposing the joint surface. Checking whether the knee joint cavity has effusion or adhesion, flattening the joint cartilage, punching the medial femoral condyle by using a 4mm cornea trephine after the joint cartilage is normal in color, removing residual cartilage fragments by using a curet and repairing the flattened bottom (A diagram of fig. 13). The patella is reset, and the joint capsule and the skin incision are sutured layer by layer. Penicillin was injected intramuscularly immediately after surgery to prevent infection for 7 consecutive days. And establishing a full-layer articular cartilage defect animal model. The diameter of the defect part is 4mm, and the depth is 1-2 mm. Daily observations and recordings of diet, stool and urine, mental status, wound infection, etc. were made for each group of animals.

Cartilage cell micro-tissue filling repair rabbit knee joint load area cartilage defect transplanting operation process of directional differentiation of human iPS cells

Referring to Boehm et al method (Boehm E,Minkus M,Scheibel M.Autologous chondrocyte implantation for treatment of focal articular cartilage defects of the humeral head.J Shoulder Elbow Surg.2020Jan;29(1):2-11)., after successfully creating a rabbit knee joint load area cartilage defect model, removing residual cartilage fragments at the damaged part by using a curet and repairing the flat bottom, uniformly flatly filling chondrocyte micro-tissues derived from iPS cell differentiation at the joint cartilage defect part, suggesting that 10-70 chondrocyte micro-tissues are transplanted per square centimeter, keeping blood dry, standing for 15min to enable cartilage pellets to be fully adhered with joint tissues (A, B chart of FIG. 13), and stitching wounds layer by layer. The materials were taken 1 month and 3 months after surgery, the material-taking parts including cartilage of the repair area, surrounding normal cartilage, subchondral bone and cancellous bone, all samples were fixed in 4% paraformaldehyde, decalcified in 10% EDTA and dehydrated.

Results

General observations and histopathological staining revealed that the cartilage defect sites of the model group were substantially free of neogenesis. Chondrocyte microtissue derived from iPS cell differentiation was transplanted to rabbit cartilage defect site, and after 1 month, defect site was covered with regenerated tissue (fig. 13, panel C). HE. The staining results of toluidine blue, safranin O fast green, A Li Xinlan and Marsonet masson show that the regenerated cartilage tissue is rich in a great amount of chondrocytes and extracellular matrixes of cartilage, such as proteoglycan, collagen fiber and the like, and has high fusion degree with surrounding natural cartilage. The proportion of the regenerated tissue area of the model control group and the chondrocyte micro-tissue treated group derived from iPS cell differentiation was 30.05% and 63.30% respectively (fig. 13, D and table 7) after Image J software analysis. This shows that the chondrocyte microstructure derived from the differentiation of the iPS cells has good filling and repairing effects on the cartilage defect of the knee joint of the rabbit.

Table 7: calculating cartilage regeneration repair proportion

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims

1. A method of preparing a chondrocyte microstructure comprising:

(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;

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:

(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.