Serum-free medium for umbilical cord mesenchymal stem cells and use method of serum-free medium

Abstract

The invention discloses a serum-free culture medium for umbilical cord mesenchymal stem cells and a use method of the serum-free culture medium. The serum-free culture medium comprises a basic culture medium and additive factors, the additive factors comprise a human platelet-derived growth factor, an epidermal growth factor, a fibroblast growth factor, human serum albumin, human transferrin, glycine, nicotinamide, folic acid, L-arginine hydrochloride, L-cystine, pyridoxine hydrochloride, riboflavin, inositol, hydrocortisone, catalase, superoxide dismutase and AWRK6; the invention further discloses a using method of the serum-free culture medium, a dynamic microenvironment of cells is simulated, and the cells are cultured in a mode of static state first and then slight oscillation. The serum-free culture medium is rich in nutrition, the culture method is standard in operation, the cell proliferation efficiency and activity can be effectively improved, the cell differentiation capacity is maintained, the cell immunoregulation capacity is enhanced, and the serum-free culture medium has wide industrial application and scientific research and clinical research prospects.

Description

Technical Field

[0001] The invention relates to the technical field of cell culture, and in particular to a serum-free culture medium for culturing umbilical cord mesenchymal stem cells and a use method thereof. Background Art

[0002] Umbilical cord mesenchymal stem cells (UC-MSCs), as an important member of the stem cell family, are derived from the early mesoderm and ectoderm of development, have multidirectional differentiation potential, and can differentiate into multiple cell types under specific conditions, such as osteoblasts, chondrocytes, adipocytes, neurons, etc., showing great potential in the field of tissue repair and regeneration. At the same time, it also has the characteristics of hematopoietic support and promotion of stem cell implantation, immune regulation and self-replication, and has important application value in hematopoietic reconstruction, immunotherapy, etc., including but not limited to the treatment of nervous system diseases, liver and kidney damage, autoimmune diseases, cardiovascular diseases, etc., and has become a research hotspot that has attracted much attention in the field of regenerative medicine.

[0003] Traditional cell culture mostly uses culture medium containing animal serum. Relatively speaking, serum-free culture medium has significant advantages, with clear composition, stable properties and controllable risks. The development of serum-free culture medium is of vital importance for the research and application of umbilical cord mesenchymal stem cells. Exosomes are nano-scale vesicles secreted by cells, which contain rich bioactive molecules, including nucleic acids (such as miRNA, mRNA), proteins and lipids.

[0004] The current mesenchymal stem cell culture medium has a single and limited selection of ingredients, poor synergistic effect, lack of specificity in added factors, difficulty in maintaining the cell microenvironment, and inability to effectively enhance cell stress resistance. The present invention is intended to solve these problems. Summary of the invention

[0005] In view of the above problems, the present invention provides a serum-free culture medium for umbilical cord mesenchymal stem cells and a method for using the same.

[0006] In a first aspect of the present invention, the present invention discloses a serum-free culture medium for umbilical cord mesenchymal stem cells, the serum-free culture medium comprising a basal culture medium and added factors, the added factors comprising exosomes, human platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, human serum albumin, human transferrin, glycine, nicotinamide, folic acid, L-arginine hydrochloride, L-cystine, pyridoxine hydrochloride, riboflavin, inositol, hydrocortisone, catalase, superoxide dismutase and AWRK6.

[0007] Among the added factors, exosomes contain a variety of bioactive molecules, which can regulate the growth, proliferation, differentiation, immunity and other processes of umbilical cord mesenchymal stem cells in the serum-free medium, participate in intercellular communication, regulate cell behavior, and provide suitable growth conditions for umbilical cord mesenchymal stem cells. Human platelet-derived growth factor, epidermal growth factor, and fibroblast growth factor are growth factor nutrients. After being added to the serum-free medium, human platelet-derived growth factor can promote cell proliferation, migration and participate in extracellular matrix synthesis, epidermal growth factor can promote epithelial cell proliferation, enhance cell survival, promote migration and regulate cell differentiation, and fibroblast growth factor can promote cell proliferation, induce angiogenesis and neuroprotective regeneration. The three can work together to provide suitable growth signals for umbilical cord mesenchymal stem cells, promote cell proliferation, survival and maintain cell differentiation ability. Human serum albumin and human transferrin are protein substances that can play a synergistic role in the culture medium. Human serum albumin can create a stable extracellular environment, which helps transferrin to better perform the function of transporting iron ions. At the same time, some substances transported by human serum albumin can assist transferrin in the cellular metabolic process, and the iron provided by transferrin may also be necessary for human serum albumin to perform certain functions. Glycine, L-arginine hydrochloride, and L-cystine are the basic units that constitute proteins. In serum-free culture medium, they can be used by cells to synthesize intracellular proteins, enzymes and other biomacromolecules, providing a material basis for cell growth and proliferation. Nicotinamide, folic acid, pyridoxine hydrochloride, riboflavin, and inositol are vitamins. After being added to serum-free culture medium, they can play an important role in various physiological processes such as cell metabolism and energy conversion. Hydrocortisone is a hormone substance that can regulate the metabolism of sugar, protein, and fat in cultured cells, enhance cell stress resistance, stabilize cell structure, regulate cell signaling pathways, and inhibit immune response and inflammation. Superoxide dismutase, catalase and AWRK6 work closely together in the serum-free culture medium. Superoxide dismutase converts superoxide anion radicals into hydrogen peroxide, catalase decomposes hydrogen peroxide into water and oxygen, and AWRK6 can efficiently remove free radicals produced by cellular metabolism by regulating the intracellular redox signaling pathway, maintain the intracellular redox balance, reduce the damage of oxidative stress to cells, and ensure the smooth operation of the intracellular signaling pathways and metabolic pathways.

[0008] In summary, the serum-free medium of the present invention is composed of a basal medium and an additive factor including exosomes, growth factors, proteins, amino acids, vitamins, hormones and enzymes / peptides. The components work synergistically. Exosomes can regulate the physiological and biochemical reactions of multiple cell processes and participate in cell communication. Growth factors promote cell proliferation, survival and differentiation. Proteins create an environment to assist metabolism. Amino acids provide a material basis. Vitamins participate in physiological processes. Hormones regulate metabolism, enhance stress, and inhibit immune inflammation. Enzymes / peptides scavenge free radicals and maintain redox balance. The serum-free medium can avoid serum-related problems, and the ingredients are clear and stable, and the risks are controllable. It can make the cultured umbilical cord mesenchymal stem cells have strong proliferation ability, high activity, good stemness, and strong immunoregulatory function.

[0009] Preferably, based on the final mass concentration in the serum-free medium, the contents of the added factors are: exosomes 80-120 μg / mL, human platelet-derived growth factor 5-10 ng / mL, epidermal growth factor 8-12 μg / L, fibroblast growth factor 8-12 μg / L, human serum albumin 2-4 g / L, human transferrin 8-12 mg / L, glycine 15-25 mg / L, nicotinamide 12-18 mg / L, folic acid 4-6 mg / mL, L-arginine hydrochloride 8-12 mg / mL, L-cystine 6-8 mg / mL, pyridoxine hydrochloride 4-6 mg / mL, riboflavin 0.3-0.5 mg / mL, inositol 6-8 mg / mL, hydrocortisone 1-2 mg / L, catalase 200-300 μg / L, superoxide dismutase 4-6×10 6 U / L, AWRK6 200~250mg / L. The umbilical cord mesenchymal stem cells cultured in the serum-free medium have higher cell viability and proliferation multiples and stronger differentiation ability than those cultured in the serum-containing medium. The reproduction multiples of the fifth-generation umbilical cord mesenchymal stem cells cultured on the 7th day were 8.15~11.71, the cell viability was 94.38~97.33%, the expression rates of the typical mesenchymal stem cell markers CD73, CD90, and CD105 were all >98%, and the expression rates of CD34, CD45, CD19, and HLA-DR were all <2%.

[0010] More preferably, based on the final mass concentration, the content of each added factor is: exosome 110 μg / mL, human platelet-derived growth factor 8 ng / mL, epidermal growth factor 11 μg / L, fibroblast growth factor 11 μg / L, human serum albumin 2 g / L, human transferrin 11 mg / L, glycine 20 mg / L, nicotinamide 15 mg / L, folic acid 5 mg / mL, L-arginine hydrochloride 10 mg / mL, L-cystine 8 mg / mL, pyridoxine hydrochloride 5 mg / mL, riboflavin 0.4 mg / mL, inositol 7 mg / mL, hydrocortisone 2 mg / L, catalase 220 μg / L, superoxide dismutase 4.5×10 6 U / L, AWRK6 240mg / L. The serum-free medium formula optimizes the content of each added factor, especially the content of exosomes, which can make the culture effect of umbilical cord mesenchymal stem cells reach the best state, and the synergistic effect between the various components is more efficient, providing cells with sufficient and balanced nutrition and growth signals, accurately regulating the signaling pathways and gene expression in cells, especially the synergy of exosomes and other added factors enhances the cell's ability to regulate immune cells, so that the cultured umbilical cord mesenchymal stem cells are significantly superior to cells cultured in serum-containing medium in terms of cell proliferation multiples, cell viability, stemness maintenance, and immune regulation.

[0011] Preferably, the exosomes are derived from umbilical cord mesenchymal stem cells themselves and / or regulatory T cells. Adding exosomes derived from umbilical cord mesenchymal stem cells themselves to the serum-free medium can promote the self-renewal of the cultured umbilical cord mesenchymal stem cells, maintain cell viability, and highly express stemness-related markers, low expression of non-stem markers, and maintain good proliferation and differentiation potential. Adding exosomes derived from regulatory T cells to the serum-free medium can optimize the cell culture immune microenvironment and enhance the immunoregulatory function of the cultured umbilical cord mesenchymal stem cells. The serum-free medium composed of the two secretions can improve the immunoregulatory ability of umbilical cord mesenchymal stem cells, significantly promote the secretion of immunosuppressive cytokines IL-10 and TGF-β, and inhibit the secretion of proinflammatory cytokines TNF-α and IL-6.

[0012] Preferably, the basal medium is any one or more of DMEM / F12 medium, α-MEM medium, and MEM medium. DMEM / F12 medium, α-MEM medium, and MEM medium contain basic nutrients and material basis necessary for cell growth, and both DMEM / F12 medium and α-MEM medium are improved from MEM medium.

[0013] Preferably, the volume ratio of the basal culture medium to the added factors is 90:1.

[0014] Preferably, the basal culture medium is DMEM / F12 culture medium. DMEM / F12 culture medium and the added factors are used to form a serum-free culture medium, and the DMEM / F12 culture medium provides a basic material environment for cell culture, and the added factors provide growth factors, vitamins, amino acids, active enzymes / peptides, nucleic acids and other nutrients for stem cell proliferation, proliferation and differentiation, which can effectively support umbilical cord mesenchymal stem cells to maintain high proliferation multiples, strong cell viability, high differentiation ability and more stable immunoregulatory ability.

[0015] In a second aspect of the present invention, a use of the serum-free culture medium described in the first aspect of the present invention is disclosed, and a method for culturing umbilical cord mesenchymal stem cells in vitro is proposed, wherein the operation steps include: Step 1: Take umbilical cord mesenchymal stem cells, wash them 2-3 times with PBS buffer, and count them at 4000-6000 cells / cm 2 The cells are inoculated into a first culture vessel containing the serum-free medium at a density of , wherein the first culture vessel is pre-coated with poly-lysine or gelatin to obtain inoculated cells. The first culture vessel is a cell culture vessel commonly used in the art, including but not limited to a cell culture flask, a cell culture plate, a cell culture dish, and a multi-well cell culture plate.

[0016] Step 2: Place the first culture dish containing the inoculated cells obtained in step 1 in a standard incubator at 37°C, 5% CO2, and 95% humidity for culture. The culture period is divided into an early stage and a late stage. The early stage culture adopts a static culture method, and the late stage culture adopts a slight shaking culture method of 50-80rpm. The serum-free medium is replaced every 3 days during the culture period. When the cell confluence reaches 70%-80%, the serum-free medium is discarded, the culture is stopped, and the confluent cells are obtained. When culturing umbilical cord mesenchymal stem cells in this method, under normal circumstances, the early stage culture time is 2 days. The early stage culture adopts static culture to provide a stable environment for the newly inoculated cells to facilitate cell adhesion. After that, it is converted to a slight shaking culture of 50-80rpm, which can provide mechanical stimulation brought by the flow of receptor fluid and tissue movement of cells in the body. By changing the rotation speed to simulate the dynamic microenvironment of cells in the body, the cells can fully exchange substances with the culture medium, timely absorb nutrients, discharge waste, regulate intracellular signal transduction pathways, enhance metabolic activity and physiological functions, make the cell growth state in vitro closer to that in vivo, and improve the quality and efficiency of cell culture.

[0017] Step 3: Subculture the confluent cells obtained in step 2, add 0.1%~0.2% trypsin-EDTA solution, gently digest at 37°C for 1~2 minutes, blow evenly to obtain a single cell suspension, and inoculate the single cell suspension and serum-free culture medium into a second culture vessel at a volume ratio of 1:3~1:4, and the second culture vessel is pre-coated with poly-lysine or gelatin to obtain primary umbilical cord mesenchymal stem cells.

[0018] Step 4: The primary umbilical cord mesenchymal stem cells obtained in step 3 are cultured using the operations of steps 1 to 3 to obtain umbilical cord mesenchymal stem cells of different culture generations.

[0019] In summary, the present invention has the following advantages: (1) The serum-free culture medium used in the present invention is composed of a basal culture medium and a variety of supplementary factors with synergistic effects. It does not contain serum, has clear ingredients, is rich in nutrients, and the components work synergistically to promote the growth, proliferation and maintenance of stemness of umbilical cord mesenchymal stem cells. It solves the problems of traditional culture medium with single supplementary factors, limited synergistic effects, and difficulty in maintaining the cell microenvironment.

[0020] (2) The present invention adds exosomes to the serum-free culture medium. The addition of exosomes derived from umbilical cord mesenchymal stem cells enables the cultured stem cells to maintain good proliferation and differentiation potential. The addition of exosomes derived from regulatory T cells can enhance the immunoregulatory function of the cultured stem cells, significantly promote the secretion of immunosuppressive cytokines IL-10 and TGF-β, and inhibit the secretion of proinflammatory cytokines TNF-α and IL-6 by the cultured stem cells, thereby solving the problem of insufficient stemness maintenance and immunoregulatory capacity of umbilical cord mesenchymal stem cells cultured in vitro.

[0021] (3) Compared with the traditional serum culture medium, the fifth generation umbilical cord mesenchymal stem cells cultured in the serum-free culture medium of the present invention had a proliferation multiple increased by up to 35.8% and a cell viability increased by up to 2.2% on the 7th day, and maintained a high level of stemness during multiple passages (the expression rates of CD73, CD90, and CD105 markers were all >98%, and the expression rates of CD34, CD45, CD19, and HLA-DR markers were all <2%). The serum-free culture medium of the present invention and its use method are consistent with or even better than the traditional serum culture medium in promoting cell proliferation and maintaining cell stemness.

[0022] (4) The present invention simulates the dynamic microenvironment of cells in vivo by adopting a technical solution of first static culture and then slight shaking culture during the culture process, thereby promoting substance exchange and signal transmission between cells and culture medium, and improving the quality and efficiency of cell culture.

[0023] (5) The present invention avoids the risks of pathogen contamination, immune response, etc. that may be brought about by culture medium containing animal serum by using a serum-free culture medium with clear ingredients, stable properties and controllable risks and a method for using the same, thereby improving the safety and reliability of cell culture.

[0024] (6) The culture method of the present invention has standardized operation, which ensures the stability and repeatability of the cell culture process, and is beneficial to industrial application, scientific research and clinical research. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The cell densities of umbilical cord mesenchymal stem cells after being cultured for 1, 3, 5, and 7 days after being subcultured to the fifth generation under the culture medium M1~M8, N1, and N2 respectively.

[0026] Figure 2 The cell proliferation multiples are 1, 3, 5, and 7 days after culturing the fifth generation of umbilical cord mesenchymal stem cells under the culture medium M1~M8, N1, and N2 conditions.

[0027] Figure 3 The cell viability of umbilical cord mesenchymal stem cells after being cultured for 1, 3, 5, and 7 days after being subcultured to the fifth generation under the culture medium M1~M8, N1, and N2 conditions.

[0028] Figure 4 The percentages of CD73, CD90 and CD105 positive cells of umbilical cord mesenchymal stem cells after being passaged to the fifth generation under culture medium M1-M8, N1 and N2 for 7 days.

[0029] Figure 5 The percentages of CD34, CD45, CD19 and HLA-DR positive cells of umbilical cord mesenchymal stem cells after being passaged to the fifth generation under culture medium M1-M8, N1 and N2 for 7 days. DETAILED DESCRIPTION

[0030] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments. Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by ordinary technicians in the field to which the present invention belongs.

[0031] Those skilled in the art should understand that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention. Without departing from the essence of the present invention, any simple modification, equivalent replacement and improvement of the technical solution of the present invention should be included in the protection scope of the present invention.

[0032] In the present invention, the components and reagents involved are conventional commercial products, or can be obtained by conventional technical means in the art. Unless otherwise specified, the materials, methods and embodiments of the present invention are only exemplary and non-limiting.

[0033] Example 1: Serum-free culture medium for umbilical cord mesenchymal stem cells The serum-free medium provided in this embodiment is composed of a basal medium and an added factor. DMEM / F12 medium is selected as the basal medium. DMEM / F12 medium can be self-prepared or directly purchased. The DMEM / F12 medium used in this embodiment is purchased from Yuanye Biotechnology Co., Ltd. (Cat. No. R20170-500ml). The added factors are recorded as MIX1 and MIX2, and the final mass concentration of each component is shown in Table 1. Only the exosome concentration is consistent in MIX1 and MIX2, and the exosomes use exosomes derived from umbilical cord mesenchymal stem cells themselves.

[0034] Table 1 Final mass concentration of each component in MIX1 and MIX2 The preparation method of the serum-free medium provided in this embodiment has the following operating steps: 1) Use 1×PBS buffer to dissolve the added factors. The dissolution order can be: salts and small molecule amino acids, vitamins and small molecule added factors, proteins and enzymes, exosomes. After adding each component, gently stir it to make it dissolve evenly to avoid excessive foaming, and the added factors MIX1 and MIX2 are prepared.

[0035] 2) The prepared MIX1 and MIX2 were respectively mixed evenly with DMEM / F12 culture medium at a volume ratio of 1:9, and after mixing, the mixtures were sterilized by filtering through a 0.1 μm filter membrane to prepare umbilical cord mesenchymal stem cell serum-free culture medium M1 and M2.

[0036] Example 2: Serum-free culture medium for umbilical cord mesenchymal stem cells with different exosome contents Exosomes have an important influence on the umbilical cord mesenchymal stem cell serum-free medium disclosed in the present invention, and serum-free medium with different exosome contents is provided in Example 2. Similar to Example 1, the serum-free medium provided in this example is composed of a basal medium and added factors, the basal medium is the same as in Example 1, and the added factors are recorded as MIX3, MIX4, MIX5 and MIX6, respectively. The final mass concentration of each component in each group of added factors is shown in Table 2, and the exosomes use exosomes derived from umbilical cord mesenchymal stem cells themselves.

[0037] Table 2 Final mass concentration of each component in MIX3~MIX6 Similarly, the preparation method of the serum-free medium provided in this embodiment comprises the following steps: 1) Use 1×PBS buffer to dissolve the added factors. The dissolution order can be: salts and small molecule amino acids, vitamins and small molecule added factors, proteins and enzymes, exosomes. After adding each component, gently stir it to make it dissolve evenly to avoid excessive foaming, and prepare the added factors MIX3, MIX4, MIX5 and MIX6.

[0038] 2) The prepared MIX3, MIX4, MIX5 and MIX6 were respectively mixed with DMEM / F12 culture medium at a volume ratio of 1:9, and after mixing, the mixture was sterilized by filtering through a 0.1 μm filter membrane to prepare umbilical cord mesenchymal stem cell serum-free culture medium M3, M4, M5 and M6.

[0039] Comparative Example 1: Serum-free medium for umbilical cord mesenchymal stem cells without exosomes In order to demonstrate the effect of exosomes on the invented serum-free medium, this comparative example provides an umbilical cord mesenchymal stem cell serum-free medium without exosomes. Similar to Example 2, the serum-free medium provided in this comparative example is composed of a basal medium and an added factor, wherein the exosome content in the added factor is 0, and the other components are consistent with the medium described in Example 2.

[0040] Similarly, according to the culture medium preparation method of Example 2, an exosome-free and serum-free culture medium N1 was prepared.

[0041] Comparative Example 2: Umbilical cord mesenchymal stem cell culture medium containing serum and no added factors This comparative example provides a serum-containing culture medium for umbilical cord mesenchymal stem cells, which is composed of fetal bovine serum and a basic culture medium and does not contain added factors. The basic culture medium is selected from DMEM / F12 culture medium.

[0042] The preparation method of the culture medium provided in this comparative example comprises the following steps: 1) Take inactivated fetal bovine serum, add it to DMEM / F12 medium at a volume ratio of 10%, and mix well to obtain serum-containing medium N2.

[0043] Example 3: Umbilical cord mesenchymal stem cell serum-free culture medium with different basal culture media The serum-free medium provided in this embodiment is composed of a basal medium and an added factor. The basal medium is selected from α-MEM medium and MEM medium, and the added factor is consistent with the added factor MIX5 in Table 2.

[0044] Similarly, the preparation method of the serum-free medium provided in this embodiment comprises the following steps: 1) Use 1× PBS buffer to dissolve the additive factors. After adding each component, gently stir to make it dissolve evenly to avoid excessive foaming, and prepare the additive factor MIX5.

[0045] 2) The prepared MIX5 was mixed evenly with α-MEM medium and MEM medium at a volume ratio of 1:9, and then sterilized by filtering through a 0.1 μm filter membrane to prepare umbilical cord mesenchymal stem cell serum-free medium M7 and M8.

[0046] Example 4: Serum-free culture medium of umbilical cord mesenchymal stem cells from different exosome sources Similar to the culture medium M5 in Example 2, this example provides a serum-free culture medium for umbilical cord mesenchymal stem cells. Except for the exosomes derived from regulatory T cells, the components and concentrations of the basal culture medium and the added factors are consistent with those of the culture medium M5.

[0047] Similarly, according to the culture medium preparation method of Example 2, a serum-free culture medium M9 was prepared.

[0048] Example 5: Proliferation rate and activity of umbilical cord mesenchymal stem cells cultured in serum-free medium In this example, the culture media M1-M8, N1 and N2 described in Examples 1-4 and Comparative Examples 1-2 are selected to culture umbilical cord mesenchymal stem cells, and the cells are cultured according to the following steps: Step 1: Take human umbilical cord mesenchymal stem cells, wash them with PBS buffer 2-3 times, and add 5000 umbilical cord mesenchymal stem cells / cm 2 The cells were uniformly inoculated into T25 cell culture flasks at a density of 100 μg / mL to obtain inoculated cells. The T25 cell culture flasks were pre-coated with poly-lysine and culture media M1-M8, N1 and N2 were added respectively. The culture media M1-M8, N1 and N2 were prepared according to the methods described in Examples 1-4 and Comparative Examples 1-2.

[0049] Step 2: Culture the T25 cell culture flask containing the inoculated cells obtained in step 1 in a standard incubator at 37°C, 5% CO2, and 95% humidity. The first two days are for preliminary culture, using a static culture method so that the cells can fully adhere to the wall. From the third day, switch to a slight shaking culture (60rpm). Replace the corresponding culture medium every 3 days. When the cell confluence reaches 80%, discard the culture medium, stop the culture, and obtain confluent cells.

[0050] Step 3: Subculture the confluent cells obtained in step 2, add an appropriate amount of 0.1% trypsin-EDTA solution, and gently digest at 37°C for 1-2 minutes. After the cells begin to round and the intercellular space increases under microscopic observation, gently blow and beat to evenly disperse the cells to form a single cell suspension. Inoculate the cell suspension into a new pre-coated T25 cell culture flask at a volume ratio of 1:3, add the corresponding fresh culture medium, and continue to culture to obtain primary umbilical cord mesenchymal stem cells.

[0051] Step 4: The umbilical cord mesenchymal stem cells obtained in step 3 are cultured using the operations of steps 1 to 3 to obtain umbilical cord mesenchymal stem cells of different culture generations.

[0052] The cell proliferation rate test was performed as follows: On the 1st, 3rd, 5th, and 7th days of the fifth generation cell culture, an appropriate amount of cell suspension was drawn from each culture flask into a centrifuge tube, the supernatant was discarded, and an appropriate amount of trypsin-EDTA solution was added to digest the cells to prepare a single cell suspension. A small amount of cell suspension was mixed with trypan blue at a ratio of 1:1, and counted using a Thermo Fisher Countess3 automatic cell counter to calculate the cell density and cell proliferation multiples. Three replicate experimental groups were set for each sample.

[0053] Cell activity was detected as follows: On the 5th and 7th days of the fifth generation of cell culture, the cells in the cell culture flask were digested with trypsin-EDTA into a single cell suspension and the cell density was adjusted to 5×10 3 / mL, inoculated in 96-well plates, 100μL per well, 5 replicates per group. Incubate in the incubator for 2 hours until the cells adhere to the wall, add 10μL CCK-8 solution to each well, and continue incubation for 3 hours. Use an enzyme marker to measure the absorbance value (OD value) of each well at a wavelength of 450nm. Calculate cell activity according to the formula, and set up 3 replicate experimental groups for each sample.

[0054] Cell activity = (OD value of experimental group - OD value of blank group) / (OD value of control group - OD value of blank group) × 100%. The blank group is the well with only culture medium and CCK-8 solution. The control group can use cells cultured with N1 culture medium.

[0055] The cell density and cell proliferation times of umbilical cord mesenchymal stem cells after culture were as follows: Figure 1 and Figure 2As shown, with the increase of culture days, the cell density and proliferation times of umbilical cord mesenchymal stem cells in each culture medium showed an upward trend. Overall, the culture medium containing added factors (M1~M8) has similar culture effects to the serum-containing culture medium (N2), among which culture media M2, M5, M6 and M8 are significantly better than N2. Taking the 7th day as an example, the cell proliferation times of culture media M2, M5, M6 and M8 are 11.28, 11.71, 10.31 and 10.78 respectively, and the cell proliferation times of culture medium N2 is 8.62, indicating that the exosomes and specific added factors added to the invented culture medium have a significant promoting effect on cell proliferation, and can effectively support the growth and division of umbilical cord mesenchymal stem cells. Figure 1 and Figure 2 As shown, among the culture media N1, M3-M6, N1 had the worst culture effect and M5 had the best culture effect, indicating that exosomes are necessary for the culture of umbilical cord mesenchymal stem cells, and the culture effect was best when the exosome concentration was 110 μg / mL.

[0056] Cell viability results were as follows Figure 3 As shown in the figure, on the 5th and 7th days, the cell viability in each culture medium was maintained at a high level, most of which were above 90%. On the 7th day, the cell viability of culture media M5, N1 and N2 were 97.33%, 92.36% and 95.28%, respectively, indicating that each culture medium can maintain the activity of cells to a certain extent and provide a relatively suitable environment for cell growth. At the same time, exosomes also play an important role in maintaining cell activity. When the exosome content is appropriate, the cell activity of the cultured cells is stronger.

[0057] Example 6: Cell surface markers of umbilical cord mesenchymal stem cells cultured in serum-free medium Example 6 The same culture medium and culture scheme as in Example 5 were used to culture umbilical cord mesenchymal stem cells, and the cell surface markers were detected as follows: the fifth generation cells were cultured to day 7, the cells were collected, and the cell concentration was adjusted to 1×10 6 / mL, take 100μL of cell suspension to flow tube, add appropriate amount of fluorescent labeled anti-CD73, CD90, CD105, CD34, CD45, CD19 and HLA-DR antibodies, detect the expression of cell surface markers by flow cytometry, and analyze the percentage of positive cells. Set up 3 replicate experimental groups for each sample.

[0058] For the positive markers CD73, CD90, and CD105, the results were as follows Figure 4As shown in the figure, the percentage of positive cells in cells cultured in each culture medium was at a high level. Among them, the expression rates of culture medium M2, M5, M6 and N2 were all >99%, the expression rate of culture medium N1 was >97%, and the other groups were >98%, indicating that the appropriate concentration of exosomes and added factors can effectively support the expression of these positive markers by umbilical cord mesenchymal stem cells, and culture medium M2, M5, and M6 can achieve better results than serum-containing culture medium N2.

[0059] For negative markers CD34, CD45, CD19, and HLA-DR, the results were as follows Figure 5 As shown, the percentage of positive cells in each culture medium is low, which is consistent with the characteristics of umbilical cord mesenchymal stem cells. Among them, the expression rate of culture medium N1 is <3%, and the other groups are <2%. The culture media M1~M8 mentioned in Examples 1~4 are better than the serum-containing culture medium N2, which can better maintain the stemness of cells and inhibit the differentiation of cells into other types of cells. However, culture medium N1 does not contain exosomes of its own origin, and has poor fine regulation of cell signaling pathways and gene expression, resulting in certain differences in cell marker expression compared with the other experimental groups.

[0060] This example detects surface markers of umbilical cord mesenchymal stem cells cultured in different culture media, indicating that the culture media disclosed in the present invention (taking M1 to M8 as an example) are comparable to serum-containing culture media (N2) in promoting cell expression of positive markers and inhibiting the expression of negative markers, and some culture media (M2, M5 and M6) are even more effective.

[0061] Example 7: Ability of umbilical cord mesenchymal stem cells cultured in serum-free medium to differentiate into osteoblasts Example 7 The same culture medium and culture scheme as in Example 5 were used to culture umbilical cord mesenchymal stem cells. The osteogenic induction medium was based on DMEM / F12 medium, and 1×10 -7 mol / L dexamethasone, 10mmol / L β-glycerophosphate sodium, 50μg / mL vitamin C, 100U / mL penicillin, and 100μg / mL streptomycin.

[0062] The differentiation induction and detection were carried out as follows: 1) On the 7th day of the fifth generation of cell culture, 1×10 4 The cells were seeded at a density of 100 / cm² in a 6-well plate, and 2 mL of osteogenic induction medium was added to each well. The culture was continued for 21 days, during which the medium was changed every 3 days. After the induction was completed, the medium was discarded and the cells were washed twice with PBS.

[0063] 2) Add 4% paraformaldehyde and fix for 30 minutes, then wash three times with PBS. Add Alizarin Red dye and stain for 30 minutes at room temperature in the dark. Wash several times with distilled water to remove excess dye.

[0064] 3) Microscopic examination and counting: Place the 6-well plate under an inverted microscope for observation, select multiple fields of view for counting, and calculate the cell differentiation efficiency according to the formula. Set up 3 replicate experimental groups for each sample.

[0065] Cell differentiation efficiency = (total number of mineralized nodule cells / total number of cells) × 100%.

[0066] The results of the induction and differentiation of the fifth generation of umbilical cord mesenchymal stem cells into osteoblasts are shown in Table 3. Exosomes and specific added factors were added to the culture medium M1~M8, and the overall cell differentiation efficiency was at a relatively high level, between 40.12% and 48.62%, which was similar to the results of the serum-containing culture medium N2, indicating that the osteogenic differentiation ability of the cultured stem cells was normal. Among them, the cell differentiation efficiency of culture medium M5 was the highest, reaching 48.62±2.50%, which was higher than that of culture medium N2, while the cell differentiation efficiency of culture medium N1 was the lowest, indicating that this culture medium has significant advantages in promoting the osteogenic differentiation of umbilical cord mesenchymal stem cells. The combination of exosomes and added factors can more effectively maintain the stemness of cells, more easily activate the signal pathways related to osteogenic differentiation in cells, and promote the transformation of cells into osteoblasts.

[0067] Table 3 Differentiation efficiency of umbilical cord mesenchymal stem cells into osteoblasts Example 8: Ability of umbilical cord mesenchymal stem cells cultured in serum-free medium to differentiate into adipocytes Example 8 The same culture medium and culture scheme as in Example 5 were used to culture umbilical cord mesenchymal stem cells. The adipogenic induction medium was based on DMEM / F12 medium, and 1×10 -6 mol / L dexamethasone, 0.5mmol / L 3-isobutyl-1-methylxanthine (IBMX), 10μg / mL insulin, 200μmol / L indomethacin, 100U / mL penicillin, and 100μg / mL streptomycin.

[0068] The differentiation induction and detection were carried out as follows: 1) On the 7th day of the fifth generation of cell culture, 1×10 4 The cells were inoculated at a density of 100 / cm² in a 6-well plate, and 2 mL of adipogenic induction medium was added to each well. The culture was continued for 14 days, during which the medium was changed every 3 days. After the induction, the medium was discarded and the cells were washed twice with PBS.

[0069] 2) Add 4% paraformaldehyde and fix for 30 minutes, then wash three times with PBS. Add Oil Red O staining solution and stain for 60 minutes at room temperature in the dark. Wash several times with 60% isopropanol to remove excess staining solution.

[0070] 3) Microscopic examination and counting: Place the 6-well plate under an inverted microscope for observation, select multiple fields of view for counting, and calculate the cell differentiation efficiency according to the formula. Set up 3 replicate experimental groups for each sample.

[0071] Cell differentiation efficiency = (total number of adipogenic staining / total number of cells) × 100%.

[0072] Similar to Example 7, the results of inducing differentiation of cultured cells into adipocytes are shown in Table 4. The adipogenic differentiation efficiency of cells cultured in medium M1~M8 is between 36.87% and 45.68%, which is higher than the effect of serum-containing medium N2. Among them, the cell differentiation efficiency of medium M5 is the highest, reaching 45.68±2.80%, and the cell differentiation efficiency of medium N1 without exosomes is the lowest, indicating that the concentration of exosomes and added factors has a great effect on promoting cell adipogenic differentiation. The combination of exosomes and added factors in medium M5 can maintain cell stemness, and in the process of inducing cell adipogenic differentiation, it is more in line with the needs of cell differentiation, and can more effectively activate key signaling molecules and transcription factors related to adipogenic differentiation, and promote the transformation of cells into adipocytes.

[0073] Table 4 Differentiation efficiency of umbilical cord mesenchymal stem cells into adipocytes Example 9: Immunomodulatory ability of umbilical cord mesenchymal stem cells cultured in serum-free medium Example 9: The culture media M5, M9, N1 and N2 described in Example 2, Example 4, Comparative Example 1 and Comparative Example 2 were selected respectively, and the umbilical cord mesenchymal stem cells were cultured using the same culture scheme as in Examples 5 to 8.

[0074] The immunomodulatory capacity of cells was tested as follows: 1) On the 7th day of the fifth generation of cell culture, the cells were collected and washed twice with PBS to adjust the cell concentration to 1×10 6 Pieces / mL.

[0075] 2) Take an appropriate amount of peripheral blood from healthy volunteers, separate peripheral blood mononuclear cells (PBMCs) using lymphocyte separation medium, and adjust the cell concentration to 2×10 6 Pieces / mL.

[0076] 3) Umbilical cord mesenchymal stem cells and PBMCs were co-cultured in a 24-well plate at a ratio of 1:10, with a total volume of 1 mL per well. A PBMCs culture group was set up as a control.

[0077] 4) After 72 hours of co-culture, the supernatant was collected and the concentrations of IL-10, TGF-β, TNF-α and IL-6 in the supernatant were detected using ELISA kits. Three replicate experimental groups were set up for each sample.

[0078] The test results of each factor are shown in Table 5. The immunoregulatory ability of umbilical cord mesenchymal stem cells cultured in M5 and M9 medium is strong, which can significantly promote the secretion of immunosuppressive cytokines IL-10 and TGF-β, and inhibit the secretion of proinflammatory cytokines TNF-α and IL-6. Among them, the immunoregulatory ability of stem cells cultured in M5 medium is the strongest, indicating that the exosomes derived from umbilical cord mesenchymal stem cells themselves are more helpful for umbilical cord mesenchymal stem cells to exert their immunoregulatory function. The results of N2 medium are second, indicating that the appropriate combination and concentration of added factors and exosomes can enhance the immunoregulatory ability of cultured stem cells, and the immunoregulatory ability of cultured cells is stronger than that of serum-containing medium. The results of N1 medium are worse, indicating that exosomes play an important role in the immunoregulatory ability of cultured cells, and adding exosome components to serum-free medium can help enhance the immunoregulatory ability of cultured stem cells.

[0079] Table 5 Secretion of IL-10, TGF-β, TNF-α and IL-6 by umbilical cord mesenchymal stem cells In summary, the serum-free culture medium for umbilical cord mesenchymal stem cells involved in the present invention is added with a variety of clear added factors such as exosomes, and has clear chemical composition, which not only avoids many problems caused by serum, but also can enhance the cell activity, value-added ability, stemness and immunoregulatory ability of the cultured stem cells. In practical applications, the serum-free culture medium of the present invention and the method of use thereof are used to culture umbilical cord mesenchymal stem cells in vitro, which can not only effectively improve the proliferation efficiency and cell viability, but also stably maintain the differentiation ability of the cells and improve the immunoregulatory ability of the cells.

[0080] Furthermore, the present invention provides ideas and methods for using the invented umbilical cord mesenchymal stem cell serum-free culture medium. It should be pointed out that the above embodiments are only preferred implementations of the present invention. For ordinary technicians in this technical field, several improvements and supplements can be made without departing from the principles of the present invention. These improvements and supplements should also be included in the protection scope of the present invention.

Claims

1. A serum-free culture medium for umbilical cord mesenchymal stem cells, characterized in that: The serum-free medium comprises a basal medium and an added factor; the added factor comprises exosomes, human platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, human serum albumin, human transferrin, glycine, nicotinamide, folic acid, L-arginine hydrochloride, L-cystine, pyridoxine hydrochloride, riboflavin, inositol, hydrocortisone, catalase, superoxide dismutase and AWRK6; Based on the final mass concentration in the serum-free medium, the contents of the added factors are: exosomes 80-120 μg / mL, human platelet-derived growth factor 5-10 ng / mL, epidermal growth factor 8-12 μg / L, fibroblast growth factor 8-12 μg / L, human serum albumin 2-4 g / L, human transferrin 8-12 mg / L, glycine 15-25 mg / L, nicotinamide 12-18 mg / L, folic acid 4-6 mg / mL, L-arginine hydrochloride 8-12 mg / mL, L-cystine 6-8 mg / mL, pyridoxine hydrochloride 4-6 mg / mL, riboflavin 0.3-0.5 mg / mL, inositol 6-8 mg / mL, hydrocortisone 1-2 mg / L, catalase 200-300 μg / L, superoxide dismutase 4-6×10 6 U / L, AWRK6 200~250mg / L.

2. The serum-free medium according to claim 1, characterized in that The contents of the added factors were as follows: exosomes 110 μg / mL, human platelet-derived growth factor 8 ng / mL, epidermal growth factor 11 μg / L, fibroblast growth factor 11 μg / L, human serum albumin 2 g / L, human transferrin 11 mg / L, glycine 20 mg / L, nicotinamide 15 mg / L, folic acid 5 mg / mL, L-arginine hydrochloride 10 mg / mL, L-cystine 8 mg / mL, pyridoxine hydrochloride 5 mg / mL, riboflavin 0.4 mg / mL, inositol 7 mg / mL, hydrocortisone 2 mg / L, catalase 220 μg / L, superoxide dismutase 4.5 × 10 6 U / L, AWRK6 240mg / L.

3. The serum-free medium according to claim 1, characterized in that The exosomes are derived from umbilical cord mesenchymal stem cells themselves and / or regulatory T cells.

4. The serum-free medium according to claim 1, characterized in that The basal culture medium is any one or more of DMEM / F12 culture medium, α-MEM culture medium, and MEM culture medium, and the volume ratio of the basal culture medium to the added factors is 90:

1.

5. The serum-free medium according to claim 1, characterized in that The basic culture medium is DMEM / F12 culture medium.

6. A method for culturing umbilical cord mesenchymal stem cells in vitro using the serum-free medium according to claim 1, characterized in that: The steps include: Step 1: Take umbilical cord mesenchymal stem cells, wash them 2-3 times with PBS buffer, and count them at 4000-6000 cells / cm 2 Inoculate the cells at a density of 100 μg / cm2 into a first culture vessel containing the serum-free medium, wherein the first culture vessel is pre-coated with poly-lysine or gelatin to obtain inoculated cells; Step 2: placing the first culture dish containing the inoculated cells obtained in step 1 in a standard incubator at 37° C., 5% CO2, and 95% humidity for culture. The culture period is divided into an early stage and a late stage. The early stage culture adopts a static culture method, and the late stage culture adopts a slight shaking culture method at 50-80 rpm. The serum-free culture medium is replaced every 3 days during the culture period. When the cell confluence reaches 70%-80%, the serum-free culture medium is discarded, the culture is stopped, and confluent cells are obtained; Step 3: Passage the confluent cells obtained in step 2, add 0.1% to 0.2% trypsin-EDTA solution, gently digest at 37°C for 1 to 2 minutes, blow evenly to obtain a single cell suspension, and inoculate the single cell suspension and serum-free culture medium into a second culture vessel at a volume ratio of 1:3 to 1:4, wherein the second culture vessel is pre-coated with poly-lysine or gelatin to obtain primary umbilical cord mesenchymal stem cells; Step 4: The primary umbilical cord mesenchymal stem cells obtained in step 3 are cultured using the operations of steps 1 to 3 to obtain umbilical cord mesenchymal stem cells of different culture generations.

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