Method and culture medium for promoting proliferation and expansion of mesenchymal stem cells

Abstract

The present application provides a method and culture medium for promoting the proliferation and expansion of mesenchymal stem cells. Specifically, the culture medium includes a basal culture medium and ethionamide, which also includes one or more of platelet factor 4, Klotho protein and taurine. The culture medium and expansion culture method provided by the present application can improve the expansion efficiency and maintain the culture of pluripotent stem cells.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, specifically to the field of cell culture technology, and in particular to a culture medium for mesenchymal stem cells and a method for amplifying and culturing mesenchymal stem cells. Background Art

[0002] Mesenchymal stem cells (MSCs) are a promising tool in tissue engineering, with the characteristics of self-regeneration, rapid proliferation, and multipotent differentiation. There are more than 6,000 clinical studies worldwide (www.clinicaltrials.gov). MSCs produce anti-inflammatory phenotypes, induce the production and expansion of immunosuppressive M2 macrophages, tolerant DCs, NK-regs, and T-regs, and effectively alleviate harmful immune responses and persistent inflammation. MSCs can be derived from different tissues of the human body, such as adipose tissue, umbilical cord or umbilical cord blood, bone marrow, etc. The umbilical cord is originally a biological waste, so there are no ethical and material issues in obtaining it. Furthermore, mesenchymal stem cells also have multidirectional differentiation ability. After specific culture, they can differentiate into adipocytes, chondrocytes, muscle cells, etc., and have application potential in tissue regeneration and repair. However, MSCs can only maintain a limited number of generations during culture, and the cell expansion efficiency is strictly affected by the composition of the culture medium. Therefore, it is of great significance to improve the expansion efficiency of MSCs, obtain sufficient amount of MSCs cells in a limited number of generations and a short time, and maintain their multipotent differentiation stemness.

[0003] xCELLigence RTCA eSight is an instrument that can monitor cell imaging data acquisition and real-time impedance measurement in real time. It can obtain a variety of quantitative parameters, such as cell exponential doubling time (CI DT ), maximum CI (CI max ), Growth time 50 (GT 50 ), where according to CI DT Whether it is greater than 50 or not can determine the differentiation potential of MSCs.

[0004] The main goal of the present invention is to utilize umbilical cord-derived mesenchymal stem cells (UC-MSCs), add cytokines to serum-free or animal-free culture medium to form MSCs culture medium, and optimize cell planting density, culture medium replacement frequency and replacement method and other culture methods to achieve effective expansion of mesenchymal stem cells, while maintaining the multipotent differentiation stemness of the cells, and achieving the goal of obtaining as many stem cells with functional characteristics as possible within a limited sample and expansion generation. Summary of the invention

[0005] The present application aims to achieve effective expansion and differentiation of mesenchymal stem cells. After continuous exploration, a mesenchymal stem cell culture medium and a specific expansion and culture method that can achieve the above-mentioned purpose are provided.

[0006] The present application provides a mesenchymal stem cell culture medium, which includes a basal culture medium and ethionamide, and further includes one or more of platelet factor 4, Klotho protein and taurine.

[0007] Furthermore, the culture medium includes: basal culture medium, ethionamide, platelet factor 4 and Klotho protein.

[0008] Furthermore, the culture medium includes: basal culture medium, ethionamide, platelet factor 4, Klotho protein and taurine.

[0009] Furthermore, the culture medium is composed of basal culture medium, ethionamide, platelet factor 4, Klotho protein and taurine.

[0010] Furthermore, the basal culture medium is a serum-free culture medium or an animal-free culture medium.

[0011] Furthermore, the serum-free culture medium includes insulin, transferrin, and fibronectin.

[0012] Furthermore, the concentration of ethionamide in the culture medium is 1-200 µM.

[0013] Further preferably, the concentration of ethionamide is 80-120 μM, more preferably 100 μM.

[0014] Furthermore, the concentration of platelet factor 4 in the culture medium is 0.1-20 µg / mL.

[0015] More preferably, the concentration of platelet factor 4 is 6-10 µg / mL.

[0016] Furthermore, the Klotho protein concentration in the culture medium is 0.1-20 µg / mL.

[0017] Further preferably, the concentration of Klotho protein is 0.5-2 µg / mL.

[0018] Furthermore, the concentration of taurine in the culture medium is 1-100 mM.

[0019] More preferably, the concentration of taurine is 10-30 mM.

[0020] The present application also provides a method for culturing mesenchymal stem cells, which comprises culturing the mesenchymal stem cells using the above-mentioned culture medium provided in the present application.

[0021] Furthermore, the method comprises: 3 / cm 2 – 2×10 4 / cm 2 The density of the cells was inoculated into the culture medium for cultivation.

[0022] Furthermore, the method includes replacing the culture medium at intervals of 1-3 days during the culture process, and the replacement method is full liquid replacement or half liquid replacement.

[0023] The present application also provides uses of the culture medium or the method for culturing mesenchymal stem cells, which include promoting the proliferation and expansion of mesenchymal stem cells, reducing the aging of mesenchymal stem cells, promoting the multifunctional differentiation of mesenchymal stem cells, and preparing mesenchymal stem cell drugs or preparations.

[0024] The above uses may be therapeutic or non-therapeutic.

[0025] Furthermore, the present application provides a method for promoting the proliferation and expansion of mesenchymal stem cells, comprising culturing mesenchymal stem cells using the culture medium or the method for culturing mesenchymal stem cells provided in the present application.

[0026] Furthermore, the present application provides a method for reducing aging of mesenchymal stem cells, comprising culturing mesenchymal stem cells using the culture medium or the method for culturing mesenchymal stem cells provided in the present application.

[0027] Furthermore, the present application provides a method for promoting multipotent differentiation of mesenchymal stem cells, comprising culturing mesenchymal stem cells using the culture medium or the method for culturing mesenchymal stem cells provided in the present application.

[0028] Furthermore, the multifunctional differentiation includes adipogenic differentiation, osteogenic differentiation, chondrogenic differentiation, etc. of mesenchymal stem cells.

[0029] Furthermore, the mesenchymal stem cells described in the present application are mesenchymal stem cells from one or more tissues selected from the group consisting of umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amnion or placenta.

[0030] Furthermore, the present application also relates to mesenchymal stem cells obtained by the culture medium or method.

[0031] Furthermore, the present application also relates to cell medicines or preparations containing the mesenchymal stem cells.

[0032] Effects of the Invention

[0033] The main purpose of this application is to add different cytokines to form a stem cell culture medium based on serum-free or animal-free culture medium, and to establish a stem cell culture method by combining the optimal planting cell density, medium replacement frequency and method, etc. By collecting cell index expansion data in real time, the optimal stem cell culture medium composition and the culture method for effectively expanding stem cells are established, which can be directly applied to clinical-level stem cell production. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The expansion index of MSCs under different serum-free medium conditions is shown.

[0035] Figure 2A , 2B It was shown that the addition of Ethionamide to GF20 medium increased the expansion of MSCs.

[0036] Figure 3 The effect of different seeding cell densities on the cell index of MSCs is shown.

[0037] Figure 4 The effects of different medium replacement methods on MSCs cell index are shown.

[0038] Figure 5A-Figure 5B The results show that different combinations of added factors can promote the expansion of MSCs and reduce senescence. Figure 5A is the proliferation cell index of MSCs, Figure 5B The senescence status of MSCs.

[0039] Figure 6 The expression of MSCs markers under different combinations of added factors was shown by flow cytometry.

[0040] Fig. 7A , 7B The results of different combinations of supplemented factors on adipogenic and osteogenic differentiation are shown. Fig. 7A For adipogenic differentiation, Figure 7B Differentiate into osteoblasts. DETAILED DESCRIPTION

[0041] The specific embodiments of the present invention will be described in more detail below with reference to the accompanying drawings. Although the specific embodiments of the present invention are shown in the accompanying drawings, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided in order to enable a more thorough understanding of the present invention and to enable the scope of the present invention to be fully communicated to those skilled in the art.

[0042] It should be noted that certain words are used in the specification and claims to refer to specific components. Those skilled in the art should understand that technicians may use different nouns to refer to the same component. This specification and claims do not use the difference in nouns as a way to distinguish components, but use the difference in the functions of the components as the criterion for distinction. As mentioned throughout the specification and claims, "including" or "comprising" is an open term, so it should be interpreted as "including but not limited to". The subsequent description of the specification is a preferred embodiment of the present invention, but the description is based on the general principles of the specification and is not intended to limit the scope of the present invention. The scope of protection of the present invention shall be determined by the attached claims.

[0043] As used herein, "substantially free" with respect to a particular component is used herein to mean that the particular component is not purposefully formulated into the composition and / or is present only as a contaminant or in trace amounts. Thus, the total amount of the particular component resulting from any accidental contamination of the composition is less than 0.05%, preferably less than 0.01%. Most preferred are compositions in which the amount of the particular component is undetectable by standard analytical methods.

[0044] As used in this specification, "a" or "an" may mean one or more. As used in the claims, when used with the word "comprising", the word "a" or "an" may mean one or more than one.

[0045] The term "or" is used in the claims to mean "and / or" unless explicitly stated to refer only to alternatives or the alternatives are mutually exclusive, although the present disclosure supports a definition referring only to alternatives and "and / or." As used herein, "another" may mean at least a second or more.

[0046] The following are definitions of some of the terms used in this article:

[0047] Ethionamide: 2-ethylpyridine-4-thiocarboxamide, chemical formula is C8H 10 N2S is mainly used in combination with other anti-tuberculosis drugs in clinical practice to enhance efficacy and prevent bacteria from developing drug resistance; it can also increase the efficiency of MSCs expansion.

[0048] Platelet factor 4 (PF4): A specific protein synthesized by platelet alpha granules that can improve brain cognitive function and prevent cell aging.

[0049] Klotho protein (Klotho): protein encoded by the Klotho gene. Klotho is a gene closely related to aging. It plays anti-aging, kidney protection, cardiovascular protection, anti-tumor and metabolic regulation roles by regulating the expression of multiple signaling pathways and target genes such as ion channels and Wnt. Klotho protein is mainly expressed in the kidneys. It is a single-chain transmembrane protein that forms a complex with the FGF23 receptor on the cell membrane, thereby exerting biological effects. It has the effects of enhancing cognitive ability and regulating platelet factors to prevent cell aging.

[0050] Taurine: 2-aminoethanesulfonic acid, with the chemical formula C2H7NO3S, is a sulfur-containing non-protein amino acid that has the functions of regulating the nervous system and promoting protein synthesis.

[0051] Cell Index (CI): A dimensionless parameter, which is obtained by calculating the impedance value measured by RTCA through a standard formula. When there is no cell adhesion, CI=0. The CI in the RTCA system is the result of the impedance induced by the electron flow to the adherent cells. CI is calculated as follows: CI=(impedance at time point n - impedance without battery) / nominal impedance value; the cell index reflects the changes in the number of cells and the state of cell adhesion. The more cells there are, the larger the cell index is, and the changes in the cell adhesion state will also affect the changes in the cell index. For example, if the cells spread out after adhesion, the cell index will also increase, or if the cells shrink after being stimulated, the changes in the cell index will also be affected.

[0052] Maximum relative cell index (RClmax): The cell index when cells reach full confluence and maximum adhesion strength.

[0053] The cell exponential doubling time (CI DT ):CI DT It is a readout of cell proliferation and behavior that combines changes in cell number, adhesion strength, and morphology. DT Greater than 50, differentiation potential is lost.

[0054] RTCA (Real Time Cellular Analysis): A real-time label-free cell analysis instrument that uses a special process to integrate a gold microelectrode sensor array at the bottom of each cell growth well in the cell culture plate to construct a cell impedance detection sensor system that can track changes in cell morphology, proliferation and differentiation in real time, dynamically and quantitatively. When cells growing on the surface of the microelectrode cause changes in the impedance of the adherent electrode interface, this change is correlated with the real-time state change of the cell. The biological information related to the physiological function of the cell can be obtained through real-time detection of the impedance value.

[0055] The present application provides a mesenchymal stem cell culture medium. In a specific embodiment, the culture medium includes a basal culture medium and ethionamide, and also includes one or more cytokines selected from platelet factor 4, Klotho protein, taurine and the like.

[0056] In a specific embodiment, the culture medium comprises basal medium, ethionamide and platelet factor 4.

[0057] In a specific embodiment, the culture medium comprises a basal medium, ethionamide and Klotho protein.

[0058] In a specific embodiment, the culture medium comprises a basal medium, ethionamide and taurine.

[0059] In a specific embodiment, the culture medium comprises basal medium, ethionamide, platelet factor 4 and Klotho protein.

[0060] In a specific embodiment, the culture medium comprises basal medium, ethionamide, platelet factor 4 and taurine.

[0061] In a specific embodiment, the culture medium comprises a basal medium, ethionamide, Klotho protein and taurine.

[0062] In a specific embodiment, the culture medium comprises basal medium, ethionamide, platelet factor 4, Klotho protein and taurine.

[0063] In some specific embodiments, the culture medium may further contain other cytokines or additives that promote cell growth.

[0064] In a specific embodiment, the culture medium consists of basal medium, ethionamide, platelet factor 4, Klotho protein and taurine.

[0065] In a specific embodiment of the present application, the concentration of ethionamide in the culture medium is 1-200 µM, preferably 80-120 µM, and more preferably 100 µM.

[0066] In a specific embodiment of the present application, the concentration of platelet factor 4 in the culture medium is 0.1-20 µg / mL, preferably 6-10 µg / mL.

[0067] In a specific embodiment of the present application, the concentration of Klotho protein in the culture medium is 0.1-20 µg / mL, preferably 0.5-2 µg / mL.

[0068] In a specific embodiment of the present application, the concentration of taurine in the culture medium is 1-100 mM, preferably 10-30 mM.

[0069] In a specific embodiment of the present application, the basal culture medium is a serum-free culture medium (SFM) or an animal-free culture medium (XFM).

[0070] In a specific embodiment, the serum-free culture medium comprises insulin, transferrin, and fibronectin.

[0071] Serum-free or animal-derived-free media provide a more stable, defined culture environment.

[0072] In a specific embodiment, the basal culture medium is GF20MSCXeno-FreeSFM culture medium (Dayou, 6914112) of Dayou. This culture medium is a GMP-grade serum-free, animal-derived component-free human mesenchymal stem cell (hMSCs) culture product.

[0073] The present application also provides a method for culturing mesenchymal stem cells, which comprises culturing the mesenchymal stem cells using the mesenchymal stem cell culture medium provided above.

[0074] In an embodiment of the present application, the method includes optimizing the initial cell planting density of the culture to achieve efficient cell expansion.

[0075] In a specific embodiment, mesenchymal stem cells are cultured at 2×10 3 / cm 2 – 2×10 4 / cm 2 The density of the cells was inoculated into the culture medium for cultivation.

[0076] In an embodiment of the present application, the method includes optimizing the frequency and method of replacing the culture medium to achieve efficient cell expansion.

[0077] In a specific embodiment, the culture medium is replaced at a frequency of 1-3 days during the culture process, and the culture medium is replaced by full or half replacement, preferably full replacement at an interval of 1 day.

[0078] The present application also provides uses of the culture medium or the method for culturing mesenchymal stem cells, which include promoting the proliferation and expansion of mesenchymal stem cells, reducing the aging of mesenchymal stem cells, promoting the multifunctional differentiation of mesenchymal stem cells, and preparing mesenchymal stem cell drugs or preparations.

[0079] In a specific embodiment, the above-mentioned use can be a therapeutic use or a non-therapeutic use.

[0080] The present application also provides a method for promoting the proliferation and expansion of mesenchymal stem cells, which specifically comprises culturing the mesenchymal stem cells using the culture medium or the method for culturing mesenchymal stem cells provided in the present application.

[0081] The present application also provides a method for reducing aging of mesenchymal stem cells, which specifically comprises culturing mesenchymal stem cells using the culture medium or the method for culturing mesenchymal stem cells provided in the present application.

[0082] The present application also provides a method for promoting multifunctional differentiation of mesenchymal stem cells, comprising culturing mesenchymal stem cells using the culture medium or the method for culturing mesenchymal stem cells provided in the present application.

[0083] In a specific embodiment, the multifunctional differentiation includes adipogenic differentiation, osteogenic differentiation, chondrogenic differentiation, etc. of mesenchymal stem cells.

[0084] The mesenchymal stem cells described in the present application may be mesenchymal stem cells from one or more tissues selected from umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amnion or placenta.

[0085] The present application also provides mesenchymal stem cells obtained by the culture medium or method.

[0086] The present application also provides a cell medicine or preparation containing the mesenchymal stem cells.

[0087] Example

[0088] The following examples are used to describe and illustrate the embodiments of the present invention in detail. However, the following contents should not be construed as limiting the present invention in any way. The reagents and instruments used in the examples are all commercially available products and can be purchased on the market unless otherwise specified.

[0089] Example 1: Proliferation of MSCs cells in serum-free medium

[0090] The use of FBS (fetal bovine serum) in clinical stem cell production carries potential risk factors, and FBS has the risk of batch instability, so clinical cell culture products are concentrated in serum-free medium (SFM) or animal-free medium (XFM). This application compares MesenCult™-ACF Plus (STEMCELL Technologies, Catalog #05448) AMMS ® The expansion efficiency of three serum-free culture media, MSC Kit 2.0 (Beijing Tongli Haiyuan Biotechnology Co., Ltd., AS-13), GF20 MSC Xeno-Free SFM (Dayou, 6914112), in culturing umbilical cord-derived mesenchymal stem cells.

[0091] The experimental steps were as follows: cryopreserved MSCs (P3 generation) were suspended in three different culture media, and then cultured at 1×10 4 Pieces / cm 2 MSCs were planted into E-PLATE at a high cell density and placed in an RTCA eSight incubator for further culture at 37°C CO2 for 6 days, with the culture medium replaced every three days. During this period, the cell growth index (Cell Index) was monitored in real time using xCELLigence RTCA eSight.

[0092] Experimental results (such as Figure 1 As shown in Figure 2, all three serum-free culture media can expand MSCs. Among them, GF20 can significantly improve the expansion efficiency of MSCs and obtain a higher maximum relative cell index (RCl). max 2.7, GT50 is 55 hours, CI DT 26; while the RCl of MSCs cells in AMMS medium max 2.1, GT50 is 80 hours, CI DT The RCl of MSCs cells in ACF Plus medium was 43. max 2.0, GT50 for 60 hours, CI DT is 36.

[0093] Example 2: Adding Ethionamide Cytokine to Proliferate MSCs

[0094] Furthermore, the present invention adds cytokines such as ethionamide (HY-B0276, MedChemExpress (MCE)), G-CSF (HY-P73523, MCE), G-CSF (10007-HNAH, Beijing Sino Biological Science and Technology Co., Ltd.), bFGF17 (HY-P72652, MCE) or bFGF17 (12342-HNAE, Beijing Sino Biological Science and Technology Co., Ltd.) to serum-free GF20 medium, suspends MSC cells (P4 generation) in the medium supplemented with these factors, and incubates at 3.6×10 3 Pieces / cm 2 The cells were planted in an E-PLATE at a high density and then placed in an RTCA eSight incubator for further culture. The cells were cultured in a 37°C CO2 incubator for more than 6 days, and the culture medium was replaced every three days. Results Figure 2A The results showed that Ethionamide alone could significantly promote the growth of MSCs, but G-CSF and bFGF-17 alone inhibited the growth of UC-MSCs.

[0095] The effect of the added concentration of Ethionamide on the proliferation of MSCs was further studied. MSCs (P4 generation) were resuspended in GF20 medium supplemented with different concentrations of Ethionamide (0 µM, 10 µM, 50 µM, 100 µM, 200 µM) and cultured at 3.6×10 3 Pieces / cm 2 Cell density: MSCs were seeded into E-PLATE and placed in RTCA eSight instrument to measure resistance every 15 minutes to monitor cell proliferation index in real time. The cells were cultured in a 37°C CO2 incubator for more than 6 days, and the culture medium was replaced every three days.

[0096] Results (such as Figure 2B As shown in the figure, with the increase of Ethionamide concentration, the cell index of MSCs increased, reaching the highest under the condition of 100μM concentration, but under the condition of high concentration of 200μM treatment, its later expansion efficiency gradually decreased.

[0097] Example 3: Effects of different cell seeding densities on the proliferation of MSCs

[0098] MSCs cells (P4) were resuspended in serum-free GF20 medium supplemented with ethionamide (100 µM) at different densities, including: 4 × 10 3 cells / cm 2 , 6×10 3 cells / cm 2 , 8×10 3 cells / cm 2 , 1.6×10 4 cells / cm 2 , and 6×10 3 cells / cm 2 The cells were resuspended in GF20 medium without ethionamide as a control, and the resuspended cells were planted in E-PLATE. The cell proliferation index was monitored in real time in the RTCA eSight instrument, with the maximum relative cell index (RCl max ) as standard.

[0099] Experimental results (such as Figure 3 As shown in Figure 2, 100 µM Ethionamide was added to the culture medium and the cell density was 4-8×10 3 cells / cm 2 Within this range, the cell index of MSCs can reach a higher RCl max， Its value is 2.4-2.7, GT50 Value 90-130 hours, CI DT The value is 10-30, but the cell density is 16×10 3 cells / cm 2 A larger RCl can be obtained in a shorter time max , its value is 2.0, GT 50 For 40 hours, CI DT is about 15; although RCl without Ethionamide max， The value is 2.0, but GT 50 130 hours, CI DT About 35.

[0100] The experimental results show that the planting density of the starting cells significantly affects the expansion efficiency of MSCs. If the starting cell density is too low, the expansion efficiency is relatively low, but if the starting cell density is too high, the obtained cell expansion multiple is low.

[0101] Example 4: Effect of MSCs medium replacement frequency on the maximum relative cell index

[0102] The nutrients in the culture medium are the basis for maintaining cell growth. During the culture process, the nutrients are gradually consumed and cell growth will be inhibited. Appropriate replacement and supplementation of the culture medium can continue to maintain cell growth. MSCs (P4 generation) were resuspended in serum-free GF20 culture medium and seeded at an initial density of 6×10 3 cells / cm 2 MSCs were seeded into E-PLATE and cultured in a 37°C CO2 incubator for more than 6 days. The culture medium was replaced or supplemented at different intervals. The resistance was measured every 15 minutes in the RTCA eSight instrument to monitor the cell proliferation index in real time.

[0103] Experimental results (such as Figure 4 As shown in the figure, under different culture replacement intervals and replacement methods (full medium replacement / half medium replacement), the order of cell proliferation efficiency from high to low is: full medium replacement at one day > medium replacement at one and a half days > full medium replacement at two days > medium replacement at two and a half days > full medium replacement at three days > medium replacement at three and a half days > no medium replacement.

[0104] Example 5: Adding factors such as Taurine, PF4 and Klotho protein can improve the expansion efficiency of MSCs and reduce cell senescence

[0105] (1) In order to further improve the expansion efficiency of MSCs cells, anti-aging related cytokines and combinations were added to the basal culture medium (GF20) supplemented with ethionamide (100µM), including: taurine (Tuarine, 20mM, HY-B0351, MCE), platelet factor 4 (8µg / mL, CXCL4 / PF4, HY-P70618, MCE), Klotho (1µg / mL, HY-P700011, MCE), Tuarine (20mM) + PF4 (8µg / mL), Tuarine (20mM) + Klotho (1µg / mL), PF4 (8µg / mL) + Klotho (1µg / mL), Taurine (20mM) + PF4 (8µg / mL) + Klotho (1µg / mL), MSCs were resuspended in culture medium supplemented with different factors. GF20 culture medium supplemented with only ethionamide (100µM) was used as the control group (Ctr). They were seeded in E-PLATE and the cell growth status was monitored in real time using the RTCA eSight instrument.

[0106] Experimental results (such as Figure 5A ) shows: CI with only Ethionamide added max is 1.8. On this basis, after further adding Taurine, PF4 or Klotho, its RCI max 1.9, 2.13, 2.2 respectively; while adding PF4+Klotho (RCI max 3.0) and Taurine+Klotho+PF4 (RCI max The maximum RCl can be obtained by combining max GT50 is around 85 hours, with no big differences between treatments.

[0107] (2) MSC cells have limited ability to divide, and they enter a state of senescence when they cannot divide. Senescent cells usually become larger in size and express β-galactosidase, which has high enzymatic activity at pH 6.0. Using X-Gal as a substrate, a dark blue product is generated under the catalysis of senescence-specific β-galactosidase. Therefore, it is easy to observe cells or tissues expressing β-galactosidase that have turned blue under an optical microscope. MSCs were divided into 1×10 4 Pieces / cm 2The cells were resuspended in the culture medium with different cytokines and combinations (the addition method and factor concentration of each group were the same as those in the experiment (1) above), namely taurine (Tuarine), platelet factor 4 (CXCL4 / PF4), Klotho, Tuarine+PF4, Tuarine+Klotho, PF4+Klotho, Taurine+PF4+Klotho, and only ethionamide was added as the control group (Ctr), and ethioside (10μM, HY-13629, MCE) was used as the positive control; the cells were seeded in 12-well plates and cultured in a 37℃ CO2 incubator. The medium was completely replaced every three days, and the cells were grown for one week until the confluence rate reached 80-90%. Then, the β-galactosidase kit (C0602, Bio-Tech) was used to detect the senescence of the cells, and the number of cells expressing blue was counted.

[0108] Experimental results (such as Figure 5B The results show that Etoposide treatment of MSCs can significantly increase cell senescence, while the groups adding the above cytokines all reduce the senescence of MSCs, especially the addition of PF4, Klotho, Taurine and the combination of PF4+Klotho and Taurine+PF4+klotho can significantly reduce the senescence of MSCs, and are better than the control group adding only Ethionamide culture medium.

[0109] Example 6: Effects of different factor treatments on MSC surface markers

[0110] Confirming the homogeneity of MSCs by flow cytometry of markers on the surface of MSCs is one of the quality control standards for measuring MSCs. For example, if the positive markers are CD73, CD90, and CD105, the number of positive cells should account for more than 95%; while the proportion of negative markers such as CD14, CD34, CD11b, CD19, HLA-DR, CD45, and CD79a should be ≤ less than 2%.

[0111] The experimental results of Example 5 show that MSCs cultured in the medium supplemented with PF4+Klotho and Taurine+PF4+Klotho based on the serum-free medium supplemented with Ethionamide have the greatest RCI max In order to further test whether the MSCs cultured in these media meet the clinical quality requirements, GF20 serum-free medium was used to add Ethionamide, Ethionamide+PF4+Klotho, Ethionamide+Taurine+PF4+Klotho at the experimental concentrations in Example 5, and 8×10 3Pieces / cm 2 MSCs (P5 generation) were suspended at a cell density and seeded in a T25 culture flask. When the cells reached a confluence of about 80%, the MSCs were digested and collected, and the expression of stem cell markers on the cell surface of the cultured MSCs was detected by flow cytometry.

[0112] Experimental results (such as Figure 6 As shown in the figure, the cell expression ratio of surface markers CD73 and CD90 on MSCs cells obtained by adding factors culture is >95%, and PF4+Klotho treatment can significantly increase the expression of CD105; while the expression of negative surface markers CD14, CD34, CD45, CD19 and HLA-DR on MSCs cells cultured with different factors is less than 2%. Therefore, supplementing PF4 and Klotho on the basis of Ethionamide GF20 serum-free culture medium can maintain MSC cells of qualified clinical quality.

[0113] Example 7: Effects of different added factors on the multifunctional differentiation characteristics of MSCs

[0114] Maintaining the stemness of MSCs, namely the properties of adipogenic, osteogenic and chondrogenic differentiation, is also one of the quality control criteria for measuring MSCs.

[0115] GF20 serum-free medium with 1×10 4 Pieces / cm 2 Resuspend MSCs (P5 generation) at the same cell density and seed them in 6-well plates. When the density reaches 90-100%, change to adipogenic differentiation medium (complete MesenCult™ Adipogenic Differentiation Medium, #05412). Change the adipogenic differentiation medium every three days for about 25-35 days. Finally, Oil Red O staining is performed to confirm the adipogenic differentiation of MSCs.

[0116] Similarly, GF20 serum-free medium supplemented with Taurine+PF4+Klotho or PF4+Klotho was added to the medium at a rate of 1×10 4 Pieces / cm 2MSCs (P5 generation) were resuspended at the same cell density and seeded in 6-well plates. When the density reached 90-98%, the medium was changed to complete MesenCult™ Adipogenic Differentiation Medium (#05465). The medium was changed every 3-4 days for about 15 days. Finally, Alizarin Red S staining was used to confirm the osteogenic differentiation of MSCs.

[0117] Fig. 7A The experimental results showed that the adipogenic ability of MSCs cells could be maintained under the treatment of different factors; Figure 7B The experimental results showed that the osteogenic differentiation of MSCs cells could be maintained under the treatment of different factors.

[0118] The above only illustrates the principle of the present application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The method and application of this application have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of this application to implement and apply the technology of this application.

Claims

1. A mesenchymal stem cell culture medium, which consists of a basal culture medium, ethionamide, platelet factor 4, Klotho protein and taurine, wherein: In the culture medium, the concentration of ethionamide is 80-120µM, the concentration of platelet factor 4 is 0.1-20µg / mL, the concentration of Klotho protein is 0.1-20µg / mL, and the concentration of taurine is 10-30 mM.

2. The culture medium according to claim 1, wherein The basic culture medium is a serum-free culture medium or an animal-free culture medium.

3. The culture medium according to claim 2, wherein The serum-free culture medium comprises insulin, transferrin and fibronectin. 4 . A method for culturing mesenchymal stem cells, comprising culturing the mesenchymal stem cells using the culture medium according to any one of claims 1 to 3 .

5. The method according to claim 4, wherein: Mesenchymal stem cells were cultured at 2×10 3 / cm 2 – 2×10 4 / cm 2 The density of the cells was inoculated into the culture medium for cultivation.

6. The method according to claim 4, wherein: During the culture process, the culture medium is replaced at intervals of 1-3 days, with the replacement method being full or half replacement.

7. Use of the culture medium according to any one of claims 1 to 3 or the method according to any one of claims 4 to 6, wherein The uses include promoting the proliferation and expansion of mesenchymal stem cells, reducing the aging of mesenchymal stem cells, promoting the multifunctional differentiation of mesenchymal stem cells, and preparing mesenchymal stem cell drugs or preparations.

8. The culture medium according to any one of claims 1 to 3, the method according to any one of claims 4 to 6, or the use according to claim 7, wherein The mesenchymal stem cells are mesenchymal stem cells from one or more tissues selected from umbilical cord, umbilical cord blood, bone marrow, fat, muscle, nerve, skin, amnion or placenta.

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