Serum-free culture medium and application thereof in mesenchymal stem cell culture

Abstract

The invention provides a non-heterogeneity serum-free culture medium for large-scale culture of human umbilical cord mesenchymal stem cells and a culture method. Specifically, the present invention provides a serum-free cell culture medium comprising a basic culture medium and additive components wherein the additive components comprise transferrin, insulin, selenium or a salt thereof, transforming growth factor-beta (TGF-beta), a platelet lysate composition, and a glutamine supplement. The invention further provides a method for culturing the mesenchymal stem cells through the culture medium and application of the mesenchymal stem cells. The culture medium disclosed by the invention has an excellent supporting effect on cell morphology, cell proliferation capacity, multiplication time and the like.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of stem cell culture, and particularly relates to a serum-free culture medium for mesenchymal stem cells (such as umbilical cord mesenchymal stem cells) and a culture method. BACKGROUND

[0002] Traditional stem cell culture uses basic culture medium plus animal-derived serum. The batch of animal-derived serum is unstable and has the risk of exogenous viral contamination. At the same time, the existing culture medium on the market has the problems of insufficient support for cell proliferation, long cell doubling time, and rapid aging, which cannot meet the demand of cell drug research and development for large-scale healthy cell production.

[0003] Patent document CN115433712B discloses a kind of umbilical cord mesenchymal stem cell proliferation culture medium and culture method: a kind of umbilical cord mesenchymal stem cell proliferation culture medium, including basic culture medium and additive, the composition of the additive is: eicosapentaenoic acid, piperine, luffa net powder, glutathione, sodium selenite, epidermal growth factor, vitamin C.Patent document CN115433712B also discloses that the culture medium composition is safe, improves the proliferation speed and cell activity of umbilical cord mesenchymal stem cells, shortens the adhesion time, makes the proliferation efficiency of umbilical cord mesenchymal stem cells higher, reduces the culture cost.Patent document CN113151165B discloses a kind of human umbilical cord mesenchymal stem cell expansion culture medium and culture method: provide a kind of human umbilical cord mesenchymal stem cell expansion culture medium, without adding serum, by basic culture medium and the following ingredients added in basic culture medium group: glutathione, alpha-tocopherol, eudesm, estradiol valerate, acetazolamide, sodium selenite, vitamin C, insulin.CN113151165B also discloses that the culture medium can improve the culture efficiency of umbilical cord mesenchymal stem cells, promotes the secretion of cytokine.Patent document CN112608891B discloses a kind of mesenchymal stem cell serum-free culture medium and its application: by basic culture medium and the added component TGF-β3 (human transforming growth factor) and ascorbic acid, can promote cell synthesis and secretion fibronectin, collagen and other extracellular matrix, and promote cell migration, make mesenchymal stem cells have good adhesion effect, and also have good improvement effect on proliferation.Patent document CN112608891B also discloses compared with other basic culture medium (such as DMEM high sugar, low sugar, a-MEM, DMEM / F12, RPMI1640 etc.), when IMDM culture medium is applied in the present application, whether cell growth speed or cell homogeneity has better effect. However, existing culture medium is difficult to carry out for example more than 10 generations long-term subculture or in the process of subculture cell proliferation ability is low, senescent cell and doubling time greatly increase, lead to the reduction of culture efficiency.Patent document CN106906182B provides a serum-free culture medium that can be used for umbilical cord mesenchymal stem cell culture, comprising a basic culture medium and an additive component added to the basic culture medium, wherein the additive component includes L-glutamine, non-essential amino acids, L-ascorbic acid, sodium selenite, fibronectin, ethanolamine, hydrocortisone, trypsin inhibitor, human transferrin, human insulin, bFGF, TGF-β1 and PDGF-BB, wherein the concentration of L-glutamine is 1-5 mM, the concentration of non-essential amino acids is 1-5 mM, the concentration of L-ascorbic acid is 32-80 mg / L, the concentration of sodium selenite is 7-20 ug / L, the concentration of fibronectin is 5-50 mg / L, the concentration of ethanolamine is 1-5 mg / L, the concentration of hydrocortisone is 5-20 mg / L, the concentration of trypsin inhibitor is 1-2 mg / L, the concentration of human transferrin is 5-20 mg / L, the concentration of human insulin is 10-30 mg / L, the concentration of bFGF is 10-30 ug / L, the concentration of TGF-β1 is 1-10 ug / L, and the concentration of PDGF-BB is 1-20 ug / L. Patent application document CN113736729A provides a UCMSC serum-free culture medium characterized by adding fibronectin, mucin and vitronectin, as well as non-essential amino acids, glutamine, recombinant human insulin, recombinant human blood albumin, recombinant human transferrin, recombinant human epidermal growth factor, recombinant human basic fibroblast growth factor, recombinant human platelet-derived growth factor, recombinant human transforming growth factor β1, recombinant human activin A, L-glutathione, L-ascorbic acid, cholesterol, linoleic acid, linolenic acid, sodium selenite and a basic culture medium. However, various complex components including expensive proteins need to be added in these culture media, and it is difficult to reduce the culture cost when large-scale culture is carried out; at the same time, the doubling time is usually greater than 25 hours during subculture, which reduces the culture efficiency and further increases the culture cost. SUMMARY

[0004] In order to meet the needs of cell production, the inventors have conducted in-depth research on cell culture systems and cell culture components, thereby providing a new serum-free cell culture medium and a cell culture method.

[0005] In some aspects, provided herein is one or more of the inventions described below.

[0006] 1. A serum-free cell culture medium comprising a basal medium and an additive component, wherein the additive component comprises (i) transferrin, (ii) insulin, (iii) selenium or a salt thereof, (iv) transforming growth factor-beta (TGF-β), (v) a platelet lysate composition, and (vi) a glutamine supplement.

[0007] 2. The serum-free cell culture medium according to item 1, wherein the transferrin is human transferrin, and / or the insulin is selected from natural human insulin or recombinant human insulin, and / or the selenium or salt thereof is sodium selenite, and / or the transforming growth factor-beta (TGF-beta) is TGF-beta 1, and / or the platelet lysate composition is human platelet lysate (hPL), and / or the glutamine supplement is selected from: L-glutamine or GlutaMAX (L-alanyl-L-glutamine dipeptide).

[0008] 3. The serum-free cell culture medium according to item 1 or 2, wherein the additive ingredients comprise human platelet lysate (hPL), L-glutamine, TGF-beta 1, recombinant human insulin, human transferrin and sodium selenite.

[0009] 4. The serum-free cell culture medium according to any one of items 1 to 3, wherein the additive ingredients consist of: transferrin, insulin, selenium or salt thereof, TGF-beta, platelet lysate composition, glutamine supplement; preferably, the additive ingredients consist of: human platelet lysate (hPL), L-glutamine, TGF-beta 1, recombinant human insulin, human transferrin and sodium selenite.

[0010] 5. The serum-free cell culture medium according to any one of items 1 to 4, wherein the concentration of TGF-beta is 1 to 8 ng / mL, preferably 3.5 to 4.5 ng / mL; the concentration of insulin is 3 to 7 pg / mL, preferably 4 to 6 pg / mL; the concentration of transferrin is 1 to 10 pg / mL, preferably 2.5 to 3.5 pg / mL; and / or the concentration of selenium or salt thereof is 0.5 to 4 ng / mL, preferably 0.75 to 1.25 ng / mL.

[0011] 6. The serum-free cell culture medium according to any one of items 2 to 4, wherein the concentration of TGF-beta is 1 to 8 ng / mL, preferably 3.5 to 4.5 ng / mL; the concentration of recombinant human insulin is 3 to 7 pg / mL, preferably 4 to 6 pg / mL, the concentration of human transferrin is 1 to 10 pg / mL, preferably 2.5 to 3.5 pg / mL, and / or the concentration of sodium selenite is 0.5 to 4 ng / mL, preferably 0.75 to 1.25 ng / mL.

[0012] 7. The serum-free cell culture medium according to item 6, wherein the concentration of TGF-beta is 4 ng / mL, the concentration of recombinant human insulin is 5 pg / mL, the concentration of human transferrin is 3 pg / mL, and the concentration of sodium selenite is 1 ng / mL.

[0013] 8. The serum-free cell culture medium according to any one of items 2-7, wherein the human platelet lysate has a volume percentage concentration of 1 vol% to 5 vol%, and the L-glutamine has a volume percentage concentration of 1 vol% to 5 vol% when added as a 200 mM 100x stock.

[0014] 9. The serum-free cell culture medium according to any one of items 1-8, wherein the basal medium is DMEM, F12 / DMEM, RPMI1640 or MEM-a medium, preferably MEM-a medium.

[0015] 10. A mesenchymal stem cell culture method comprising the step of culturing mesenchymal stem cells in the serum-free cell culture medium according to any one of items 1-9.

[0016] 11. The method according to item 10, wherein the mesenchymal stem cells are umbilical cord mesenchymal stem cells, such as human umbilical cord mesenchymal stem cells.

[0017] 12. The method according to item 10 or 11, wherein the method has any one of the following characteristics: 1) the cells are cultured for 8 passages, 9 passages, 10 passages, 11 passages, 12 passages, 13 passages, 14 passages, 15 passages, 16 passages, 17 passages, 18 passages, 19 passages, 20 passages or more; 2) the cell viability is above 90% during the culture passages, 3) the cell expansion is kept above 6-fold during the culture passages, 4) the cell doubling time is kept below 25 h during the culture passages, 5) the number of cells is 10 20 ,10 21 ,10 22 ,10 23 ,10 24 ,10 25 or more after the culture passages, and 6) the senescence percentage of the cells is less than 1.2%, less than 1.1%, less than 0.7%, less than 0.5%, preferably less than 0.1% for the cells cultured to P5, and less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, preferably less than 1% for the cells cultured to P10.

[0018] 13. Use of the serum-free cell culture medium according to any one of items 1-9 for culturing mesenchymal stem cells, preferably umbilical cord mesenchymal stem cells, such as human umbilical cord mesenchymal stem cells.

[0019] In some aspects, the present application advantageously has any one or more of the following advantages:

[0020] 1. The present application solves the problem of exogenous viruses from animal-derived serum by constructing a serum-free culture medium, and the cell proliferation ability is higher than that in serum-containing culture medium by optimizing the culture medium formula and cell culture process.

[0021] 2、The culture medium of the present application can maintain good proliferation ability and cell activity of cells at high passages by optimizing the culture medium formula and cell culture process, and the cell doubling time and cell aging ratio are significantly better than those of the existing culture medium on the market.

[0022] 3、To ensure the production of large-scale healthy cells, the present application provides a culture method of umbilical cord mesenchymal stem cells, which can improve cell yield, ensure stable cell culture quality, and effectively reduce the cost of umbilical cord mesenchymal stem cell culture in vitro.

[0023] 4、Compared with the culture medium in the prior art, the culture medium of the present application has fewer added components and simple batch production process, and is more conducive to industrialized production.

[0024] 5、The human umbilical cord mesenchymal stem cells cultured by the culture medium in the prior art are generally passaged to 15 generations, and the proliferation ability and cell activity of the cells will greatly decrease after the cell number reaches a certain range. Surprisingly, the human umbilical cord mesenchymal stem cells cultured by the culture medium according to the present application can be passaged to 20 generations or more, and the cell number can reach 10 25 or more, and the cells can still maintain good proliferation ability and cell activity. Therefore, the culture medium according to the present application is more conducive to large-scale culture of cells and meets the needs of industrialized production.

[0025] The comparative analysis of the serum-free culture medium and various serum-containing culture media has shown that the serum-free culture medium of the present application has better supporting effect on cell morphology, cell proliferation ability, doubling time, etc. Meanwhile, the present application has obtained a culture medium and culture method with excellent culture effect through detailed research on the components and proportions of the serum-free culture medium and comparative analysis. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 : Cell morphology pictures of Comparative Examples 1-3 and Examples 1-5.

[0027] Figure 2 : Comparison of P2 to P5 cell viability, expansion fold, and doubling time of Comparative Examples 1-3 and Examples 1-5.

[0028] Figure 3: Cell morphology pictures of Comparative Examples 1-3 and Examples 1-3. Figure 3A : Cell morphology pictures of Comparative Examples 1-3 and Example 1. Figure 3BCell morphology pictures and cell aspect ratio data graphs of Examples 2 & 3 (results shown in cell aspect ratio graphs are expressed as mean ± SEM. Data shown are representative of at least three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Statistical significance of differences between groups was determined by Student t test).

[0029] Figures 4A-4B Cell viability, expansion fold, doubling time of Comparative Examples 1-3 and Examples 1-3. Figure 4A Cell viability, expansion fold, doubling time of Comparative Examples 1-3 and Example 1. Figure 4B Cell viability, expansion fold, doubling time of Comparative Example 3 and Examples 1-3.

[0030] Figure 5 Cumulative expansion curves of Comparative Examples 1-3 and Example 1.

[0031] Figure 6 Cumulative expansion curves of Comparative Example 3 and Examples 1-3.

[0032] Figures 7A-7B Cell senescence detection of Comparative Examples 1-3 and Examples 1-3, where the corresponding % β-gal is shown below each cell picture. Figure 7A Cell senescence detection of Comparative Examples 1-3. Figure 7B Cell senescence detection of Examples 1-3.

[0033] Figure 8 Growth curve comparison of Example 1 with Comparative Examples 1-3 and Examples 2 & 3, respectively (results shown are expressed as mean ± SEM. Data shown are representative of at least three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. Statistical significance of differences between groups was determined by Student t test). DETAILED DESCRIPTION

[0034] Culture medium formulation

[0035] Cell growth generally requires the presence of serum. Currently, bovine serum is commonly added to serum culture medium. As known to those skilled in the art, serum-free medium refers to a medium without the addition of serum, but in which serum components such as growth factors and hormones, adhesion factors, binding proteins, nutrients such as trace elements or low molecular weight nutrients, and enzyme inhibitors, etc. can be added. Serum-free medium has relatively clear composition, simple preparation process, and can reduce the risk of exogenous viruses from animal-derived serum. Serum-free medium can be prepared by adding supplemental ingredients to a nutritionally complete basal medium. In some embodiments, the serum-free medium formulation of the present application can include a basal medium and added ingredients.

[0036] The basal medium can be prepared by artificial synthesis or using commercially available medium. Available commercially available medium includes but is not limited to Dulbecco's Modified Eagle's Medium (DMEM), Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, Ham's medium, Ham's F-12 medium, F12 / DMEM medium, α-Minimal Essential Medium (α-MEM or MEM-α), Glasgow's Minimal Essential Medium (G-MEM), and Isocove's Modified Dulbecco's Medium (IMDM). In some embodiments, the basal medium can be a commonly used basal medium in the art, such as DMEM, F12 / DMEM, MEM-α, RPMI 1640, etc. In some embodiments, the basal medium of the present application can be MEM-α medium.

[0037] In some embodiments, the additive components can include nutrients such as trace elements, vitamins, lipids, and amino acids, etc. Glycine, L-glutamine, L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-serine, L-threonine, L-tryptophan, L-tyrosine, and L-valine are the primary amino acids for protein synthesis. By selecting a higher concentration, it helps to promote nucleic acid and protein synthesis and cell proliferation during stem cell developmental growth. In some embodiments, the additive components include glutamine supplement selected from L-glutamine or GlutaMAX (L-alanyl-L-glutamine dipeptide). In some embodiments, the additive components include L-glutamine, the final concentration of L-glutamine ranges from 2 to 10 mM, such as 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM. In some embodiments, the added L-glutamine is a ready-to-use 200 mM stock solution (100x), and the volume percentage concentration of the addition is 1 vol% to 5 vol%.

[0038] Vitamins are antioxidants, and the available vitamins include, but are not limited to, ascorbic acid phosphate, choline chloride, D-biotin (included in DMEM high glucose medium), D-calcium pantothenate, vitamin E, folic acid, inositol, nicotinamide, riboflavin, thiamine hydrochloride, vitamin B12 (included in DMEM high glucose medium). Selecting a higher concentration of vitamins can remove metabolic products produced by high cell proliferation.

[0039] In some embodiments, the additive ingredient includes one or more growth factors and hormones. Hormones can be classified into polypeptide hormones and carrier hormones. Polypeptide hormones include, but are not limited to, insulin, growth factors, glucagon, etc. Steroid hormones include, but are not limited to, progesterone, hydrocortisone, estradiol, etc. Growth factors refer to protein physiological active substances that promote cell division, growth and differentiation. In some embodiments, the one or more cell growth factors can be, for example: fibroblast growth factor (FGF, including FGF7, FGF10, bFGF, FGF4), platelet-derived growth factor (PDGF, including PDGF-aa, PDGFbb), epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), transforming growth factor (including TGF-a, TGF-b such as TGF-b1 and TGF-b3), bone morphogenetic protein (BMP, including BMP-2, BMP-5, BMP-6, BMP-7), vascular endothelial growth factor (VEGF), colony-stimulating factor (CSF), keratinocyte growth factor (KGF), wnt signaling pathway agonist, interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, placenta growth factor (P1GF), stromal cell-derived factor (SDF, including SDF-1a, SDF-1b, etc.). Transforming growth factor beta (TGF-b) is a multifunctional peptide that controls proliferation, differentiation and other functions in many cell types. Epidermal growth factor (EGF) stimulates cell proliferation, differentiation and survival. Basic fibroblast growth factor (FGF-b) promotes angiogenesis and binds to heparin widely stimulates various cells. Platelet-derived growth factor (PDGF) is a dimeric glycoprotein that regulates cell growth and division and promotes angiogenesis. Stromal cell-derived factor-1a (SDF-1a) activates leukocytes and promotes angiogenesis. Vascular endothelial growth factor (VEGF) helps vasculogenesis and growth. In some embodiments, the additive ingredient includes transforming growth factor (TGF-b). In some embodiments, the additive ingredient includes TGF-b1.

[0040] In some embodiments, the additive component includes an amount of insulin. Insulin is a hormone with multiple biological effects and is an important cell survival factor. In addition to regulating carbohydrate metabolism, insulin promotes the synthesis of fat, reduces the breakdown of lipids, and promotes the uptake of amino acids into cells. It also regulates transcription, changes the content of mRNA, and stimulates proliferation, increases DNA synthesis, and cell replication. Insulin plays an important role in cell growth and proliferation, both in physiological and pathological conditions. Insulin can be derived from natural cells or can be produced recombinantly. In some embodiments, the additive component includes purified natural insulin or recombinant human insulin. In some embodiments, the human insulin can be recombinant human insulin.

[0041] Animal platelet lysate compositions, such as human platelet lysate (hPL), contain various growth factors. In some embodiments, the additive component includes a biologically active fraction derived from mammalian platelets, which comprises a platelet lysate composition. In some embodiments, the platelet lysate composition is human platelet lysate (hPL). Human platelet lysate (hPL) is a turbid, yellowish liquid obtained after freezing / thawing of human platelets. Freezing / thawing of platelets releases a large number of growth factors required for cell expansion, including, for example, TGF-βΙ, EGF, FGF-β, PDGF-AA, PDGF-BB, SDF-Ια, and VEGF, among others.

[0042] In some embodiments, the additive component includes an iron source or an iron transporter. Exemplary iron sources include, but are not limited to, ferrous and ferric salts, such as ferrous sulfate, ferrous citrate, ferric citrate, ferric nitrate, ferric sulfate; ferric ammonium compounds, such as ferric ammonium citrate, ferric ammonium oxalate, ferric ammonium fumarate, ferric ammonium malate, and ferric ammonium succinate. Exemplary iron transporters include, but are not limited to, transferrin and lactoferrin. In some embodiments, the iron transporter is lactoferrin. In some embodiments, the iron transporter is transferrin. In some embodiments, the transferrin is purified natural or recombinant human transferrin.

[0043] In some embodiments, the additive component includes a trace element selected from the group consisting of selenium, molybdate, chromium, cobalt, nickel, zinc, copper, manganese, barium, gallium, lithium, tin, titanium, bromine, iodine, vanadium, germanium, molybdenum, silicon, iron, fluorine, silver, rubidium, zirconium, cadmium, and aluminum. In some embodiments, the additive component includes a selenium compound, such as sodium selenite.

[0044] In some embodiments, the additive ingredients can include human platelet lysate, L-glutamine (200 mM) 100x with TGF-β1, recombinant human insulin, human transferrin, sodium selenite (sometimes also referred to herein as "4 essential factors"). In some embodiments, the culture media described herein can be used to culture stem cells, such as mesenchymal stem cells, and particularly umbilical cord mesenchymal stem cells.

[0045] In some embodiments, the serum-free culture media of the present application can consist only of a basal media and additive ingredients. In some embodiments, the additive ingredients can consist only of human platelet lysate (hPL), L-glutamine, transforming growth factor-β1 (TGF-β1), recombinant human insulin, human transferrin, and sodium selenite.

[0046] In some embodiments, the human platelet lysate can have a volume percent concentration of 1 vol% to 5 vol%, such as 1 vol%, 2 vol%, 3 vol%, 4 vol%, 5 vol%, or any concentration therebetween.

[0047] In some embodiments, the L-glutamine (200 mM) 100x can have a volume percent concentration of 1 vol% to 5 vol%, such as 1 vol%, 2 vol%, 3 vol%, 4 vol%, 5 vol%, or any concentration therebetween.

[0048] In some embodiments, the TGF-β1 can have a concentration of 1 ng / mL to 8 ng / mL, such as 1 ng / mL, 1.5 ng / mL, 2 ng / mL, 2.5 ng / mL, 3 ng / mL, 3.5 ng / mL, 4 ng / mL, 4.5 ng / mL, 5 ng / mL, 5.5 ng / mL, 6 ng / mL, 6.5 ng / mL, 7 ng / mL, 7.5 ng / mL, 8 ng / mL, or any concentration therebetween.

[0049] In some embodiments, the recombinant human insulin can have a concentration of 3 μg / mL to 7 μg / mL, such as 3 μg / mL, 3.5 μg / mL, 4 μg / mL, 4.5 μg / mL, 5 μg / mL, 5.5 μg / mL, 6 μg / mL, 6.5 μg / mL, 7 μg / mL, or any concentration therebetween.

[0050] In some embodiments, the concentration of the human transferrin can be: 1-10 μg / mL, for example, 1 μg / mL, 1.5 μg / mL, 2 μg / mL, 2.5 μg / mL, 3 μg / mL, 3.5 μg / mL, 4 μg / mL, 4.5 μg / mL, 5 μg / mL, 5.5 μg / mL, 6 μg / mL, 6.5 μg / mL, 7 μg / mL, 7.5 μg / mL, 8 μg / mL, 8.5 μg / mL, 9 μg / mL, 9.5 μg / mL, 10 μg / mL, or any concentration therebetween.

[0051] In some embodiments, the concentration of the sodium selenite can be: 0.5-4 ng / mL, for example, 0.5 ng / mL, 0.75 ng / mL, 1 ng / mL, 1.25 ng / mL, 1.5 ng / mL, 1.75 ng / mL, 2 ng / mL, 2.25 ng / mL, 2.5 ng / mL, 2.75 ng / mL, 3 ng / mL, 3.25 ng / mL, 3.5 ng / mL, 3.75 ng / mL, 4 ng / mL, or any concentration therebetween.

[0052] In some embodiments, the optimal concentration of the TGF-β1 is 4 ng / mL.

[0053] In some embodiments, the optimal concentration of the recombinant human insulin is 5 μg / mL.

[0054] In some embodiments, the optimal concentration of the human transferrin is 3 μg / mL.

[0055] In some embodiments, the optimal concentration of the sodium selenite is 1 ng / mL.

[0056] In some embodiments, the culture medium, method or use of the present application can possess any one of the following features: 1) the cells are cultured for 8 passages, 9 passages, 10 passages, 11 passages, 12 passages, 13 passages, 14 passages, 15 passages, 16 passages, 17 passages, 18 passages, 19 passages, 20 passages or more; 2) the cell viability in the subculture is above 90%; 3) the cell expansion fold in the subculture is maintained at above 6; 4) the cell doubling time in the subculture is maintained at below 25 h; 5) after the subculture, the cell number is 10 20 ,10 21 ,10 22 ,10 23 ,10 24 ,10 25or more, and 6) passaging to P5 cells with senescence less than 1.2%, less than 1.1%, less than 0.7%, less than 0.5%, preferably less than 0.1%, and to P10 cells with senescence less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, preferably less than 1%.

[0057] The culture medium of the present application is composed of a basic medium, additives and "essential factors", which is conducive to promoting cell proliferation, maintaining high activity of cells and reducing the proportion of cell senescence. Compared with traditional serum culture system and existing serum-free system, it is free of heterogeneity and animal origin, avoiding the instability of animal origin components and batches. The four essential factors in the present application can significantly improve the support of serum-free culture medium for cell proliferation and reduce cell senescence.

[0058] (1) Human platelet lysate (hPL): Platelets contain many different cell growth factors, including PDGF, VEGF, EGF, etc. It has been proven that the cell growth factors secreted by platelets can support the growth of various cells. Since hPL is derived from humans, there is no need to worry about viruses from cows or immune reactions to foreign proteins from cows. No serious side effects have been observed in current clinical applications.

[0059] (2) L-glutamine (200 mM) 100x: Glutamine can support the growth of cells with high energy needs and synthesize a large amount of proteins and nucleic acids. It can serve as another energy source for cells that rapidly divide and cannot effectively use glucose.

[0060] (3) Transforming growth factor-β (TGF-β): TGF-β is a multifunctional protein that can affect the growth, differentiation, apoptosis and immune regulation of various cells. Transforming growth factor-β can bind to transforming growth factor-β receptors on the cell surface to activate its receptors. Transforming growth factor-β receptors are serine / threonine kinase receptors. Its signal transmission can be through the SMAD signaling pathway and / or the DAXX signaling pathway.

[0061] (4) Recombinant human insulin: Insulin is a serum-free medium formulation component for all primary cells and cell lines. It is used in serum-free medium to promote cell growth and increase fatty acid and glycogen synthesis.

[0062] (5) Human transferrin: Transferrin can reversibly bind to trivalent iron ions by interacting with transferrin receptors (Transferrin Receptor, TFR), regulating iron ion transport and metabolism, maintaining the homeostasis of intracellular iron and various metal ions such as chromium, manganese, cadmium and nickel, and maintaining cell growth and proliferation.

[0063] (6) Sodium selenite: Selenium is an essential trace element necessary for maintaining various physiological functions of life. In a cell culture system, sodium selenite (SS) protects cells from oxidative damage by reducing the production of free radicals and inhibiting lipid peroxidation.

[0064] Cell culture process:

[0065] Cell preparation: Primary culture method

[0066] The umbilical cord tissue was soaked in 1% gentamicin-containing normal saline for injection, and the umbilical cord tissue was cut into 1.5-2 cm long pieces using a sterile ophthalmic scissors after high-pressure steam sterilization. The umbilical cord tissue was washed twice with normal saline for injection, and the residual blood stains on the outer surface, arteries, and veins of the umbilical cord were cleaned with a sterile toothless forceps after high pressure. Then the umbilical cord tissue was soaked in 75% medical alcohol for 1 min, and the alcohol was squeezed out of the umbilical cord to completely disinfect the arteries and veins. After disinfection, the umbilical cord tissue was washed twice with 1% gentamicin-containing normal saline for injection, and then transferred to a clean culture dish. The two arteries and one vein in the umbilical cord tissue were removed using a sterile ophthalmic scissors and a sterile toothed forceps, and then the Wharton's jelly was peeled off and transferred to a 50 mL centrifuge tube for weighing about 1 g. The weighed umbilical cord tissue was cut into 0.5 cm 2 pieces using a sterile ophthalmic scissors, resuspended with 10 mL of culture medium, and transferred to a T75 culture bottle. The umbilical cord tissue was evenly distributed in the culture bottle by gently shaking the bottle, and then placed in a 37°C, 5% CO2 incubator for culture, denoted as P0.

[0067] Cell culture:

[0068] The harvested cells were counted, centrifuged at 1300 rpm for 6 min (9 up and 9 down) at room temperature, and then resuspended with an appropriate amount of culture medium according to the cell number. The cell inoculation density was controlled at 5000-10000 cells / cm 2 , and the optimal inoculation density was 6600 cells / cm 2 . Large-scale expansion was performed using cell culture bottles, and the cell culture medium was used in an amount of 3 mL / T25 bottle, 10 mL / T75 bottle, and 20 mL / T175 bottle. After cell inoculation according to the above cell inoculation density and culture medium amount, the resuscitation inoculation culture cells were cultured for 75-80 h, and the passage inoculation culture cells were cultured for 68-72 h.

[0069] Comparative Example 1:

[0070] A serum-containing medium for culturing umbilical cord mesenchymal stem cells, comprising RPMI1640 medium and an additive. The additive comprises fetal bovine serum at a volume percentage concentration of 10 vol%, and glutamine at a volume percentage concentration of 1 vol%.

[0071] Comparative Example 2

[0072] A serum-containing medium for culturing umbilical cord mesenchymal stem cells, comprising MEM-α medium and an additive. The additive comprises fetal bovine serum at a volume percentage concentration of 10 vol%, and L-glutamine at a volume percentage concentration of 1 vol%.

[0073] Comparative Example 3

[0074] A serum-free medium for large-scale culturing of human umbilical cord mesenchymal stem cells, comprising MEM-α medium and an additive. The additive comprises human platelet lysate at a volume percentage concentration of 5 vol%, L-glutamine at a volume percentage concentration of 1 vol%, and no "4 essential factors".

[0075] Example 1

[0076] A serum-free medium for large-scale culturing of human umbilical cord mesenchymal stem cells, comprising MEM-α medium and an additive. The additive comprises human platelet lysate at a volume percentage concentration of 5 vol%, L-glutamine at a volume percentage concentration of 1 vol%, TGF-β at a dosage of 4 ng / mL, recombinant human insulin at a dosage of 5 μg / mL, human transferrin at a dosage of 3 μg / mL, and sodium selenite at a dosage of 1 ng / mL.

[0077] Example 2

[0078] Example 2 provides a serum-free medium for large-scale culturing of human umbilical cord mesenchymal stem cells, and the difference compared with Comparative Example 3 is that TGF-β is at a dosage of 1 ng / mL, recombinant human insulin is at a dosage of 3 μg / mL, human transferrin is at a dosage of 1 μg / mL, and sodium selenite is at a dosage of 0.5 ng / mL.

[0079] Example 3

[0080] Example 3 provides a medium for large-scale culturing of human umbilical cord mesenchymal stem cells, and the difference compared with Comparative Example 3 is that TGF-β is at a dosage of 8 ng / mL, recombinant human insulin is at a dosage of 7 μg / mL, human transferrin is at a dosage of 10 μg / mL, and sodium selenite is at a dosage of 4 ng / mL.

[0081] Example 4

[0082] A serum-free culture medium for large-scale culture of human umbilical cord mesenchymal stem cells contains DMEM culture medium and additives. The additive component group: the volume percentage concentration of human platelet lysate is 5vol%, the volume percentage concentration of L-glutamine is 1vol%, the amount of TGF-β is 4ng / mL, the amount of recombinant human insulin is 5μg / mL, the amount of human transferrin is 3μg / mL, and the amount of sodium selenite is 1ng / mL.

[0083] Example 5

[0084] A serum-free culture medium for large-scale culture of human umbilical cord mesenchymal stem cells contains F12 / DMEM culture medium and additives. The additive component group: the volume percentage concentration of human platelet lysate is 5vol%, the volume percentage concentration of L-glutamine is 1vol%, the amount of TGF-β is 4ng / mL, the amount of recombinant human insulin is 5μg / mL, the amount of human transferrin is 3μg / mL, and the amount of sodium selenite is 1ng / mL.

[0085] Test example

[0086] Experimental method

[0087] The medium of the above examples and comparative examples is used to culture human umbilical cord mesenchymal stem cells. The specific process is as follows:

[0088] I. Primary separation and culture of umbilical cord-derived mesenchymal stem cells

[0089] The umbilical cord tissue is soaked in normal saline for injection containing 1% penicillin / streptomycin, and the umbilical cord tissue is cut into 1.5-2 cm long blocks using sterile ophthalmic scissors after high-pressure steam sterilization. The umbilical cord tissue is washed twice with normal saline for injection, and the residual blood stains on the outer surface, arteries and veins of the umbilical cord are cleaned with sterile forceps after high pressure. Then the umbilical cord tissue is soaked in 75% medical alcohol for 1 min, and the alcohol is continuously squeezed out of the umbilical cord to completely disinfect the arteries and veins. After disinfection, the umbilical cord tissue is washed twice with normal saline for injection containing 1% penicillin / streptomycin, and then transferred to a clean culture dish. The two arteries and one vein in the umbilical cord tissue are removed using sterile ophthalmic scissors and sterile forceps, and then the Wharton's jelly is peeled off and transferred to a 50mL centrifuge tube for weighing about 1g. The weighed umbilical cord tissue is cut into 0.5cm 2 size using sterile ophthalmic scissors, resuspended with 10mL medium, transferred to a T75 culture, and placed in a 37℃, 5% CO2 incubator for culture, denoted as P0.

[0090] After 120h of culture, the supernatant and unattached tissue in the culture bottle were poured into a 50mL centrifuge tube, centrifuged at 1300rpm for 5min at room temperature, and the supernatant was discarded. 10mL of medium was added to resuspend the umbilical cord tissue without dispersing the fine tissue fragments and residual blood cell precipitates at the bottom. The umbilical cord tissue was transferred to a culture bottle and placed in a 37℃, 5% CO2 incubator for culture. After 120h of complete medium exchange, semi-medium exchange was performed by removing 5mL of old medium from the culture bottle and adding 5mL of new medium. After 96h-120h of culture, the cells were subcultured according to the confluence rate.

[0091] II. Subculture of umbilical cord-derived mesenchymal stem cells

[0092] The old medium was removed, and the residual medium and floating tissue in the T75 culture bottle were washed with 10mL of normal saline for injection. After washing, 3mL of 0.05% Trypsin EDTA was added, and the culture bottle was shaken and placed in a 37℃ incubator for 1min of digestion. The culture bottle was removed and gently tapped to ensure complete cell detachment. 3mL of medium was added to the culture bottle to stop the digestion. The cell suspension was filtered through a 100μm disposable filter and transferred to a 15mL centrifuge tube. 5mL of normal saline for injection was added to the culture bottle to wash the residual cells, which were then transferred to the same 15mL centrifuge tube. The tube was centrifuged at 1300rpm for 5min at room temperature. After centrifugation, the supernatant was discarded, and the cell pellet was dispersed by tapping the bottom of the centrifuge tube. 5mL of medium was added to resuspend the cells, and 100μL of the suspension was removed for counting using the trypan blue counting method with a countess cell counter. After counting, the cells were inoculated into a T175 bottle containing 25mL of medium at a seeding density of 5000-10000 cells / cm 2 After 68-70h of culture, the cells were photographed and subcultured.

[0093] (1) The state of cells at different generations was recorded by optical microscopy.

[0094] (2) The cell viability and number at each generation were recorded based on the counting results of each generation, and the cell doubling time was calculated according to the formula TD=t×lg2 / (lgNt-lgN0) (TD is the cell doubling time, t is the culture time, Nt is the OD value at t hours of culture, and N0 is the OD value after inoculation).

[0095] (3) The cumulative P20 cells were calculated under the same cell inoculation amount based on the expansion fold obtained from cell counting at each generation.

[0096] (4) The proportion of senescent cells was measured by a β-galactosidase detection kit.

[0097] Experimental results I

[0098] The P2 cells recovered from the P1 generation were cultured for 4 generations, and the cell morphology, cell viability, expansion fold, and doubling time were observed. The experimental results are shown in Table 1. Figure 1 and Figure 2 .

[0099] It can be seen from Figure 1 and Figure 2 that:

[0100] (1) By observing the cell morphology, the long spindle-shaped cells can be cultured in Comparative Examples 1-3 and Examples 1-5.

[0101] (2) By observing the cell viability, the cell viability of Comparative Examples 1-3 and Examples 1-5 is above 92%.

[0102] (3) By observing the expansion fold and doubling time, the cells in Comparative Examples 1-3 and Examples 1-5 can proliferate normally. In addition, it can be seen that, in the continuous culture for 4 generations, the expansion fold of the other groups is higher and the doubling time is shorter than that of Comparative Example 1.

[0103] From the experimental results, it can be seen that the added components of the present application, especially the 4 essential factors, have no selectivity for the basic culture medium including DMEM, F12 / DMEM, RPMI1640 or MEM-α, and can achieve high-quality culture of mesenchymal stem cells.

[0104] Experimental results II

[0105] The P2 cells recovered from the P1 generation were cultured for more than 10 generations, and the cell morphology, cell viability, expansion fold, and doubling time were observed.

[0106] 2.1 Cell morphology

[0107] The cell morphology results and the cell length-width ratio were observed by optical microscope photography and comparison, as shown in Figure 3.

[0108] It can be seen from Figure 3 that:

[0109] (1) The P2 cells recovered from the P1 generation have a thick and long spindle-shaped cell morphology compared to Comparative Example 3 and Examples 1-3, and the cell number under the same magnification is less, and the length-width ratio of Comparative Examples 1&2 is significantly lower than that of Example 1.

[0110] (2) Cultured to P5 generation, the cell morphology of Comparative Examples 1-3 and Example 1-3 were long spindle-shaped, and the cell aspect ratio of Comparative Examples 1 & 2 was significantly lower than that of Example 1.

[0111] (3) Cultured to P10 generation, the cell morphology of Comparative Example 1 was irregular round and robust long spindle-shaped, the cell morphology of Comparative Example 2 was robust long spindle-shaped, the cell morphology of Comparative Examples 3 and Example 1-3 were long spindle-shaped, and the cell aspect ratio of Comparative Examples 1 & 2 was also significantly lower than that of Example 1.

[0112] In summary, the results of Comparative Examples 1 & 2, Comparative Example 3 and Example 1-3 demonstrated that the serum-free medium had a better culture effect on umbilical cord mesenchymal stem cells.

[0113] 2.2 Cell viability, expansion fold, doubling time

[0114] The experimental results are shown in Table 2.2. Figures 4A-4B .

[0115] It can be seen that: Figures 4A-4B

[0116] (1) By cell viability, the cell viability of Example 1 could be maintained above 90% in the culture of up to 20 generations, the cell viability of Comparative Example 1 was below 90% after being cultured to P8 generation, the cell viability of Comparative Example 2 was below 90% after being cultured to P13 generation, the cell viability of Comparative Example 3 was below 90% after being cultured to P15 generation, and the cell viability of Example 2 and Example 3 was below 90% after being cultured to P19 generation.

[0117] (2) By expansion fold, the cell expansion fold of Example 1 could be maintained above 6 in the culture of up to 20 generations, the expansion fold of Comparative Example 1 was less than 6 during the culture, the expansion fold of Comparative Example 2 was less than 6 after being cultured to P8 generation, the expansion fold of Comparative Example 3 was less than 6 after being cultured to P12 generation, and the expansion fold of Example 2 and Example 3 was less than 6 after being cultured to P15 generation.

[0118] (3) By doubling time, the doubling time of Example 1 could be maintained below 25h in the culture of up to 20 generations, the doubling time of Comparative Example 1 was greater than 30h during the culture, the doubling time of Comparative Example 2 was greater than 25h after being cultured to P5 generation, the doubling time of Comparative Example 3 was greater than 25h after being cultured to P8 generation, the doubling time of Example 2 was greater than 25h after being cultured to P13 generation, and the doubling time of Example 3 was greater than 25h after being cultured to P15 generation.

[0119] ​In summary, compared with Comparative Examples 1 & 2 and existing culture media, the exemplary culture media of the present invention, such as Comparative Example 3 and Examples 1-3, demonstrate that serum-free culture media have better culture effects on umbilical cord mesenchymal stem cells. By comparing Comparative Example 3 with Example 1, the addition of the "4 essential factors" helps to improve cell activity. By comparing Example 3 with Examples 2 & 3, an appropriate concentration of the "4 essential factors" can significantly improve cell proliferation and cell activity.

[0120] 2.3 Cell Count

[0121] The amplification fold obtained from cell counting at each generation was controlled at 1×10⁻⁶. 7 One P1 generation cell was continuously cultured until the P20 generation. The total number of cells was counted. See the experimental results below. Figure 5 and Figure 6 .

[0122] Depend on Figure 5 A comparison of Example 1 with Comparative Examples 1-3 shows that:

[0123] (1) After being cultured to generation P11, cells in Comparative Example 1 did not proliferate;

[0124] (2) After being cultured to the P5 generation, the number of cells in Comparative Examples 2 & 3 and Example 1 differed by about 10 times, with Example 1 > Comparative Example 3 > Comparative Example 2;

[0125] (3) After culturing to generation P20, the cell counts of Comparative Examples 2 & 3 and Example 1 differed significantly, with Example 1 having 10 cells. 25 Comparative Example 3 had 10 cells. 21 Comparative Example 2 had 10 cells. 20 .

[0126] Depend on Figure 6 Comparing Comparative Example 3 with Examples 1-3, it can be seen that:

[0127] (1) The cells were cultured to the P5 generation, and the number of cells in Comparative Example 3 was similar to that in Examples 1-3;

[0128] (2) Cultured to P10 generation, cell number Example 3 > Example 1 > Example 2 > Comparative Example 3;

[0129] (3) Cultured to passage P15, cell number in Example 1 > Example 3 > Example 2 > Comparative Example 3, with 10 cells in Example 1. 21.5 Example 3: Cell count was 10. 21 .

[0130] (4) After culturing to passage P20, the cell number in Example 1 > Example 3 > Example 2 > Comparative Example 3 showed a significant difference. The cell number in Example 1 was 10. 25, the cell number of Example 3 was 10 23.5 , the cell number of Example 2 was 10 21.5 , the cell number of Comparative Example 3 was 10 21 .

[0131] In summary, the cell proliferation amount of P5 generation was significantly different between the serum-free culture and the fetal bovine serum culture. Whether adding the "4 essential factors" had a significant influence on the proliferation ability of the cells, and the optimal concentration of the "4 essential factors" could make the cells be cultured to P20 generation and still maintain high cell activity.

[0132] 2.4 Cell senescence

[0133] The proportion of senescent cells was measured by a cell senescence beta-galactosidase staining kit (Bi Yun Tian, item number: C0602). The experimental results are shown in Figures 7A-7B .

[0134] From Figures 7A-7B it can be seen that:

[0135] (1) The proportion of senescent cells of P2 generation cells of Example 1-3 and Comparative Example 1-3 cultured by P1 generation resuscitation was 0.00%.

[0136] (2) The proportion of senescent cells of P5 generation cells cultured by P1 generation resuscitation was Comparative Example 1> Comparative Example 2> Comparative Example 3> Example 2> Example 3> Example 1.

[0137] (3) The proportion of senescent cells of P10 generation cells cultured by P1 generation resuscitation was Comparative Example 1> Comparative Example 2> Comparative Example 3> Example 3> Example 2> Example 1.

[0138] 2.5 P5 generation cell growth curve

[0139] P4 generation cells of Example 1-3 and Comparative Example 1-3 were taken respectively, and 1.5×10 4 cells / mL was added to 1 mL of the culture medium of the corresponding group in each well, and was added to a 12-well plate, 24 wells were plated in each group, and was placed in a 37℃, 5% CO2 incubator for culture. The time was calculated from the inoculation, and 3 wells were taken from each group at 24h, 48h, 72h, 96h, 108h, 120h, 144h, and 168h to calculate the cell amount. The cell growth curve was drawn with time as the abscissa and cell number as the ordinate, as shown in Figure 8 From the figure, it can be seen that the cell number of the culture medium of the application (Example 1-3) was significantly more than 3×10 5 .

[0140] From Figure 8 it can also be seen that:

[0141] (1) The growth curves of Examples 1-3 and Comparative Examples 1-3 are all "S" type curves, which proves that the cells cultured in each group of medium meet the normal cell growth characteristics.

[0142] (2) Compared with Comparative Examples 1-3, Example 1 has a significant difference in cell number from 96h, and Example 3 has a stronger proliferation ability.

[0143] (3) Compared with Example 1, Examples 2&3 have approximately the same proliferation ability, but there is a difference in cell number at 168h, which proves that Example 1 can maintain cell activity for a longer time.

[0144] In summary, Examples 1 and 2&3 all have strong proliferation ability, but Example 1 can maintain better cell activity in the same medium for 7 days (168h).

[0145] Conclusion: From the experimental results, it can be seen that compared with fetal bovine serum culture, serum-free culture has differences in P5 generation and significant differences in P10 generation, and serum-free medium is more suitable for cell culture. Through comparison between Comparative Example 3 and Examples 1-3, the medium without adding "4 essential factors" has a higher proportion of senescent cells at P5 and P10 than the medium with "4 essential factors", and the medium with the best concentration of "4 essential factors" helps to reduce the proportion of senescent cells.

Claims

1. A serum-free cell culture medium comprising a basal medium and additives, wherein the additives include (i) transferrin, (ii) insulin, (iii) selenium or a salt thereof, (iv) transforming growth factor-β (TGF-β), (v) a platelet lysate composition, and (vi) a glutamine supplement.

2. The serum-free cell culture medium according to claim 1, wherein the transferrin is human transferrin, and / or the insulin is selected from natural insulin or recombinant human insulin, and / or the selenium or its salt is sodium selenite, and / or the transforming growth factor-β (TGF-β) is TGF-β1, and / or the platelet lysate composition is human platelet lysate (hPL), and / or the glutamine supplement is selected from L-glutamine or GlutaMAX (L-alanyl-L-glutamine dipeptide).

3. The serum-free cell culture medium according to claim 1 or 2, wherein the added component comprises: Human platelet lysate (hPL), L-glutamine, TGF-β1, recombinant human insulin, human transferrin, and sodium selenite.

4. The serum-free cell culture medium according to claims 1-3, wherein the added component comprises the following components: Transferrin, insulin, selenium or its salts, TGF-β, platelet lysate composition, glutamine supplement; preferably, the added ingredients consist of: human platelet lysate (hPL), L-glutamine, TGF-β1, recombinant human insulin, human transferrin and sodium selenite.

5. The serum-free cell culture medium according to any one of claims 1-4, wherein the concentration of TGF-β is 1-8 ng / mL, preferably 3.5-4.5 ng / mL; the concentration of insulin is 3-7 μg / mL, preferably 4-6 μg / mL; the concentration of transferrin is 1-10 μg / mL, preferably 2.5-3.5 μg / mL; and / or the concentration of selenium or its salt is 0.5-4 ng / mL, preferably 0.75-1.25 ng / mL.

6. The serum-free cell culture medium according to any one of claims 2-4, wherein the concentration of TGF-β1 is 1-8 ng / mL, preferably 3.5-4.5 ng / mL; the concentration of recombinant human insulin is 3-7 μg / mL, preferably 4-6 μg / mL; the concentration of human transferrin is 1-10 μg / mL, preferably 2.5-3.5 μg / mL; and / or the concentration of sodium selenite is 0.5-4 ng / mL, preferably 0.75-1.25 ng / mL.

7. The serum-free cell culture medium according to claim 6, wherein the concentration of TGF-β1 is 4 ng / mL, the concentration of recombinant human insulin is 5 μg / mL, the concentration of human transferrin is 3 μg / mL, and the concentration of sodium selenite is 1 ng / mL.

8. The serum-free cell culture medium according to any one of claims 2-7, wherein the human platelet lysate concentration is 1 vol% to 5 vol%, and the L-glutamine concentration is 1 vol% to 5 vol% when added as 200 mM 100× stock solution.

9. The serum-free cell culture medium according to any one of claims 1-8, wherein the basal culture medium is DMEM, F12 / DMEM, RPMI1640 or MEM-α medium, preferably MEM-α medium.

10. A method for culturing mesenchymal stem cells, comprising the step of culturing mesenchymal stem cells using the serum-free cell culture medium according to any one of claims 1-9.

11. The method of claim 10, wherein the mesenchymal stem cells are umbilical cord mesenchymal stem cells, such as human umbilical cord mesenchymal stem cells.

12. The method according to claim 10 or 11, wherein the method comprises any of the following features: 1) the culture is passaged for 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more generations; 2) the cell viability during passage culture is above 90%; 3) the cell fold increase during passage culture is maintained at above 6-fold; 4) the cell doubling time during passage culture is maintained below 25 hours; 5) after passage culture, the cell number is 102. 20 10 21 10 22 10 23 10 24 10 25 Or more, and 6) the percentage of senescent cells in the P5 passage is less than 1.2%, less than 1.1%, less than 0.7%, less than 0.5%, preferably less than 0.1%, and the percentage of senescent cells in the P10 passage is less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, preferably less than 1%.

13. Use of the serum-free cell culture medium according to any one of claims 1-9 for culturing mesenchymal stem cells, preferably the mesenchymal stem cells being umbilical cord mesenchymal stem cells, such as human umbilical cord mesenchymal stem cells.

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