Serum-free mesenchymal stem cell culture method and application thereof

Abstract

The application discloses a serum-free mesenchymal stem cell culture method and application thereof, and particularly discloses a serum-free mesenchymal stem cell culture medium, which comprises a first-stage culture solution, a second-stage culture solution and a third-stage culture solution; the first-stage culture solution comprises a basic culture medium, human platelet lysate and transforming growth factor-beta 1; the second-stage culture solution comprises the basic culture medium, the human platelet lysate, basic fibroblast growth factor, insulin and ascorbic acid; and the third-stage culture solution comprises the basic culture medium, the human platelet lysate, vascular endothelial growth factor, heparin and interferon gamma. The culture medium comprises three serum-free culture solution systems suitable for different stages of in-vitro expansion of mesenchymal stem cells, and can not only promote the proliferation efficiency of the mesenchymal stem cells in in-vitro culture, but also maintain the stable phenotype of the mesenchymal stem cells and improve the cell purity.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a serum-free mesenchymal stem cell culture method and its application. Background Technology

[0002] Mesenchymal stem cells (MSCs) are a type of pluripotent stem cell derived from the mesoderm, also known as multipotent stromal cells. They are mainly distributed in various connective tissues and organ stroma, including bone marrow, adipose tissue, perinatal tissues (such as the umbilical cord, placenta, amnion, and amniotic fluid), and mucous membranes. These cells possess self-renewal, multi-lineage differentiation potential (they can differentiate into various cell types such as fat, bone, and cartilage under specific conditions), and significant immunomodulatory functions, thus attracting considerable attention in regenerative medicine and disease treatment research.

[0003] The culture media used for in vitro culture of mesenchymal stem cells generally require serum supplementation, which introduces the risk of contamination with exogenous viruses and pathogenic factors during cell culture. Furthermore, due to the numerous unknown components in serum and inconsistencies in bioactive factors between different batches, product and experimental results are poorly reproducible. Residual serum can also easily cause allergic reactions in recipients, rendering it unsuitable for clinical research. Therefore, to overcome the various drawbacks of serum, it is essential to find serum substitutes or adopt serum-free culture media in the cell culture process.

[0004] In existing technologies, traditional two-dimensional culture systems lack simulation of the in vivo microenvironment, resulting in poor cell adhesion, slow proliferation rate, and a tendency to senescence (such as β-cell aging) after long-term culture. Increased galactosidase activity and decreased differentiation efficiency (osteogenic / adipogenic induction efficiency is often below 80%). Meanwhile, traditional digestion methods (such as single-enzyme digestion) do not adequately dissociate umbilical cord tissue, resulting in low cell yield. Conventional culture conditions (such as normoxic environment and lack of mechanical stimulation) cannot effectively maintain high expression of stem cell surface markers (such as CD73, CD90, and CD105) and easily activate hematopoietic cell markers (such as CD34 and CD45), affecting cell purity. Summary of the Invention

[0005] The present invention aims to at least solve one of the technical problems existing in the prior art. To this end, the present invention proposes a serum-free mesenchymal stem cell culture medium, which includes three serum-free culture medium systems adapted to different stages of in vitro expansion of mesenchymal stem cells. This not only promotes the proliferation efficiency of mesenchymal stem cells in in vitro culture, but also maintains their stable phenotype and improves cell purity. By supplementing with human platelet lysis buffer, the use of animal serum is avoided, reducing the risk of contamination by exogenous viruses and pathogenic factors.

[0006] The present invention also proposes a serum-free method for culturing mesenchymal stem cells.

[0007] The present invention also proposes the application of the above-mentioned serum-free mesenchymal stem cell culture medium and serum-free mesenchymal stem cell culture method.

[0008] According to a first aspect of the present invention, a serum-free mesenchymal stem cell culture medium is provided, the serum-free mesenchymal stem cell culture medium comprising a first-stage culture medium, a second-stage culture medium and a third-stage culture medium; The first stage culture medium includes basal culture medium, human platelet lysis buffer, and transforming growth factor-β1; The second-stage culture medium includes basal culture medium, human platelet lysis buffer, basic fibroblast growth factor, insulin, and ascorbic acid; The third-stage culture medium includes basal culture medium, human platelet lysate, vascular endothelial growth factor, heparin, and interferon-γ.

[0009] In some embodiments of the present invention, the concentration of human platelet lysis buffer in the culture medium of the first stage is 5% to 15% (v / v).

[0010] In some embodiments of the present invention, the concentration of human platelet lysis buffer in the first-stage culture medium is 6% to 10% (v / v).

[0011] Human platelet lysate, as a core supplement, is rich in various growth factors and is key to replacing animal serum. Using human platelet lysate avoids the risk of contamination by exogenous viruses and pathogenic factors during cell culture.

[0012] In some embodiments of the present invention, the concentration of transforming growth factor-β1 in the culture medium of the first stage is 1~10 ng / mL.

[0013] In some embodiments of the present invention, the concentration of transforming growth factor-β1 in the culture medium of the first stage is 1~5 ng / mL.

[0014] Transforming growth factor-β1 (TGF-β1) plays a crucial role in the in vitro proliferation of mesenchymal stem cells (MSCs), especially in serum-free culture systems. Its core mechanism involves specifically activating FAK phosphorylation, thereby initiating the Akt-mTOR-S6K1 proliferation pathway, ultimately accelerating the G1 / S phase cell cycle transition and promoting cell proliferation. Specifically, as a multifunctional cytokine, TGF-β1 first binds to TGF-β receptor II (TβRII) on the MSC cell membrane, inducing a conformational change and forming a dimer. TβRII further recruits and phosphorylates the GS domain of TGF-β receptor I (TβRI), activating its serine / threonine kinase activity. This process initiates the non-canonical Smad signaling pathway, shifting towards pro-proliferative signal transduction.

[0015] In some embodiments of the present invention, the concentration of human platelet lysis buffer in the second-stage culture medium is 5% to 15% (v / v).

[0016] In some embodiments of the present invention, the concentration of human platelet lysis buffer in the second-stage culture medium is 6% to 10% (v / v).

[0017] In some embodiments of the present invention, the concentration of basic fibroblast growth factor in the second-stage culture medium is 5-15 ng / mL.

[0018] In some embodiments of the present invention, the concentration of basic fibroblast growth factor in the second-stage culture medium is 5-10 ng / mL.

[0019] In some embodiments of the present invention, the concentration of insulin in the second-stage culture medium is 1~10 μg / mL.

[0020] In some embodiments of the present invention, the concentration of insulin in the second-stage culture medium is 3~7 μg / mL.

[0021] Basic fibroblast growth factor (bFGF) and insulin independently activate and synergistically enhance two core proliferative signaling pathways, Ras-Raf-MEK-ERK and PI3K-Akt-mTOR, propelling cells from the G0 / G1 phase into the S phase, accelerating cell cycle progression, and inhibiting apoptosis, thereby achieving efficient and stable stem cell expansion. Specifically, bFGF, as a heparin-binding growth factor, first binds to heparan sulfate proteoglycan (HSPG) on the cell surface to form a stable complex before binding to fibroblast growth factor receptors (FGFR1-4, RTK family). Receptor dimerization triggers autophosphorylation of the intracellular tyrosine kinase domain, generating multiple phosphorylated tyrosine sites, serving as docking platforms for downstream signaling molecules. Insulin binds to the insulin receptor (IR, dimer RTK), triggering receptor autophosphorylation and activating intracellular kinase activity PMC, which activates IR phosphorylates insulin receptor substrates (IRS-1 / 2), generating multiple phosphorylated tyrosine sites to recruit downstream signaling molecules.

[0022] bFGF, via FRS2, and insulin, via IRS-1 / 2, converge in the Ras-Raf-MEK-ERK cascade, resulting in a significantly higher ERK1 / 2 phosphorylation level than that of stimulation alone. bFGF binds to PI3K via PLCγ, and insulin binds to PI3K via IRS, thus doubly activating PI3K, producing more PIP3, and enhancing Akt activation.

[0023] In some embodiments of the present invention, the concentration of ascorbic acid in the second-stage culture medium is 50~120 μg / mL.

[0024] In some embodiments of the present invention, the concentration of ascorbic acid in the second-stage culture medium is 70-90 μg / mL.

[0025] Ascorbic acid has antioxidant properties and can maintain collagen synthesis.

[0026] In some embodiments of the present invention, the concentration of human platelet lysis buffer in the third-stage culture medium is 5% to 15% (v / v).

[0027] In some embodiments of the present invention, the concentration of human platelet lysis buffer in the third-stage culture medium is 6% to 10% (v / v).

[0028] In some embodiments of the present invention, the concentration of vascular endothelial growth factor in the third-stage culture medium is 10-30 ng / mL.

[0029] In some embodiments of the present invention, the concentration of vascular endothelial growth factor in the third-stage culture medium is 10-20 ng / mL.

[0030] In some embodiments of the present invention, the concentration of heparin in the third-stage culture medium is 0.1~3 IU / mL.

[0031] In some embodiments of the present invention, the concentration of heparin in the third-stage culture medium is 0.5~2 IU / mL.

[0032] Vascular endothelial growth factor (VEGF) and heparin significantly reduce the procoagulant activity of mesenchymal stem cells (MSCs) by synergistically regulating transcription factors such as NF-κB, inhibiting TF gene transcription and protein expression, and enhancing the expression of TFPI anticoagulant factor. At the same time, they promote MSC proliferation and maintain the expression of core surface markers such as CD73 / CD90 / CD105 by activating the VEGFR2-PI3K / Akt / ERK signaling pathway, providing key assurance for safe clinical infusion.

[0033] In some embodiments of the present invention, the concentration of interferon γ in the third-stage culture medium is 20-50 ng / mL.

[0034] In some embodiments of the present invention, the concentration of interferon γ in the third-stage culture medium is 25~35 ng / mL.

[0035] Short-term IFN-γ stimulation can increase the expression of immunomodulatory factors (such as IDO and PGE2) in MSCs, enhancing their therapeutic potential. Under low concentration or short-term stimulation conditions, IFN-γ promotes proliferation through the following mechanisms: activating the PI3K / Akt pathway and enhancing cell survival signals; and upregulating the expression of the anti-apoptotic protein Bcl-2, thereby reducing apoptosis.

[0036] In some embodiments of the present invention, the basal culture medium includes DMEM culture medium and / or α-MEM culture medium.

[0037] According to a second aspect of the present invention, a method for culturing serum-free mesenchymal stem cells is provided, wherein the serum-free mesenchymal stem cell culture medium described in the first aspect of the present invention is used for culturing, and the method includes the following steps: S1: Pretreatment of primary tissue; S2: Mix the pretreated tissue blocks with the enzymatic hydrolysate for enzymatic separation; S3: Centrifuge to collect the cells from the enzymatic digestion system in step S2, resuspend the cells using the culture medium from the first stage, and seed the cell suspension into a culture plate coated with RGD peptides and culture for 1-3 days; S4: Replace the entire culture medium in step S3 with the second-stage culture medium and culture for 4-8 days, replacing half of the second-stage culture medium every 2 days during this period; finally, replace the entire medium with the third-stage culture medium and continue culturing for 1-3 days to obtain primary mesenchymal stem cells.

[0038] In some embodiments of the present invention, the primary tissue in step S1 may be derived from at least one of the following: umbilical cord, amnion, fat, and placental tissue.

[0039] In some embodiments of the present invention, the enzymatic hydrolysate in step S2 includes 0.1-2 g / L collagenase type IV and 0.1-2 g / L hyaluronidase.

[0040] In some embodiments of the present invention, the solvent of the enzymatic hydrolysate in step S2 is DMEM medium and / or α-MEM medium.

[0041] In some embodiments of the present invention, the enzymatic digestion and separation conditions in step S2 include: digestion at 35°C to 39°C with shaking at 40 to 60 rpm for 30 to 60 minutes.

[0042] In some embodiments of the present invention, before centrifuging and collecting cells in step S3, the enzymatic hydrolysis system is passed through a 60-80 μm cell sieve.

[0043] In some embodiments of the present invention, the centrifugation conditions in step S3 include centrifugation at 400-600 g for 6-10 min at 2°C-6°C.

[0044] In some embodiments of the present invention, the cell density of the cell suspension in step S3 is 0.8 × 10⁻⁶. 4 pcs / cm 2 ~1.6×10 4 pcs / cm 2 .

[0045] In some embodiments of the present invention, the coating method for the culture plate coated with RGD peptide in step S3 includes: Add 1-3 mL of basal culture medium containing 2-8 μg / mL RGD peptide to the culture plate, incubate overnight at 2℃-6℃, discard the coating solution the next day and wash, then inoculate cells.

[0046] RGD peptide, short for Arginylglycylaspartic Acid, is also known as L-arginylglycylaspartic-L-aspartic acid. Its structure is a tripeptide derived from the amino acid sequence Arg-Gly-Asp (RGD) in the fibroblast attachment domain, a key sequence fragment for cell surface protein binding. This invention pre-coating culture plates with RGD peptide provides a clear adhesion signal, synergistically accelerating cell cycle entry with endogenous pathways.

[0047] In some embodiments of the present invention, the basal culture medium includes DMEM culture medium and / or α-MEM culture medium.

[0048] According to a third aspect of the present invention, the application of the serum-free mesenchymal stem cell culture medium described in the first aspect of the present invention or the serum-free mesenchymal stem cell culture method described in the second aspect of the present invention in the in vitro culture of mesenchymal stem cells is proposed.

[0049] In some embodiments of the present invention, the in vitro cultured mesenchymal stem cells include primary isolated mesenchymal stem cells.

[0050] In some embodiments of the present invention, the primary isolated mesenchymal stem cells are derived from at least one of the following sources: umbilical cord, amnion, fat, and placental tissue.

[0051] The present invention has at least the following beneficial effects: The serum-free mesenchymal stem cell culture medium provided by this invention does not involve the addition of animal serum, thus avoiding the risk of contamination by exogenous viruses and pathogenic factors during cell culture. This culture medium includes three serum-free culture medium systems adapted to different stages of in vitro mesenchymal stem cell expansion, which not only promotes the proliferation efficiency of mesenchymal stem cells in in vitro culture but also maintains their stable phenotype and improves cell purity.

[0052] The serum-free mesenchymal stem cell (MSC) culture method provided by this invention actively intervenes in the TGF-β1 / FAK / Akt-mTOR pathway, which has been proven crucial for MSC proliferation. By adding TGF-β1 and optimizing the adhesion environment in the early stages, it aims to efficiently drive cells into a proliferative state from the outset. The subsequent second and third stages are specifically designed according to the needs of cell proliferation and differentiation, supplementing corresponding growth factors. Through synergistic regulation of key signaling pathways, the proliferation efficiency and clinical applicability of MSCs are simultaneously optimized. Detailed Implementation

[0053] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0054] Unless otherwise specified, all instruments and reagents used in the following examples are commercially available instruments and reagents that were purchased through conventional channels.

[0055] Example 1 This embodiment provides a serum-free mesenchymal stem cell culture medium, which includes three serum-free culture medium systems for different stages of in vitro expansion of mesenchymal stem cells. The specific formulations of the three culture medium systems are as follows: The first stage culture medium was based on DMEM medium, with the following components added to the final concentration: 8% (v / v) human platelet lysis buffer (hPL) and 3 ng / mL transforming growth factor-β1 (TGF-β1).

[0056] The second-stage culture medium was based on DMEM medium, with the following components added to final concentrations: 8% (v / v) hPL, 7 ng / mL basic fibroblast growth factor (bFGF), 5 μg / mL insulin, and 80 μg / mL ascorbic acid.

[0057] The third stage culture medium was based on DMEM medium, with the following components added to the final concentration: 8% (v / v) hPL, 15 ng / mL vascular endothelial growth factor (VEGF), 1 IU / mL heparin and 30 ng / mL interferon-γ (IFN-γ).

[0058] Example 2 This embodiment provides a serum-free mesenchymal stem cell culture method, which uses the serum-free mesenchymal stem cell culture medium provided in Example 1. The specific steps are as follows: 1. Umbilical cord tissue pretreatment: Fresh human umbilical cord tissue donated by volunteers was thoroughly washed with D-Hanks balanced salt solution to remove surface blood contamination. Blood vessels and the outer membrane of Wharton's jelly were then removed using surgical scissors to obtain Wharton's jelly tissue. The tissue was rinsed three times with D-PBSA buffer containing 1% (v / v) penicillin-dextrin antibodies for 5 minutes each time, and then minced into 2 mm pieces. 3 The left and right tissue blocks.

[0059] 2. Enzymatic separation: The tissue blocks obtained in step 1 were placed in the enzymatic digestion solution and digested at 37°C with shaking at 50 rpm for 50 min. The enzymatic digestion solution was DMEM / F12 medium containing 1 g / L collagenase type IV and 0.5 g / L hyaluronidase.

[0060] 3. Primary inoculation (Phase 1): After step 2 terminates digestion, the enzymatic digestion system is passed through a 70 μm cell sieve, centrifuged at 500 g for 8 min at 4°C to collect cells, and resuspended in the first-stage culture medium provided in Example 1 to a concentration of 1.2 × 10⁻⁶ cells / mL. 4 pcs / cm 2The RGD peptides were pre-coated into the wells of a 6-well culture plate, and then the cell suspension was seeded into the culture plate.

[0061] The above method for coating RGD peptides is as follows: Add 2 mL of DMEM medium containing 5 μg / mL RGD peptides to a 6-well culture plate, incubate at 4°C overnight, discard the coating solution the next day, wash 3 times with PBS buffer, and then wash once with DMEM medium before seeding cells.

[0062] 4. Primary culture (second stage): The cells seeded in step 3 were cultured in an incubator at 37°C, 5% CO2, and 95% humidity for 2 days. Then, the medium was completely replaced with the second-stage culture medium provided in Example 1, and the cells were cultured for another 6 days, with half of the second-stage culture medium replaced every 2 days. Finally, the medium was completely replaced with the third-stage culture medium provided in Example 1, and the cells were cultured for another 2 days to obtain primary mesenchymal stem cells.

[0063] Comparative Example 1 This comparative example provides a serum-free mesenchymal stem cell culture method. The only difference between this method and Example 2 is that the step of coating RGD peptide in primary seeding in step 3 is omitted. All other steps are the same as in Example 2.

[0064] Comparative Example 2 This comparative example provides a serum-free mesenchymal stem cell culture method. The only difference between this method and Example 2 is that TGF-β1 in the first-stage culture medium used for primary seeding in step 3 is omitted. All other steps are the same as in Example 2.

[0065] Comparative Example 3 This comparative example provides a serum-free mesenchymal stem cell culture method. The only difference between this method and Example 2 is that the bFGF in the second-stage culture medium used for primary culture in step 4 is omitted. All other steps are the same as in Example 2.

[0066] Comparative Example 4 This comparative example provides a serum-free mesenchymal stem cell culture method. The only difference between this method and Example 2 is that the insulin in the second-stage culture medium used for primary culture in step 4 is omitted. All other steps are the same as in Example 2.

[0067] Comparative Example 5 This comparative example provides a serum-free mesenchymal stem cell culture method. The only difference between this method and Example 2 is that ascorbic acid in the second-stage culture medium used for primary culture in step 4 is omitted. All other steps are the same as in Example 2.

[0068] Comparative Example 6 This comparative example provides a serum-free mesenchymal stem cell culture method. The only difference between this method and Example 2 is that VEGF in the third-stage culture medium used for primary culture in step 4 is omitted. All other steps are the same as in Example 2.

[0069] Comparative Example 7 This comparative example provides a serum-free mesenchymal stem cell culture method. The only difference between this method and Example 2 is that heparin in the third-stage culture medium used in the primary culture in step 4 is omitted. All other steps are the same as in Example 2.

[0070] Comparative Example 8 This comparative example provides a serum-free mesenchymal stem cell culture method. The only difference between this method and Example 2 is that the IFN-γ in the third-stage culture medium used for primary culture in step 4 is omitted. All other steps are the same as in Example 2.

[0071] Comparative Example 9 This comparative example provides a serum-free mesenchymal stem cell culture method. The only difference between this method and Example 2 is that in the primary culture in step 4, the third-stage culture medium that is completely replaced at the end is replaced with the second-stage culture medium that is completely replaced. All other steps are the same as in Example 2.

[0072] Comparative Example 10 This comparative example provides a serum-free mesenchymal stem cell culture method. The only difference between this method and Example 2 is that the first-stage culture medium used in the primary seeding step 3 is replaced with the second-stage culture medium, and the coating of RGD peptide is omitted. All other steps are the same as in Example 2.

[0073] Comparative Example 11 This comparative example provides a serum-free mesenchymal stem cell culture method. The only difference between this method and Example 2 is that all culture systems used in steps 3 and 4 use the second-stage culture medium, while the remaining steps are the same as in Example 2.

[0074] Test case This experiment tested the proliferative activity and surface marker expression of mesenchymal stem cells obtained by the serum-free mesenchymal stem cell culture methods provided in Example 2 and Comparative Examples 1-11. The specific experimental methods and results are as follows: 1. Proliferation activity test Using the serum-free mesenchymal stem cell culture methods provided in Example 2 and Comparative Examples 1-11, P0 generation mesenchymal stem cells were isolated and cultured from human umbilical cord tissue for proliferation activity testing and cell viability testing.

[0075] Mesenchymal stem cells from each P0 generation were collected, counted, and their cell expansion fold was calculated. The cell viability of the P0 generation mesenchymal stem cells was detected using trypan blue staining, and the results are shown in Table 1.

[0076] The formula for calculating cell expansion fold is: Cell expansion fold = Number of cells in passage P0 / Initial number of cells inoculated.

[0077] The formula for calculating cell viability is: Cell viability (%) = Number of viable cells / Total number of cells × 100%.

[0078] Table 1 Results of proliferation activity test

[0079] As shown in Table 1, the mesenchymal stem cells obtained in Example 2 of the present invention have a much greater cell proliferation capacity than those in Comparative Examples 1-11, and the cell viability also reaches 99.10%.

[0080] In the comparative example: Due to the lack of RGD peptide coating, the culture environment of Comparative Example 1 lacked adhesion signals, which prevented the cell cycle from progressing. Therefore, the cell expansion fold was slightly lower than that of Example 2. In Comparative Example 2, due to the lack of TGF-β1 in the culture medium during the first stage, FAK phosphorylation could not be promoted, and the proliferation-related signaling pathways could not be fully activated. Therefore, the cell expansion rate was significantly lower than that in Example 2. In Comparative Examples 3 and 4, the mitotic signals in the mesenchymal stem cells obtained from the second-stage culture medium were not fully activated due to the lack of bFGF and insulin, respectively, resulting in a significantly lower cell expansion rate compared to Example 2. Comparative Example 5: Ascorbic acid was missing in the culture medium during the second stage. Ascorbic acid can resist oxidation and maintain collagen synthesis. Without ascorbic acid, the cell proliferation rate and cell viability were affected. In Comparative Examples 6 and 7, due to the lack of VEGF and heparin in the third-stage culture medium, umbilical cord-derived mesenchymal stem cells expressed high levels of tissue factors (TF, CD142), which may lead to the risk of thrombosis after mesenchymal stem cell infusion, and cell proliferation capacity and cell viability are also affected. Comparative Example 8 showed that the lack of IFN-γ in the third-stage culture medium led to a decrease in cell migration ability and anti-apoptotic ability, which in turn affected cell proliferation ability and cell viability. Comparative Example 9 showed decreased stability and proliferation activity due to the replacement of the third-stage culture medium with the second-stage culture medium, which resulted in the lack of VEGF, heparin, and IFN-γ in the last two days of culture. In Comparative Example 10, since the first-stage culture medium was replaced with the second-stage culture medium, the initial stage of culture is the cell adhesion and activation stage. In the absence of TGF-β1 and RGD peptides, the primary cells could not quickly start the cell adhesion and proliferation program, and the Akt-mTOR-S6K1 proliferation pathway was difficult to activate. Therefore, the cell proliferation fold obtained was much lower than that in Example 2. Comparative Example 11, due to the use of the second-stage culture medium throughout the process, did not clearly specify the appropriate serum-free culture medium system for different stages of in vitro expansion of mesenchymal cells. As a result, the adhesion, activation and proliferation of mesenchymal stem cells were affected. Using only the second-stage culture medium throughout the process could not achieve the excellent proliferation-promoting effect of Example 2.

[0081] 2. Expression of surface markers Mesenchymal stem cells were isolated and cultured from human umbilical cord tissue using the mesenchymal stem cell culture methods provided in Example 2 and Comparative Examples 1-11, respectively. The cells were continuously passaged to P4, and then collected for flow cytometry analysis. Surface markers detected included positive markers CD73, CD90, and CD105, and negative markers CD34, CD45, CD14, CD19, and HLA-DR. Positive markers required high expression (≥95%), while negative markers required no expression (≤2%). The results are shown in Table 2.

[0082] Table 2 Positive rate of surface markers (%)

[0083] As shown in Table 2, the mesenchymal stem cells obtained in Example 2 and Comparative Examples 1-8 of this invention exhibit stable stem cell characteristics, with positive rates of CD90, CD105, and CD73 all exceeding 95%, and positive rates of CD19, CD14, CD34, CD45, and HLA also showing good results. The DR positivity rate was below 2% for all of them. However, the comparative examples 9-11 had unstable cell phenotypes because the culture medium system was adjusted for different culture stages. In particular, comparative examples 9 and 11 did not use the third-stage culture medium in the final harvest stage. The proportion of positive cells for the negative markers exceeded 2%, indicating that the third-stage culture medium provided in Example 1 of this invention plays a key role in the phenotypic stability of mesenchymal stem cells in vitro.

[0084] The embodiments of the present invention have been described in detail above. However, the present invention is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the spirit of the present invention. Furthermore, unless otherwise specified, the embodiments of the present invention and the features thereof can be combined with each other.

Claims

1. A serum-free mesenchymal stem cell culture medium, characterized in that, The serum-free mesenchymal stem cell culture medium includes a first-stage culture medium, a second-stage culture medium, and a third-stage culture medium; The first stage culture medium includes basal culture medium, human platelet lysis buffer, and transforming growth factor-β1; The second-stage culture medium includes basal culture medium, human platelet lysis buffer, basic fibroblast growth factor, insulin, and ascorbic acid; The third-stage culture medium includes basal culture medium, human platelet lysate, vascular endothelial growth factor, heparin, and interferon-γ.

2. The serum-free mesenchymal stem cell culture medium according to claim 1, characterized in that, The concentration of human platelet lysis buffer in the culture medium during the first stage was 5% to 15% (v / v). Preferably, the concentration of transforming growth factor-β1 in the culture medium during the first stage is 1~10 ng / mL.

3. The serum-free mesenchymal stem cell culture medium according to claim 1, characterized in that, The concentration of human platelet lysis buffer in the culture medium during the second stage is 5%~15% (v / v). Preferably, the concentration of basic fibroblast growth factor in the culture medium during the second stage is 5-15 ng / mL; Preferably, the concentration of insulin in the culture medium during the second stage is 1~10 μg / mL; Preferably, the concentration of ascorbic acid in the culture medium of the second stage is 50~120 μg / mL.

4. The serum-free mesenchymal stem cell culture medium according to claim 1, characterized in that, The concentration of human platelet lysis buffer in the culture medium of the third stage is 5%~15% (v / v). Preferably, the concentration of vascular endothelial growth factor in the third-stage culture medium is 10-30 ng / mL; Preferably, the concentration of heparin in the third-stage culture medium is 0.1~3 IU / mL; Preferably, the concentration of interferon γ in the third-stage culture medium is 20~50 ng / mL.

5. A serum-free method for culturing mesenchymal stem cells, characterized in that, The serum-free mesenchymal stem cell culture method uses the serum-free mesenchymal stem cell culture medium according to any one of claims 1 to 4, and includes the following steps: S1: Pretreatment of primary tissue; S2: Mix the pretreated tissue blocks with the enzymatic hydrolysate for enzymatic separation; S3: Centrifuge to collect the cells from the enzymatic digestion system in step S2, resuspend the cells using the culture medium from the first stage, and seed the cell suspension into a culture plate coated with RGD peptides and culture for 1-3 days; S4: Replace the entire culture medium in step S3 with the second-stage culture medium and culture for 4-8 days, replacing half of the second-stage culture medium every 2 days during this period; finally, replace the entire medium with the third-stage culture medium and continue culturing for 1-3 days to obtain primary mesenchymal stem cells.

6. The method for culturing serum-free mesenchymal stem cells according to claim 5, characterized in that, The enzymatic hydrolysate in step S2 includes 0.1-2 g / L collagenase type IV and 0.1-2 g / L hyaluronidase; Preferably, the enzymatic digestion and separation conditions in step S2 include: digestion at 35℃~39℃ with shaking at 40~60 rpm for 30~60 min.

7. The method for culturing serum-free mesenchymal stem cells according to claim 5, characterized in that, Before centrifuging and collecting cells as described in step S3, the enzymatic digestion system will be passed through a 60-80 μm cell sieve.

8. The method for culturing serum-free mesenchymal stem cells according to claim 5, characterized in that, The centrifugation conditions described in step S3 include centrifugation at 400-600 g for 6-10 min at 2℃-6℃; Preferably, the cell density of the cell suspension in step S3 is 0.8 × 10⁻⁶. 4 pcs / cm 2 ~1.6×10 4 pcs / cm 2 .

9. The method for culturing serum-free mesenchymal stem cells according to claim 5, characterized in that, The coating method for the culture plate coated with RGD peptides in step S3 includes: Add 1-3 mL of basal culture medium containing 2-8 μg / mL RGD peptide to the culture plate, incubate overnight at 2℃-6℃, discard the coating solution the next day and wash, then inoculate cells.

10. The use of the serum-free mesenchymal stem cell culture medium of any one of claims 1 to 4 or the serum-free mesenchymal stem cell culture method of any one of claims 5 to 9 in the in vitro culture of mesenchymal stem cells.

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