阅读说明：本技术 脐带间充质干细胞无血清培养基及其制备方法 (Serum-free medium for umbilical cord mesenchymal stem cells and preparation method thereof ) 是由 张印 于 2020-06-02 设计创作，主要内容包括：本发明涉及干细胞培养技术领域,涉及一种脐带间充质干细胞无血清培养基及其制备方法,包括DMEM低糖培养基,还包括人血清白蛋白、转铁蛋白、表皮细胞生长因子、转化生长因子、胰岛素、橄榄油、丹参素钠和银杏酸。本发明制备的培养基可显著提高脐带间充质干细胞的增殖数量和增殖率,并且脐带间充质干细胞不易分化产生表型改变,较好的保护脐带间充质干细胞,并且脐带间充质干细胞贴壁的效果佳,成本较低,易于工艺化生产。(The invention relates to the technical field of stem cell culture, and relates to a serum-free culture medium for umbilical cord mesenchymal stem cells and a preparation method thereof. The culture medium prepared by the invention can obviously improve the proliferation quantity and the proliferation rate of the umbilical cord mesenchymal stem cells, the umbilical cord mesenchymal stem cells are not easy to differentiate to generate phenotype change, the umbilical cord mesenchymal stem cells are well protected, the wall-adhering effect of the umbilical cord mesenchymal stem cells is good, the cost is low, and the process production is easy.)

1. The serum-free culture medium for the umbilical cord mesenchymal stem cells is characterized by comprising a DMEM low-sugar culture medium, and further comprising human serum albumin, transferrin, epidermal growth factors, transforming growth factors, insulin, olive oil, sodium danshensu and ginkgolic acid.

2. The serum-free medium for umbilical cord mesenchymal stem cells according to claim 1, wherein the concentration of human serum albumin is 1-5 mg/mL, the concentration of transferrin is 20-50 mg/mL, the concentration of epidermal growth factor is 10-30 ng/mL, the concentration of transforming growth factor is 20-40 ng/mL, the concentration of insulin is 15-25 μ g/mL, the concentration of olive oil is 1-5 mg/mL, the concentration of sodium danshensu is 10-30 μ g/mL and the concentration of ginkgolic acid is 5-15 μ g/mL.

3. The serum-free medium for umbilical cord mesenchymal stem cells according to claim 2, wherein the concentration of human serum albumin is 1-4 mg/mL, the concentration of transferrin is 20-40 mg/mL, the concentration of epidermal growth factor is 15-30 ng/mL, the concentration of transforming growth factor is 20-35 ng/mL, the concentration of insulin is 15-20 μ g/mL, the concentration of olive oil is 1-3 mg/mL, the concentration of sodium danshensu is 15-30 μ g/mL and the concentration of ginkgolic acid is 5-10 μ g/mL.

4. The serum-free medium for umbilical cord mesenchymal stem cells according to claim 2, wherein the concentration of human serum albumin is 3.5mg/mL, the concentration of transferrin is 25mg/mL, the concentration of epidermal growth factor is 26ng/mL, the concentration of transforming growth factor is 30ng/mL, the concentration of insulin is 18 μ g/mL, the concentration of olive oil is 2mg/mL, the concentration of sodium danshensu is 24 μ g/mL and the concentration of ginkgolic acid is 8 μ g/mL.

5. The serum-free medium for umbilical cord mesenchymal stem cells according to claim 1, wherein the insulin is human insulin and the transforming growth factor is transforming growth factor-beta 1.

6. The serum-free medium for umbilical cord mesenchymal stem cells according to claim 1, further comprising L-ascorbic acid-2 phosphoric acid at a concentration of 0.01 to 0.02 μmol/mL and trypsin at a concentration of 0.15 to 0.25 mg/mL.

7. The preparation method of the serum-free culture medium for umbilical cord mesenchymal stem cells according to claim 6, which comprises the following steps:

s1, taking a DMEM low-sugar culture medium, human serum albumin, transferrin, epidermal growth factor, transforming growth factor, insulin, olive oil, sodium danshensu, ginkgolic acid, L-ascorbic acid-2 phosphoric acid and trypsin, uniformly stirring, and filtering and sterilizing by using a filter membrane to obtain the serum-free culture medium for the umbilical cord mesenchymal stem cells.

8. The method for preparing the serum-free culture medium for the umbilical cord mesenchymal stem cells according to claim 7, wherein the pore size of the filter membrane is 0.1-0.5 μm.

9. The method for preparing the serum-free culture medium of the umbilical cord mesenchymal stem cells according to claim 7, wherein the pH value of the serum-free culture medium of the umbilical cord mesenchymal stem cells is 6.5-7.8.

10. The use of the serum-free medium for umbilical cord mesenchymal stem cells according to any one of claims 1 to 6 in the culture of umbilical cord mesenchymal stem cells.

Technical Field

The invention relates to the technical field of stem cell culture, in particular to a serum-free culture medium for umbilical cord mesenchymal stem cells and a preparation method thereof.

Background

The umbilical cord mesenchymal stem cell refers to a multifunctional stem cell existing in umbilical cord tissues of newborn, which can be differentiated into a plurality of tissue cells, a culture system taking human umbilical cord blood serum as a main body can be used for successfully amplifying the human umbilical cord mesenchymal stem cell, the growth of the human umbilical cord mesenchymal stem cell depends on the existence of blood serum, such as fetal bovine serum or newborn bovine serum, in a culture medium without adding blood serum, most cells (including stem cells) cannot grow, proliferate or differentiate, the blood serum can provide a plurality of nutrients necessary for the growth of a plurality of cells (including stem cells), such as insulin, adrenocortical hormone, steroid hormone (estradiol, testosterone, progesterone) and the like, and also provides binding proteins carrying various important low-molecular-weight substances, such as albumin carrying vitamins, fat, hormones and the like, transferrin carrying iron and the like.

Serum is a very complex mixture, the components can be hundreds, the exact components, contents and action mechanisms of the serum are still unclear at present, the quality difference among the serum batches is large, the influence of the sources of the raw materials of manufacturers is large, the consistency of each batch is difficult to ensure, the standardization of laboratory research or product production is not facilitated, the serum can contain unknown or undetected harmful microorganisms such as mycoplasma and viruses, and the like, and the harmful microorganisms can have potential influence on cells, and substances such as antibodies, complements, bacterial toxins and the like in the serum can also have adverse influence on the growth of the cells, and even cause the death of the cells.

However, the clinical application of stem cells requires the culture with a serum-free medium, the culture effect of the serum-free medium in the current market is far inferior to that of the serum medium, and the Chinese patent application CN105420182A discloses a serum-free umbilical cord mesenchymal stem cell culture medium, which comprises a DMEM medium, a basic fibroblast growth factor, an epidermal growth factor, insulin, a leukemia inhibitory factor, trypsin, 2-mercaptoethanol, sodium selenite, fibronectin and the like, and achieves better adherence performance by adding higher-content fibronectin, but the proliferation rate of cells is slower; chinese patent application CN102634482A discloses a serum-free complete medium for mesenchymal stem cells, which consists of a basal medium and additive components, wherein the basal medium is DMEM/F12, the additive components consist of recombinant insulin, human serum albumin, human transferrin, cholesterol, sodium selenite, ferric nitrate and dextran, and the medium does not contain serum, so as to avoid the defects of serum containing the serum medium, such as instability in batches, cytotoxicity and a large amount of heterologous proteins, but the cultured stem cells are easy to differentiate to change phenotype, and the serum-free medium for umbilical cord mesenchymal stem cells sold in some markets has the defects of high price, unsatisfactory adherence performance and the like.

Therefore, there is a need for a serum-free culture medium for umbilical cord mesenchymal stem cells, which has a fast proliferation rate, is not easy to differentiate, has a low cost and is easy to industrialize, and a preparation method thereof.

Disclosure of Invention

Aiming at the defects in the prior art, one of the purposes of the invention is to provide the serum-free culture medium for the umbilical cord mesenchymal stem cells, the proliferation rate of the umbilical cord mesenchymal stem cells can be obviously improved by adding the sodium danshensu and the ginkgolic acid, the umbilical cord mesenchymal stem cells are not easy to differentiate to generate phenotype change, the umbilical cord mesenchymal stem cells are well protected, the effect of enhancing the wall adhesion by adding the olive oil is also added, the cost is lower, and the process production is easy.

The above object of the present invention is achieved by the following technical solutions:

a serum-free culture medium for umbilical cord mesenchymal stem cells comprises a DMEM low-sugar culture medium, and further comprises human serum albumin, transferrin, epidermal growth factor, transforming growth factor, insulin, olive oil, sodium danshensu and ginkgolic acid.

Preferably, the concentration of the human serum albumin is 1-5 mg/mL, the concentration of the transferrin is 20-50 mg/mL, the concentration of the epidermal growth factor is 10-30 ng/mL, the concentration of the transforming growth factor is 20-40 ng/mL, the concentration of the insulin is 15-25 mug/mL, the concentration of the olive oil is 1-5 mg/mL, the concentration of the danshensu sodium is 10-30 mug/mL, and the concentration of the ginkgolic acid is 5-15 mug/mL.

Preferably, the concentration of the human serum albumin is 1-4 mg/mL, the concentration of the transferrin is 20-40 mg/mL, the concentration of the epidermal growth factor is 15-30 ng/mL, the concentration of the transforming growth factor is 20-35 ng/mL, the concentration of the insulin is 15-20 mug/mL, the concentration of the olive oil is 1-3 mg/mL, the concentration of the danshensu sodium is 15-30 mug/mL, and the concentration of the ginkgolic acid is 5-10 mug/mL.

More preferably, the concentration of the human serum albumin is 3.5mg/mL, the concentration of the transferrin is 25mg/mL, the concentration of the epidermal growth factor is 26ng/mL, the concentration of the transforming growth factor is 30ng/mL, the concentration of the insulin is 18 mug/mL, the concentration of the olive oil is 2mg/mL, the concentration of the salvianic acid A sodium is 24 mug/mL and the concentration of the ginkgoic acid is 8 mug/mL.

Preferably, the insulin is human insulin, and the transforming growth factor is transforming growth factor-beta 1.

Preferably, the serum-free culture medium for the umbilical cord mesenchymal stem cells further comprises L-ascorbic acid-2 phosphoric acid with the concentration of 0.01-0.02 mu mol/mL and trypsin with the concentration of 0.15-0.25 mg/mL.

The second objective of the invention is to provide a preparation method of a serum-free culture medium for umbilical cord mesenchymal stem cells, which comprises the following steps:

s1, taking a DMEM low-sugar culture medium, human serum albumin, transferrin, epidermal growth factor, transforming growth factor, insulin, olive oil, sodium danshensu, ginkgolic acid, L-ascorbic acid-2 phosphoric acid and trypsin, uniformly stirring, and filtering and sterilizing by using a filter membrane to obtain the serum-free culture medium for the umbilical cord mesenchymal stem cells.

Preferably, the aperture of the filter membrane is 0.1-0.5 μm.

Preferably, the pH value of the serum-free culture medium for the umbilical cord mesenchymal stem cells is 6.5-7.8.

The invention also provides application of the serum-free culture medium for the umbilical cord mesenchymal stem cells in culture of the umbilical cord mesenchymal stem cells.

The method is based on a DMEM low-sugar culture medium, and adds a certain amount of sodium danshensu and ginkgolic acid while adding nutritional ingredients such as human serum albumin, transferrin, epidermal growth factors, transforming growth factors, insulin and the like, so that the proliferation rate of the umbilical cord mesenchymal stem cells can be obviously improved, the phenotypic shape of the umbilical cord mesenchymal stem cells is kept from differentiating, the quality of the umbilical cord mesenchymal stem cells is improved, and the fact that a small amount of olive oil is added in the culture process can help the cells to adhere to the wall is found in the culture process, so that the proliferation effect of the umbilical cord mesenchymal stem cells is better, and the adipogenic and osteogenic induced differentiation potential is good.

The DMEM low-sugar medium provides basic substances such as saccharides, L-glutamine and inorganic salt and provides an energy source for umbilical cord mesenchymal stem cells, human serum albumin can replace calf serum and is a nutrient source for growth of the umbilical cord mesenchymal stem cells, proper osmotic pressure of the medium can be provided, osmotic pressure balance of cell growth is met, transferrin provides needed iron for cell internalization and cell metabolism and plays a role in iron transfer, epidermal cell growth factors and transforming growth factors play a role in survival maintenance and proliferation stimulation of the umbilical cord mesenchymal stem cells, and insulin can improve anabolic capacity of the umbilical cord mesenchymal stem cells and stimulate growth and reproduction of the cells.

In summary, the invention includes at least one of the following beneficial technical effects:

1. the umbilical cord mesenchymal stem cell serum-free culture medium provided by the invention has clear components, eliminates pathogen pollution from animal sources, has better reproducibility and controllability and serum culture medium effect, is lower in cost, and is suitable for industrial production;

2. the invention provides a serum-free culture medium for umbilical cord mesenchymal stem cells, which is added with sodium danshensu and ginkgolic acid, so that the proliferation rate of the umbilical cord mesenchymal stem cells can be obviously improved, the phenotypic shape of the umbilical cord mesenchymal stem cells is kept from differentiating, the quality of the umbilical cord mesenchymal stem cells is improved, and the multidirectional differentiation potential of the umbilical cord mesenchymal stem cells is maintained;

3. the invention provides a serum-free culture medium for umbilical cord mesenchymal stem cells, which can help cells adhere to the wall by adding a small amount of olive oil in the culture process, so that the proliferation effect of the umbilical cord mesenchymal stem cells is better, and the differentiation potential of adipogenesis and osteogenesis is better.

Detailed Description

The present invention will be described in further detail below.

In the present invention, the components and reagents involved are all conventional commercial products, or can be obtained by means of conventional techniques in the field. DMEM low-sugar medium was sourced from semer feishel technologies (china) ltd.