Serum-free culture medium for human mesenchymal stem cells and application of serum-free culture medium

Abstract

The invention discloses a serum-free culture medium for human mesenchymal stem cells and application of the serum-free culture medium. The invention firstly discloses the serum-free culture medium for the human mesenchymal stem cells. The serum-free culture medium comprises a basic culture medium and supplementary ingredients, wherein the basic culture medium is a mixed solution of IMDM and high-glucose DMEM, and the supplementary components comprise VB12, insulin, hydrocortisone, progesterone, putrescine, transferrin, human serum albumin, ethanolamine, ascorbic acid, phorbol 12-myristate 13-acetate, TGF-beta, bFGF, EGF, PDGF-BB, IGF1, beta-mercaptoethanol, fibronectin and laminin. The invention further discloses application of the culture medium in human mesenchymal stem cell culture. The serum-free culture medium for the human mesenchymal stem cells has the advantages of no heterology, no need of coating, definite chemical components, small difference between batches, strong cell amplification capability, facilitation of cell biology research, and suitableness for large-scale culture of clinical stem cells.

Description

Technical Field

[0001] The present invention relates to the technical field of stem cell culture, and more specifically, to a human mesenchymal stem cell serum-free culture medium and application thereof. Background Art

[0002] Mesenchymal stem cells (MSC) are multipotent adult stem cells with self-renewal ability and multidirectional differentiation potential. They are derived from the mesoderm and can differentiate into a variety of cell types, such as chondrocytes, adipocytes, and osteoblasts. Compared with other stem cells, mesenchymal stem cells have the characteristics of immune regulation, multidirectional differentiation potential, easy access, rapid in vitro proliferation, small loss of activity after freezing, low immunogenicity, and no toxic side effects, making mesenchymal stem cells an ideal type of cell therapy. Since Lazarus et al. first reported the clinical study of mesenchymal stem cells in Bone Marrow Transplant in 1995, it has been widely used in the clinical treatment of various diseases.

[0003] The traditional culture method of mesenchymal stem cells is to use a culture medium containing animal serum for culture. However, as a mixture with unclear chemical composition, serum has a large difference in product quality between different batches and different manufacturers, which will have a certain degree of impact on the stability and repeatability of scientific research results. At the same time, due to its heterogeneity and the possibility of containing some unknown or undetected viruses and other microorganisms, it is not conducive to clinical treatment applications such as infusion. In order to solve the animal-derived components in the culture medium and facilitate the application of cells in clinical treatment, some research groups and companies have begun to use human serum, platelet lysate, etc. instead of animal serum to culture mesenchymal stem cells. Current studies have shown that the use of platelet lysate as a serum substitute can maintain the rapid proliferation of cells in vitro, ensure cell stemness and maintain immune regulation ability, but these blood products also have obvious batch differences and are easily affected by factors such as donor age. In addition, due to the limited source of human blood and the high price, the large-scale preparation of clinical mesenchymal stem cells is limited, which is not conducive to industrial production. At present, some companies are also developing a new generation of serum-free culture media. These culture media have clear chemical compositions and do not contain animal-derived ingredients. However, these culture media are relatively expensive. At the same time, during the culture process, most of them require the culture vessels to be coated, which is cumbersome to operate and has relatively poor proliferation effects. This is not conducive to the large-scale clinical-level culture of umbilical cord mesenchymal stem cells.

[0004] Therefore, it is very necessary to provide a serum-free culture medium with clear chemical composition, no animal origin, no need for encapsulation, and that can meet the needs of rapid proliferation of mesenchymal stem cells in vitro. Summary of the invention

[0005] One object of the present invention is to provide a serum-free culture medium for human mesenchymal stem cells, which has a clear chemical composition, is animal-free, easy to operate, and has small batch-to-batch differences. It not only solves the technical problem that a culture medium with a clear chemical composition needs to be coated during the culture process, but also overcomes the problem of low amplification efficiency of a culture medium with a clear chemical composition, and is conducive to the realization of clinical-grade large-scale culture of mesenchymal stem cells.

[0006] Another object of the present invention is to provide application of the above human mesenchymal stem cell serum-free culture medium.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] The present invention first provides a human mesenchymal stem cell serum-free culture medium, which comprises a basic culture medium and supplementary components;

[0009] The basal culture medium is a mixture of IMDM and high-glucose DMEM, and the supplementary ingredients include VB12, insulin, hydrocortisone, progesterone, putrescine, transferrin, human serum albumin, ethanolamine, ascorbic acid, phorbol 12-myristate 13-acetate, TGF-β, bFGF, EGF, PDGF-BB, IGF1, β-mercaptoethanol, fibronectin and laminin.

[0010] The mixed solution of IMDM and high-glucose DMEM prepared in a certain volume ratio can improve the ratio of nutrients such as amino acids, inorganic salts, sugars, etc. in the basic culture medium, and effectively promote the proliferation of mesenchymal stem cells; the addition of supplementary components such as hydrocortisone and ascorbic acid is beneficial to the proliferation of cells and the stability of adhesion; VB12 can promote the metabolism of carbohydrates, fats and proteins, and also has the functions of activating amino acids, promoting nucleic acid biosynthesis and protein synthesis, which is beneficial to the proliferation of mesenchymal stem cells; human serum albumin protects cells from mechanical damage; transferrin provides cells with required iron and promotes growth; putrescine can provide cells with amines; ethyl Alcoholamines can participate in the synthesis of spermine and promote cell proliferation; insulin can promote cells' utilization of glucose and play a key regulatory role in cell metabolism; progesterone has the effect of promoting cell growth; the combined use of TGF-β, bFGF, EGF, PDGF-BB and IGF1 growth factors can maintain normal cell morphology and rapid expansion; phorbol 12-myristate 13-acetate can promote cell proliferation; β-mercaptoethanol acts as an antioxidant to protect cells; fibronectin and laminin can be added to the culture medium in a certain proportion to stabilize cell adhesion without coating the culture dish.

[0011] Furthermore, the volume ratio of the IMDM to high-glucose DMEM is 1:1-2:1; preferably, the volume ratio of the IMDM to high-glucose DMEM is 1:1.

[0012] Further, the final concentrations of the components in the supplementary ingredients in the human mesenchymal stem cell serum-free medium are: VB12 concentration of 0.1-10 μg / ml, insulin concentration of 5-25 mg / l, hydrocortisone concentration of 0.01-0.1 mg / l, progesterone concentration of 0.01-1 mg / l, putrescine concentration of 0.01-100 mg / l, transferrin concentration of 2-30 mg / l, human serum albumin concentration of 1000-5000 mg / l, ethanolamine concentration of 0.0 5-5 mg / l, the concentration of ascorbic acid is 1-1000 μg / ml, the concentration of phorbol 12-myristate 13-acetate is 0.01-1 mg / l, the concentration of TGF-β is 0.001-1 mg / l, the concentration of bFGF is 0.001-1 mg / l, the concentration of EGF is 0.001-1 mg / l, the concentration of PDGF-BB is 0.001-1 mg / l, the concentration of IGF1 is 0.001-1 mg / l, and the concentration of β-mercaptoethanol is 1-10 mg / l;

[0013] Preferably, the final concentrations of the components in the supplementary ingredients in the human mesenchymal stem cell serum-free medium are: VB12 concentration is 0.5-5 μg / ml, insulin concentration is 10-20 mg / l, hydrocortisone concentration is 0.01-0.05 mg / l, progesterone concentration is 0.05-1 mg / l, putrescine concentration is 1-20 mg / l, transferrin concentration is 20-30 mg / l, human serum albumin concentration is 2000-4000 mg / l, and ethanolamine concentration is 1-5 mg / l. / l, the concentration of ascorbic acid is 1-100μg / ml, the concentration of phorbol 12-myristate 13-acetate is 0.05-1mg / l, the concentration of TGF-β is 0.01-0.1mg / l, the concentration of bFGF is 0.01-0.1mg / l, the concentration of EGF is 0.01-0.1mg / l, the concentration of PDGF-BB is 0.01-0.1mg / l, the concentration of IGF1 is 0.001-0.01mg / l, and the concentration of β-mercaptoethanol is 1-5mg / l.

[0014] Furthermore, the final concentrations of the components in the supplementary ingredients in the human mesenchymal stem cell serum-free culture medium are: the concentration of fibronectin is 0.1-10 mg / l, the concentration of laminin is 0.1-10 mg / l, and the concentration ratio of fibronectin to laminin is 1:1-3:2; preferably, the final concentrations of the components in the supplementary ingredients in the human mesenchymal stem cell serum-free culture medium are: the concentration of fibronectin is 3-10 mg / l, the concentration of laminin is 1-5 mg / l, and the concentration ratio of fibronectin to laminin is 3:2.

[0015] Furthermore, the supplementary component also includes at least one of IL-1β and IL-6; the final concentration of IL-1β in the human mesenchymal stem cell serum-free culture medium is 0.001-1 mg / l, and the final concentration of IL-6 in the human mesenchymal stem cell serum-free culture medium is 0.001-1 mg / l; preferably, the final concentration of IL-1β in the human mesenchymal stem cell serum-free culture medium is 0.001-0.01 mg / l, and the final concentration of IL-6 in the human mesenchymal stem cell serum-free culture medium is 0.001-0.01 mg / l.

[0016] Furthermore, the pH of the human mesenchymal stem cell serum-free culture medium is 7.3-7.4, the osmotic pressure is 280-320 mOsm, and the endotoxin is less than 0.5 EU / ml.

[0017] The present invention also provides an application of the human mesenchymal stem cell serum-free culture medium in human mesenchymal stem cell culture.

[0018] In a specific embodiment of the present invention, the human mesenchymal stem cells are human umbilical cord mesenchymal stem cells.

[0019] The beneficial effects of the present invention are as follows:

[0020] The supplementary components in the serum-free culture medium for human mesenchymal stem cells of the present invention can replace serum or serum substitutes to culture mesenchymal stem cells, and are superior to similar products of current serum-free culture medium with clear chemical components; the addition of a certain proportion of fibronectin and laminin can meet the requirement that the culture dish does not need to be coated during the culture of mesenchymal stem cells; the ratio of nutrients such as amino acids and vitamins is more suitable for the culture of mesenchymal stem cells, and can be matched with serum or serum substitutes to obtain better cell proliferation effect.

[0021] The human mesenchymal stem cell serum-free culture medium of the present invention is xeno-free, does not require coating, has clear chemical composition, is easy to operate, has small differences between batches, has strong cell expansion ability, greatly simplifies the operation steps, is conducive to cell biology research, is more suitable for large-scale cultivation of clinical-grade stem cells, and has good industrial prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The specific implementation modes of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0023] Figure 1 These are light microscopic images of umbilical cord mesenchymal stem cells P5 expanded and cultured in serum-free medium of human mesenchymal stem cells of Example 1 and Example 2 at different time points.

[0024] Figure 2 The cell proliferation curves of mesenchymal stem cells cultured to P5 are shown in Figure 2, when the basal culture medium of the present invention is used in combination with serum and platelet lysate (the mesenchymal stem cell culture medium of Comparative Examples 1 and 3) and DMEM / F12 is used in combination with serum and platelet lysate (the mesenchymal stem cell culture medium of Comparative Examples 2 and 4).

[0025] Figure 3 This is a comparison chart of the cumulative cell population doublings of mesenchymal stem cells cultured in the serum-free culture medium of Examples 1 and 2 of the present invention, the control serum-containing culture medium of Comparative Example 2, and the control serum-free culture medium of Comparative Example 5.

[0026] Figure 4 This is a comparison chart of the cumulative cell population doublings of mesenchymal stem cells cultured in the serum-free medium for human mesenchymal stem cells of Example 1 of the present invention and Comparative Example 6.

[0027] Figure 5 This is a diagram of the adipogenic, osteogenic and chondrogenic differentiation of umbilical cord mesenchymal stem cells continuously cultured in serum-free medium of human mesenchymal stem cells according to Examples 1 and 2 of the present invention to P10. DETAILED DESCRIPTION

[0028] In order to more clearly illustrate the present invention, the present invention is further described below in conjunction with preferred embodiments and accompanying drawings. Similar components in the accompanying drawings are represented by the same reference numerals. It should be understood by those skilled in the art that the content specifically described below is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0029] The consumables and reagents used in the present invention are all common commercial products and can be purchased on the market. Specifically, the purchase sources and product numbers of the components used in the present invention are shown in Table 1:

[0030] Table 1 Purchase sources and product numbers of the components used in the present invention

[0031] Serial number Components company Part Number 1 IMDM powder Thermo 12200036 2 High glucose DMEM TransGen FI101-01 3 β-Mercaptoethanol Thermo 21985023 4 Putrescine Aladdin D106194 5 Hydrocortisone Aladdin H657409 6 insulin Shanghai Guchen Biotechnology GCF2568-1 7 Progesterone Sigma P0130 8 Transferrin Sigma T8158 9 Human serum albumin Sigma SAE0160 10 Ethanolamine Sigma 15014 11 Phorbol 12-myristate 13-acetate Sigma P8139 12 Fibronectin R&D 1918-FN-02M 13 Laminin R&D 2308-VN-050 14 TGF-β1 peprotech 100-21C 15 FGF peprotech 100-18B 16 EGF peprotech AF-100-15 17 PDGF-BB peprotech 100-14B 18 IGF1 peprotech 100-11 19 IL-1β peprotech 200-01B 20 IL-6 peprotech 216-16

[0032] Example 1 A serum-free culture medium for human mesenchymal stem cells

[0033] The human mesenchymal stem cell serum-free medium of this embodiment is composed of a basal medium and supplementary components, wherein the basal medium is a mixture of IMDM and high-glucose DMEM in a volume ratio of 1:1; the supplementary components and their final concentrations in the human mesenchymal stem cell serum-free medium are shown in Table 2:

[0034] Table 2 Supplementary ingredients and their contents

[0035] Serial number Components Final concentration mg / L 1 VB12 3 2 insulin 15 3 Hydrocortisone 0.04 4 Progesterone 0.08 5 Putrescine 10 6 Transferrin 25 7 Human serum albumin 3000 8 Ethanolamine 3 9 ascorbic acid 50 10 Phorbol 12-myristate 13-acetate 0.06 11 TGF-β 0.02 12 FGF 0.015 13 EGF 0.02 14 PDGF-BB 0.01 15 IGF1 0.002 16 β-Mercaptoethanol 3 17 Fibronectin 6 18 Laminin 4 19 IL-1β 0.004 20 IL-6 0.005

[0036] The components were fully mixed, the pH of the culture medium was adjusted to 7.32, the osmotic pressure was adjusted to 315 mOsm, and the culture medium was filtered through a 0.22 μm filter, and the endotoxin content was less than 0.5 EU / ml.

[0037] Example 2 A serum-free culture medium for human mesenchymal stem cells

[0038] The human mesenchymal stem cell serum-free medium of this embodiment is composed of a basal medium and supplementary components, wherein the basal medium is a mixture of IMDM and high-glucose DMEM in a volume ratio of 2:1; the supplementary components and their final concentrations in the human mesenchymal stem cell serum-free medium are shown in Table 3:

[0039] Table 3 Supplementary ingredients and their contents

[0040]

[0041]

[0042] The components were fully mixed, the pH of the culture medium was adjusted to 7.36, the osmotic pressure was adjusted to 310 mOsm, and the culture medium was filtered through a 0.22 μm filter, and the endotoxin content was less than 0.5 EU / ml.

[0043] Comparative Example 1 A serum-containing mesenchymal stem cell culture medium

[0044] A serum-containing mesenchymal stem cell culture medium, the culture medium consists of the basic culture medium in Example 1 and fetal bovine serum, and the volume content of the fetal bovine serum in the mesenchymal stem cell culture medium is 10%.

[0045] Comparative Example 2: A serum-containing mesenchymal stem cell culture medium (control serum-containing culture medium)

[0046] A serum-containing mesenchymal stem cell culture medium consists of DMEM / F12 and fetal bovine serum, wherein the volume content of the fetal bovine serum in the mesenchymal stem cell culture medium is 10%.

[0047] Comparative Example 3: A mesenchymal stem cell culture medium containing platelet lysate

[0048] A mesenchymal stem cell culture medium containing platelet lysate, the culture medium consists of the basic culture medium in Example 1 and the platelet lysate, and the volume content of the platelet lysate in the mesenchymal stem cell culture medium is 5%.

[0049] Comparative Example 4: A mesenchymal stem cell culture medium containing platelet lysate

[0050] A mesenchymal stem cell culture medium containing platelet lysate, the culture medium consists of DMEM / F12 and platelet lysate, and the volume content of the platelet lysate in the mesenchymal stem cell culture medium is 5%.

[0051] Comparative Example 5: A serum-free mesenchymal stem cell culture medium (control serum-free culture medium)

[0052] A serum-free mesenchymal stem cell culture medium, the culture medium is StemPro TM MSC SFM XenoFree. The culture medium is composed of Stem TM MSC SFM basal medium, StemPro TM MSC SFM XenoFree supplement and L-glutamine, the volume ratio of the three ingredients is 84:15:1; when used, you need to use CELLstart TM CTS TM Substrate coating.

[0053] Comparative Example 6: A serum-free mesenchymal stem cell culture medium

[0054] A human mesenchymal stem cell serum-free culture medium, comprising a basal culture medium and supplementary components, wherein the basal culture medium is a mixture of IMDM and high-glucose DMEM in a volume ratio of 1:2; the supplementary components and their final concentrations in the human mesenchymal stem cell serum-free culture medium are consistent with Table 2 in Example 1.

[0055] Experimental Example 1 Cultivation of human umbilical cord mesenchymal stem cells

[0056] The culture medium of Examples 1 and 2 and Comparative Examples 1-6 was used to separate primary human umbilical cord mesenchymal stem cells, respectively, and the specific steps were as follows:

[0057] (1) The umbilical cord of the newborn was collected under sterile conditions, the two ends of the umbilical cord were ligated with silk thread, and then immersed in PBS precooled at 4°C;

[0058] (2) Remove the umbilical cord and cut off the two ends of the umbilical cord 1 cm away from where the two ends of the umbilical cord are tied. Rinse with PBS several times and cut the umbilical cord into small pieces of about 2 cm. Discard the deformed blood vessels and areas with more blood clots.

[0059] (3) After removing the outer layer of the umbilical cord, cut the umbilical vein, tear off the inner membrane and pull the umbilical artery directly out;

[0060] (4) After cleaning the blood vessels and epithelium, wash the Wharton's jelly 2-3 times with PBS and cut into pieces of 1-3 mm. 3 About the size, evenly spread in the culture dish;

[0061] (5) Avoiding the tissue blocks, slowly dripping the culture medium of any one of Examples 1 and 2 and Comparative Examples 1-6 into the culture dish with the tissue blocks attached, so that the culture medium soaks into the tissue blocks, and then placing them in a CO2 incubator for culture. After 1-2 days of culture, add 2-3 mL of complete culture medium to the culture dish, and then replace the liquid every 3 days;

[0062] (6) After about 6-9 days of culture, cells can be observed crawling out. At this time, the cells crawling out from the edge of the tissue block are UC-MSCs, recorded as P0; thereafter, the culture medium is replaced every 3 days, 7 mL each time.

[0063] (7) When the confluence of the crawled cells reaches 80%-100%, discard the tissue, wash twice with PBS, then add 1 mL of Recombinant Trypsin-EDTA Solution (1×) (TransGen, FG302) preheated at 37°C, and let stand in a CO2 incubator for 2 minutes. When the cells are almost completely detached from the bottom of the dish, add 1 mL of the culture medium of any one of Examples 1, 2 and Comparative Examples 1-6, gently blow and mix, transfer the cell suspension to a 15 mL centrifuge tube, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant. Resuspend the cell pellet with 1 mL of the culture medium of any one of Examples 1, 2 and Comparative Examples 1-6, count the cells, and calculate the number of cells at 10,000 cells / cm 2 The cells were inoculated into a new culture dish at a density of 100 μg / cm2, recorded as P1, and supplemented with an appropriate amount of the culture medium of any one of Examples 1, 2 and Comparative Examples 1-6 to continue culturing; the subsequent cell passaging method was consistent with this, and the cells were continuously cultured to P5. Under an optical microscope, the light microscopic images of the cells cultured in Example 1, Example 2, Comparative Examples 2 and 5 at different time points (first day D1, second day D2 and third day D3) of P5 were as follows: Figure 1 shown.

[0064] from Figure 1 It can be seen that the umbilical cord mesenchymal stem cells cultured in the human mesenchymal stem cell serum-free culture medium in Examples 1 and 2 of the present invention have a long spindle-shaped morphology, and the cells are smaller and in better condition than those in the serum-containing culture medium in Comparative Example 2; and the cells are smaller in morphology and proliferate faster than those in the serum-free culture medium in Comparative Example 5.

[0065] Experimental Example 2 Comparative Experiment on Basic Culture Medium for In Vitro Proliferation Ability of Human Umbilical Cord Mesenchymal Stem Cells

[0066] The cells cultured in the four culture media of Experimental Example 1 and Comparative Examples 1-4 were taken and cultured continuously until P5. 4 / ml density was inoculated into 12-well plates, 1ml of the corresponding culture medium was added to each well, and the plates were cultured in a 37°C, 5% CO2 incubator. Starting from the second day, 3 wells of each group were taken every day to calculate the cell amount. The measurement was continued for 6 days, and a growth curve was drawn with the culture time as the horizontal axis and the total number of cell proliferation as the vertical axis. Figure 2 shown.

[0067] from Figure 2 It can be seen that when culturing human umbilical cord mesenchymal stem cells, the present invention uses a basal culture medium (Comparative Examples 1 and 3) compared to a commercially available basal culture medium (Comparative Examples 2 and 4) in combination with serum or serum substitutes, which can achieve better cell proliferation effects.

[0068] Experimental Example 3 Comparative Experiment on the Ex vivo Proliferation Ability of Human Umbilical Cord Mesenchymal Stem Cells

[0069] The cells cultured in the four groups of Experimental Example 1, Example 1, Example 2, Comparative Example 2 and Comparative Example 5 were taken at 1×10 4 / cm 2 The cells were seeded into six-well plates at a density of 1.50 μg / mL and cultured for twelve generations. The live cell data of each generation were recorded and the cumulative population doubling level (CPDL) was calculated to compare the in vitro expansion capacity of human umbilical cord mesenchymal stem cells by different culture media. Figure 3 shown.

[0070] from Figure 3 It can be seen that the expansion capacity of the culture medium of Example 1 and Example 2 of the present invention is significantly better than that of the serum-containing culture medium of Comparative Example 2 and the serum-free culture medium of Comparative Example 5.

[0071] Experimental Example 4 Comparative Experiment 2 on the Ex vivo Proliferation Ability of Human Umbilical Cord Mesenchymal Stem Cells

[0072] The cells cultured in the two groups of Experimental Example 1, Example 1 and Comparative Example 6 were taken at 1×10 4 / cm 2 The cells were seeded into six-well plates at a density of 1.50 μg / cm2 and cultured for seven generations. The live cell data of each generation were recorded and the cumulative population doubling level (CPDL) was calculated to compare the in vitro expansion capacity of human umbilical cord mesenchymal stem cells by different culture media. Figure 4 shown.

[0073] from Figure 4It can be seen that the expansion ability of the culture medium of Example 1 of the present invention is better than that of the culture medium of Comparative Example 6, indicating that the volume ratio of IDMD to high-glucose DMEM between 1:1 and 2:1 is better than the concentration outside the ratio.

[0074] Experimental Example 5 Comparative experiment on identification of surface markers of human umbilical cord mesenchymal stem cells by flow cytometry

[0075] The cells cultured in the four groups of Example 1, Example 2, Comparative Example 2 and Comparative Example 5 in Experimental Example 1 were respectively cultured continuously to P10 using their respective culture media, and the cell purity was analyzed according to the following method:

[0076] 1) Cell washing: discard the old cell culture medium and add appropriate amount of PBS to wash once

[0077] 2) Cell digestion: Discard PBS and add appropriate amount of Recombinant Trypsin-EDTA Solution (1×) (TransGen, FG302), and place in a CO2 incubator for 0.5-2 minutes. When the cells are almost completely detached from the bottom of the dish, add an equal volume of complete culture medium to neutralize. Then transfer the cell suspension to a centrifuge tube, centrifuge at 1000 rpm for 5 minutes, and discard the supernatant.

[0078] 3) Cell counting: Resuspend the cell pellet with 1 mL of PBS, count the cells, and adjust the cell concentration to 7 × 10 6 / / mL.

[0079] 4) Mix the cell suspension thoroughly, prepare 13 EP tubes, and add 100 μL of cell suspension to each tube.

[0080] Add the corresponding flow cytometry antibody to each EP tube and incubate in the dark for 30-60 minutes. The antibodies and amounts added to each tube are shown in Table 4:

[0081] Table 4 Antibodies and Addition Amounts

[0082]

[0083]

[0084] 5) After antibody incubation, wash each tube of cells three times with PBS and centrifuge at 1500 rpm for 5 min.

[0085] 6) Resuspend the cells with 300 μL PBS and transfer to the flow tube, waiting for the detection on the machine

[0086] Three replicates were performed for each culture medium, and the flow cytometry results are shown in Table 5:

[0087] Table 5 Expression of cell surface markers in umbilical cord mesenchymal stem cells cultured to P10

[0088]

[0089] The results showed that the cells continuously cultured using the four culture media of Example 1, Example 2, Comparative Example 2 and Comparative Example 5 all expressed normal surface markers. Therefore, the human mesenchymal stem cell serum-free culture medium of Example 1 and Example 2 of the present invention can ensure normal expression of cell surface markers while maintaining continuous cell passage.

[0090] Experimental Example 6 Detection of the Adipogenic, Osteogenic and Chondrogenic Differentiation Ability of Human Umbilical Cord Mesenchymal Stem Cells

[0091] The cells cultured in serum-free medium of human mesenchymal stem cells in Experimental Example 1, Example 1 and Example 2 were used to test the adipogenic, osteogenic and chondrogenic differentiation abilities. The specific test was performed using the product of Beijing Quanshijin Biotechnology Co., Ltd. Human Mesenchymal Stromal Cell Adipogenic Differentiation Medium(MM201-01), Human Mesenchymal Stromal Cell OsteogenicDifferentiation Medium(MM301-01), Human Mesenchymal Stromal CellChondrogenic Differentiation Medium (MM401-01) was used for testing according to its instructions. The results were as follows Figure 5 shown.

[0092] The results showed that mesenchymal stem cells continuously cultured to the tenth generation using the human mesenchymal stem cell serum-free culture medium of Example 1 and Example 2 could still maintain the multidirectional differentiation potential of mesenchymal stem cells.

[0093] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not limitations on the implementation methods of the present invention. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made based on the above description. It is impossible to list all the implementation methods here. All obvious changes or modifications derived from the technical solution of the present invention are still within the protection scope of the present invention.

Claims

1. A serum-free culture medium for human mesenchymal stem cells, characterized in that: The human mesenchymal stem cell serum-free culture medium comprises a basic culture medium and supplementary components; The basal culture medium is a mixture of IMDM and high-glucose DMEM, and the supplementary ingredients include VB12, insulin, hydrocortisone, progesterone, putrescine, transferrin, human serum albumin, ethanolamine, ascorbic acid, phorbol 12-myristate 13-acetate, TGF-β, bFGF, EGF, PDGF-BB, IGF1, β-mercaptoethanol, fibronectin and laminin.

2. The human mesenchymal stem cell serum-free culture medium according to claim 1, characterized in that The volume ratio of the IMDM to high-glucose DMEM is 1:1-2:

1.

3. The human mesenchymal stem cell serum-free culture medium according to claim 2, characterized in that The volume ratio of the IMDM and high-glucose DMEM is 1:

1.

4. The human mesenchymal stem cell serum-free culture medium according to claim 1, characterized in that The final concentrations of the components in the supplementary ingredients in the human mesenchymal stem cell serum-free medium are: VB12 concentration of 0.1-10 μg / ml, insulin concentration of 5-25 mg / l, hydrocortisone concentration of 0.01-0.1 mg / l, progesterone concentration of 0.01-1 mg / l, putrescine concentration of 0.01-100 mg / l, transferrin concentration of 2-30 mg / l, human serum albumin concentration of 1000-5000 mg / l, ethanolamine concentration of 0.05-100 mg / l, and transferrin concentration of 2-30 mg / l. 5mg / l, the concentration of ascorbic acid is 1-1000μg / ml, the concentration of phorbol 12-myristate 13-acetate is 0.01-1mg / l, the concentration of TGF-β is 0.001-1mg / l, the concentration of bFGF is 0.001-1mg / l, the concentration of EGF is 0.001-1mg / l, the concentration of PDGF-BB is 0.001-1mg / l, the concentration of IGF1 is 0.001-1mg / l, and the concentration of β-mercaptoethanol is 1-10mg / l.

5. The human mesenchymal stem cell serum-free culture medium according to claim 4, characterized in that The final concentrations of the components in the supplementary ingredients in the mesenchymal stem cell serum-free medium are: VB12 concentration of 0.5-5 μg / ml, insulin concentration of 10-20 mg / l, hydrocortisone concentration of 0.01-0.05 mg / l, progesterone concentration of 0.05-1 mg / l, putrescine concentration of 1-20 mg / l, transferrin concentration of 20-30 mg / l, human serum albumin concentration of 2000-4000 mg / l, and ethanolamine concentration of 1-5 mg / l. , the concentration of ascorbic acid is 1-100 μg / ml, the concentration of phorbol 12-myristate 13-acetate is 0.05-1 mg / l, the concentration of TGF-β is 0.01-0.1 mg / l, the concentration of bFGF is 0.01-0.1 mg / l, the concentration of EGF is 0.01-0.1 mg / l, the concentration of PDGF-BB is 0.01-0.1 mg / l, the concentration of IGF1 is 0.01-0.1 mg / l, and the concentration of β-mercaptoethanol is 1-5 mg / l.

6. The human mesenchymal stem cell serum-free culture medium according to claim 1, characterized in that The final concentrations of the components in the supplementary ingredients in the human mesenchymal stem cell serum-free culture medium are: the concentration of fibronectin is 0.1-10 mg / l, the concentration of laminin is 0.1-10 mg / l, and the concentration ratio of fibronectin to laminin is 1:1-3:

2.

7. The human mesenchymal stem cell serum-free culture medium according to claim 6, characterized in that The final concentrations of the components in the supplementary ingredients in the human mesenchymal stem cell serum-free culture medium are: the concentration of fibronectin is 3-10 mg / l, the concentration of laminin is 1-5 mg / l, and the concentration ratio of fibronectin to laminin is 3:

2.

8. The human mesenchymal stem cell serum-free culture medium according to claim 1, characterized in that The supplementary components also include at least one of IL-1β and IL-6; preferably, the final concentration of IL-1β in the human mesenchymal stem cell serum-free culture medium is 0.001-1 mg / l, and the final concentration of IL-6 in the human mesenchymal stem cell serum-free culture medium is 0.001-1 mg / l; more preferably, the final concentration of IL-1β in the human mesenchymal stem cell serum-free culture medium is 0.001-0.01 mg / l, and the final concentration of IL-6 in the human mesenchymal stem cell serum-free culture medium is 0.001-0.01 mg / l.

9. The human mesenchymal stem cell serum-free culture medium according to claim 1, characterized in that The pH of the human mesenchymal stem cell serum-free culture medium is 7.3-7.4, the osmotic pressure is 280-320 mOsm, and the endotoxin is less than 0.5 EU / ml.

10. Use of the human mesenchymal stem cell serum-free culture medium according to any one of claims 1 to 9 in culturing human mesenchymal stem cells.

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