Cartilage induction culture medium and application thereof in preparation of cartilage cells derived from bone marrow stem cells

Abstract

The invention relates to the technical field of stem cell biology, in particular to a cartilage induction culture medium and application thereof in preparation of cartilage cells derived from bone marrow stem cells. The cartilage induction culture medium comprises a DMEM / F-12 culture medium, fetal calf serum, penicillin, streptomycin, sodium pyruvate, an insulin-transferrin-selenium additive, dexamethasone, L-ascorbic acid, L-proline, a transforming growth factor-beta1 and docetaxel. The cartilage induction culture medium provided by the invention can obviously improve the chondrogenic differentiation efficiency of bone marrow mesenchymal stem cells (BMSCs), is beneficial to shortening the in-vitro preparation period of bone marrow stem cell-derived cartilage cells, and avoids the loss of cartilage cell phenotypes in the stem cell in-vitro culture process to the greatest extent; the method has important application value in preparation research, development and clinical transformation of cartilage cell transplantation.

Description

Technical Field

[0001] The invention relates to the field of stem cell biotechnology, in particular to a cartilage induction culture medium and application thereof in the preparation of cartilage cells derived from bone marrow stem cells. Background Art

[0002] Articular cartilage damage caused by trauma, aging, and osteoarthritis seriously affects the physiological structure and function of joints and surrounding tissues, leading to joint pain and degeneration of mobility. As the first generation of tissue engineering cartilage regeneration and repair technology, autologous chondrocyte implantation (ACI) is an effective solution for the clinical treatment of early articular cartilage damage. However, the limited source of ACI donor cells, the loss of chondrocyte phenotypes caused by in vitro expansion, and secondary damage to donor tissues are the main challenges faced by the clinical application of ACI technology. Bone marrow-derived mesenchymal stem cells (BMSCs) are an important cell source for autologous or allogeneic chondrocyte transplantation due to their excellent self-renewal and bone / cartilage differentiation abilities. At present, the preparation process of bone marrow stem cell-derived chondrocytes mainly includes the primary isolation and expansion of BMSCs, induced differentiation and culture, etc. Among them, the low purity of primary separation of BMSCs, long in vitro expansion time, and low efficiency of chondrogenic induction differentiation have become the main bottlenecks for the large-scale preparation and clinical transformation of bone marrow stem cell-derived chondrocytes. Summary of the invention

[0003] In order to shorten the in vitro preparation period of allogeneic bone marrow stem cell-derived chondrocyte preparations, the present invention aims to provide a chondrogenic induction culture medium and its application in the preparation of bone marrow stem cell-derived chondrocytes.

[0004] In the present invention, the chondrocyte culture method refers to the formation of seed cells with a chondrocyte phenotype after in vitro purification, amplification, and differentiation induction culture of mesenchymal stem cells obtained by whole bone marrow adherence separation; the chondrocyte induction medium refers to a novel chondrocyte induction medium prepared by adding a special component to the classic chondrocyte induction medium; the primary separation and purification refers to a method for obtaining BMSCs by density gradient centrifugation of whole bone marrow blood and in vitro adherence culture; the chondrocyte induction culture is the use of the chondrocyte induction medium provided by the present invention to perform chondrocyte differentiation culture on BMSCs.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] The first object of the present invention is to provide a chondrogenic induction culture medium, which comprises DMEM / F-12 culture medium, 10% fetal bovine serum, 100U / mL penicillin, 100μg / mL streptomycin, 1mmol / L sodium pyruvate, 1% insulin-transferrin-selenium additive, 40ng / mL dexamethasone, 50μg / mL L-ascorbic acid, 46μg / mL L-proline, 10ng / mL transforming growth factor-β1 and 2.5ng / mL to 10ng / mL docetaxel.

[0007] In one embodiment of the present invention, the culture medium includes DMEM / F-12 medium, 10% fetal bovine serum, 100U / mL penicillin, 100μg / mL streptomycin, 1mmol / L sodium pyruvate, 1% insulin-transferrin-selenium additive, 40ng / mL dexamethasone, 50μg / mL L-ascorbic acid, 46μg / mL L-proline, 10ng / mL transforming growth factor-β1 and 2.5nmol / L docetaxel.

[0008] In one embodiment of the present invention, the culture medium includes DMEM / F-12 medium, 10% fetal bovine serum, 100U / mL penicillin, 100μg / mL streptomycin, 1mmol / L sodium pyruvate, 1% insulin-transferrin-selenium additive, 40ng / mL dexamethasone, 50μg / mL L-ascorbic acid, 46μg / mL L-proline, 10ng / mL transforming growth factor-β1 and 10nmol / L docetaxel.

[0009] The second object of the present invention is to provide a use of a chondrogenic induction medium in the preparation of chondrocytes derived from bone marrow stem cells.

[0010] The third object of the present invention is to provide a chondrocyte derived from bone marrow stem cells, characterized in that it is prepared using the above-mentioned chondrogenic induction culture medium.

[0011] The fourth object of the present invention is to provide a method for preparing chondrocytes derived from bone marrow stem cells, comprising the following steps:

[0012] (S1) Mixing the bone marrow whole blood dilution with Percoll cell separation medium and then performing density gradient centrifugation to collect the middle nucleated cell layer, then resuspending the middle nucleated cell layer with stem cell growth medium and performing two-dimensional culture, and after the cell confluence reaches 80% to 90%, performing subculture and expansion culture to obtain BMSCs;

[0013] (S2) Inducing differentiation and culturing the BMSCs prepared in step (S1) using the above-mentioned chondrogenic induction medium to prepare chondrocytes derived from bone marrow stem cells.

[0014] In one embodiment of the present invention, in step (S1), the volume ratio of bone marrow whole blood diluent to Percoll cell separation fluid is 0.8-1.2:1;

[0015] Preferably, the volume ratio of the bone marrow whole blood diluent to the Percoll cell separation fluid is 1.

[0016] In one embodiment of the present invention, in step (S1), the density of the Percoll cell separation solution is 1.017 g / mL to 1.073 g / mL;

[0017] Preferably, the density of the Percoll cell separation solution is 1.073 g / mL.

[0018] In one embodiment of the present invention, in (S1), the BMSCs are obtained by subculturing and amplifying to the third to fifth generations; preferably, the BMSCs are subculturing and amplifying to the third generation;

[0019] The cell density ratio of the generations in the subculture amplification culture is 1:2 to 1:4; preferably, the cell density ratio of the generations in the subculture amplification culture process is 1:2.

[0020] In one embodiment of the present invention, in step (S2), during the induction differentiation culture, the required initial cell density is 0.5×10 4 cell / cm 2 ~2×10 4 cell / cm 2 Preferably, the starting cell density is 1×10 4 cell / cm 2 .

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The chondrogenic induction culture medium provided by the present invention can significantly improve the chondrogenic differentiation efficiency of BMSCs, help shorten the in vitro preparation period of bone marrow stem cell-derived chondrocytes, and avoid the loss of chondrocyte phenotype during in vitro stem cell culture to the greatest extent, and has important application value for the preparation development and clinical transformation of chondrocyte transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1The figures show the cell morphology observation and cell proliferation activity detection results of BMSCs during the induced differentiation culture process after primary separation and purification; (A) Schematic diagram of the induced differentiation culture process and identification method of primary chondrocytes derived from BMSCs; (B) Result of chondrocyte proliferation activity during induced differentiation culture; (C) Result of chondrocyte proliferation morphology during induced differentiation culture; Among them, *p<0.1, **p<0.01, ***p<0.001.

[0024] Figure 2 This is the result of toluidine blue staining identification of the differentiated phenotype of chondrocytes during the induced differentiation culture process.

[0025] Figure 3 This is the result of immunofluorescence staining identification of the differentiation phenotype of chondrocytes during the induced differentiation culture process.

[0026] Figure 4 The figure shows the results of real-time fluorescence quantitative PCR detection of chondrocyte markers during the induction of differentiation culture; among them, **p<0.01, ***p<0.001. DETAILED DESCRIPTION

[0027] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments.

[0028] In the following examples, unless otherwise specified, all reagents used are commercially available reagents, and all detection means and methods used are conventional means and methods in the art.

[0029] Example 1

[0030] This embodiment provides a chondrogenic induction medium suitable for primary culture of bone marrow stem cells and chondrocyte differentiation induction, and the specific configuration method is as follows:

[0031] Add 10 mL of fetal bovine serum and 1 mL of penicillin-streptomycin solution (100X) to 89 mL of DMEM / F12 basal medium, filter and sterilize using a 0.22 μm sterile filter to obtain stem cell growth medium, and refrigerate at 4°C for later use;

[0032] 1 mL 100 mM sodium pyruvate, 1 mL insulin-transferrin-selenium additive (100X), 2 mL 2 μg / mL dexamethasone, 2 mL 2.5 mg / mL L-ascorbic acid, 2 mL 2.3 mg / mL L-proline, and 0.2 mL 5 μg / mL TGF-β1 were added to 91.8 mL stem cell growth medium, filtered and sterilized to obtain common chondrocyte medium (chon-M), and refrigerated at 4°C for later use;

[0033] 81 μL of 1.24 μmol / L docetaxel was added to 50 mL of chondrogenic differentiation medium, and the chondrogenic differentiation medium (chon-M / 2.5 nM) containing 2.5 nmol / L docetaxel was obtained by filtration and sterilization, and then refrigerated at 4°C for later use;

[0034] 403 μL of 1.24 mmol / L docetaxel was added to 50 mL of chondrogenic differentiation medium, and the chondrogenic differentiation medium (chon-M / 10nM) containing 10 nmol / L docetaxel was obtained by filtration and sterilization, and then refrigerated at 4°C for later use.

[0035] Example 2

[0036] This embodiment provides a method for preparing chondrocytes derived from bone marrow stem cells, which specifically comprises the following steps:

[0037] Femoral and tibia tissues of young Sprague-Dawley rats were cut under sterile conditions, and soft tissues such as hair and muscle were removed. The bone scales at both ends were cut off, and an appropriate amount of DMEM / F12 basal medium was drawn into a disposable medical syringe to elute the bone marrow cavity several times until the eluent was no longer suspended. The eluent (i.e., "bone marrow whole blood dilution") was collected and an equal volume of Percoll cell separation solution was added to it. After density gradient centrifugation, the middle nucleated cell layer was collected and inoculated on a 10 cm 2 The cells were placed in a culture dish and cultured in a constant temperature incubator (37°C, 5% CO2);

[0038] pass Figure 1 It can be found that the obtained BMSCs are long spindle-shaped during primary culture, and the cell outline is clear and obvious;

[0039] Furthermore, when the confluence of BMSCs reached 80% to 90%, the BMSCs were digested with trypsin, the cell pellets were collected, and the cells were resuspended in stem cell growth medium at a rate of 1×10 4 cell / cm 2 The cells were inoculated into a cell culture dish at a density of , and after the cells adhered to the wall, the medium was replaced with the chon-M / 2.5nM or chon-M / 10nM medium prepared in Example 1 and continued to be incubated (wherein the chon-M medium was used as the control group);

[0040] pass Figure 1 It can be found that during the induction differentiation culture process, the cell morphology and its proliferation activity were not significantly affected.

[0041] Example 3

[0042] In this example, toluidine blue staining was used to identify the chondrocyte differentiation effect in Example 2.

[0043] The identification method of this embodiment is to take out the cell smear and wash it with phosphate buffered saline (PBS), fix it with 4% paraformaldehyde solution for 30 minutes, wash away the residual paraformaldehyde, and then use 0.1% toluidine blue dye solution for 10 minutes, wash it with distilled water and observe it under a microscope ( Figure 2 ).

[0044] pass Figure 2 It can be found that after inducing differentiation culture using chon-M / 2.5nM or chon-M / 10nM culture medium, the obtained chondrocytes showed a more obvious aggregation growth trend, and the toluidine blue stained area showed obvious cartilage pit-like structure.

[0045] Example 4

[0046] In this example, the expression of chondrocyte markers during the induction differentiation culture in Example 2 was evaluated by immunofluorescence staining.

[0047] The cell smear was taken out and washed with PBS buffer, and then fixed with 4% paraformaldehyde solution for 30 minutes. After washing away the residual paraformaldehyde, goat serum blocking solution containing 0.1% Triton X-100 was added for blocking. After blocking for 1 hour, different primary antibodies (rabbit Collagen II polyclonal antibody, rabbit Collagen X monoclonal antibody) were added respectively, and incubated overnight at 4°C. After washing away the excess primary antibody, Alexa 555-labeled secondary antibody was added, incubated at room temperature in the dark for 1 hour, counterstained with DAPI staining solution for 5 minutes, and observed under a microscope after sealing with glycerol.

[0048] pass Figure 3 It can be found that with the extension of the induction culture time, the expressions of cartilage matrix specific markers Collagen II and Collagen X increased, indicating that the chondrocyte induction medium containing docetaxel provided in Example 1 can effectively induce BMSCs into chondrocytes; in addition, with the increase of the docetaxel content in the chon-M medium, the expression of the hyaline cartilage phenotype marker Collagen II showed an increasing trend at the early stage of differentiation, while the expression of the fibrocartilage phenotype marker Collagen X was inhibited, indicating that the addition of docetaxel is conducive to the generation of hyaline cartilage phenotype.

[0049] Example 5

[0050] In this example, RT-qPCR was used to quantitatively analyze the expression levels of chondrocyte markers during the induction differentiation culture process in Example 2.

[0051] The analysis method of this example is to extract RNA using a total RNA extraction kit (stored at -80°C for future use), and reverse transcribe mRNA into cDNA according to the reaction system, operation steps and reverse transcription conditions shown in Table 1.

[0052] Table 1 RNA reverse transcription related reagents, reaction system and reverse transcription program settings

[0053]

[0054] Furthermore, the obtained reverse transcription product was diluted 10 times, and then a quantitative PCR reaction was performed according to the reaction system and amplification program shown in Table 2, and the melting curve was recorded and analyzed.

[0055] Table 2 Quantitative PCR reaction system and amplification procedure

[0056]

[0057] Table 3 Primer sequences

[0058]

[0059] pass Figure 4 It can be found that compared with the traditional chon-M culture medium, after 7 days of induction culture using chon-M / 2.5nM and chon-M / 10nM culture media, the expression levels of chondrocyte Collagen II, SOX9, and ACAN markers were significantly increased, proving that the chondrocyte induction culture method and the new induction culture medium provided by the present invention have higher chondrocyte induction efficiency.

[0060] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the explanation of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A chondrogenic induction medium, characterized in that Including DMEM / F-12 culture medium, 10% fetal bovine serum, 100U / mL penicillin, 100μg / mL streptomycin, 1mmol / L sodium pyruvate, 1% insulin-transferrin-selenium additive, 40ng / mL dexamethasone, 50μg / mL L-ascorbic acid, 46μg / mL L-proline, 10ng / mL transforming growth factor-β1 and 2.5ng / mL~10ng / mL docetaxel.

2. A chondrogenic induction medium according to claim 1, characterized in that The culture medium includes DMEM / F-12 culture medium, 10% fetal bovine serum, 100U / mL penicillin, 100μg / mL streptomycin, 1mmol / L sodium pyruvate, 1% insulin-transferrin-selenium additive, 40ng / mL dexamethasone, 50μg / mL L-ascorbic acid, 46μg / mL L-proline, 10ng / mL transforming growth factor-β1 and 2.5nmol / L docetaxel.

3. A chondrogenic induction medium according to claim 1, characterized in that The culture medium includes DMEM / F-12 culture medium, 10% fetal bovine serum, 100U / mL penicillin, 100μg / mL streptomycin, 1mmol / L sodium pyruvate, 1% insulin-transferrin-selenium additive, 40ng / mL dexamethasone, 50μg / mL L-ascorbic acid, 46μg / mL L-proline, 10ng / mL transforming growth factor-β1 and 10nmol / L docetaxel.

4. Use of the chondrogenic induction medium according to any one of claims 1 to 3 in the preparation of chondrocytes derived from bone marrow stem cells.

5. A chondrocyte derived from bone marrow stem cells, characterized in that: The chondrogenic induction medium is prepared using any one of claims 1 to 3.

6. A method for preparing chondrocytes derived from bone marrow stem cells, characterized in that: The following steps are involved: (S1) Mixing the bone marrow whole blood dilution with Percoll cell separation medium and then performing density gradient centrifugation to collect the middle nucleated cell layer, then resuspending the middle nucleated cell layer with stem cell growth medium and performing two-dimensional culture, and performing subculture and expansion culture after the cell confluence reaches 80% to 90% to obtain BMSCs; (S2) Inducing differentiation and culturing the BMSCs prepared in step (S1) using the chondrology induction culture medium described in any one of claims 1 to 3 to prepare chondrocytes derived from bone marrow stem cells.

7. The method for preparing chondrocytes derived from bone marrow stem cells according to claim 6, characterized in that: In step (S1), the volume ratio of bone marrow whole blood diluent to Percoll cell separation fluid is 0.8-1.2:

1.

8. The method for preparing chondrocytes derived from bone marrow stem cells according to claim 7, characterized in that: The density of Percoll cell separation fluid is 1.017 g / mL to 1.073 g / mL.

9. The method for preparing chondrocytes derived from bone marrow stem cells according to claim 6, characterized in that: In step (S1), the cells are cultured to the third to fifth generations to obtain BMSCs. Among them, the intergenerational cell density ratio of subculture expansion culture is 1:2 to 1:

4.

10. The method for preparing chondrocytes derived from bone marrow stem cells according to claim 6, characterized in that: In step (S2), during the differentiation induction culture, the initial cell density was 0.5×10 4 cell / cm 2 ~2×10 4 cell / cm 2 .

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