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CN111440764A - Serum-free culture medium of mesenchymal stem cells and clinical-grade large-scale culture method of mesenchymal stem cells - Google Patents

Serum-free culture medium of mesenchymal stem cells and clinical-grade large-scale culture method of mesenchymal stem cells Download PDF

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CN111440764A
CN111440764A CN202010110451.6A CN202010110451A CN111440764A CN 111440764 A CN111440764 A CN 111440764A CN 202010110451 A CN202010110451 A CN 202010110451A CN 111440764 A CN111440764 A CN 111440764A
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parts
serum
culture
stem cells
acid
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CN111440764B (en
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陈海佳
陈东煌
戚康艺
姜交华
岳坤
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Guangdong Guoke Cell Technology Co ltd
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Guangdong Guoke Cell Technology Co ltd
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Abstract

The invention relates to the technical field of stem cell culture, in particular to a serum-free culture medium of mesenchymal stem cells and a clinical-grade large-scale culture method of the mesenchymal stem cells. The mesenchymal stem cell serum-free culture medium provided by the invention comprises amino acids, vitamins, inorganic salts, a pH buffering agent, lipids, nucleic acids and other substances, can provide sufficient nutrients for the growth and proliferation of cells, and is more suitable for MSCs serum-free conditions and high-density culture, so that a good culture effect is obtained. The invention also provides a supplementary factor which comprises hormone, growth factor, adherence factor, antioxidant ingredient, lipid, soybean pancreatin inhibitor and soybean hydrolysate. The supplement factor is matched with the serum-free culture medium for use, so that the stem cells can be rapidly proliferated and keep good dryness under the condition of no animal-derived component addition.

Description

Serum-free culture medium of mesenchymal stem cells and clinical-grade large-scale culture method of mesenchymal stem cells
Technical Field
The invention relates to the technical field of stem cell culture, in particular to a serum-free culture medium of mesenchymal stem cells and a clinical-grade large-scale culture method of the mesenchymal stem cells.
Background
The stem cell industry is a typical strategic emerging industry, has high technical, high barrier and high risk, and has huge development potential, until 2019, 16 stem cell products are sold in the world, wherein Mesenchymal Stem Cells (MSCs) are the most abundant products due to various advantages, the most vigorous stem cell types are developed, such as Alofisel which is a new drug for treating complicated perianal fistula and is produced by TiGenix corporation approved by European Commission (EC), MSTECEME LL which is produced by MSCs from bone marrow source produced by Meisoblat corporation for treating MSGVHD which is produced by Mesorolast corporation for treating MSMCCs from bone marrow source, MSTEE LL which is produced by KF, FULLOBIANY approved first FCB-Pharmic corporation for treating acute myocardial autopsy, MSCs which is produced by Korean Mesorblast corporation for treating MSMCCs from MSE MCCs, MSE CA, and MSCs which are produced by MRC from MRC, and has extensive application prospect of autologous tissue differentiation and production of autologous Mesenchymal stem cells, and autologous tissue regeneration of bone marrow tissue regeneration and organ, and tissue regeneration of MSCs from Osbecker.
The cell culture medium is a key material for cell culture, provides a proper in vitro growth environment and sufficient nutrition for cells, and the performance of the cell culture medium greatly determines the cell proliferation performance and the cell function, quality and safety. The cell culture medium is developed through a serum-containing medium and a serum-free medium, and most of the mammalian cells used for producing the antibodies and the recombinant proteins are cultured by using the serum-free medium. However, in serum-free culture media for MSCs, the quality of products varies throughout the current domestic market, resulting in varying quality of MSCs products, and it is difficult to ensure the safety and effectiveness of MSCs products. The domestic MSCs serum-free culture medium market is basically monopolized by foreign brands with high price, and the MSCs serum-free culture medium market cannot reach the application standard of large-scale cell preparation in the aspects of cell amplification efficiency, primary culture and subculture, dry maintenance and other performance indexes, raw material control and the like. Therefore, the research and the production of high-quality domestic clinical-grade MSCs serum-free culture medium, and the large-scale preparation of MSCs have become the strategic basis of the MSCs industry development.
Most of the culture media used in the existing mesenchymal stem cell culture methods contain animal serum, such as the most common Fetal Bovine Serum (FBS). FBS is complex in composition, contains heterogeneous proteins, and is likely to carry viruses or to be infected with mycoplasma. On the other hand, the FBS has large batch difference and unstable source, and has large influence on the process of amplifying the MSCs in vitro on a large scale. At present, research shows that MSCs phagocytize proteins in a culture medium during culture, and the culture medium contains bovine serum albumin, so that anti-bovine albumin antibodies generated in a recipient can cause immune response, and the MSCs are ineffective or especially ineffective after repeated infusion treatment. Therefore, more and more researchers and enterprises are beginning to develop alternatives to FBSs. Currently, commercial serum-free, protein-free MSCs media, represented by Gibico, Stemcell, Takara, etc., are the predominant products on the market. Some MSCs can not realize high-density culture process in the existing serum-free culture medium; some of them support cell growth well, but may cause the cell to express the product with reduced ability or even loss; in addition, the formulation of commercial serum-free media is often commercially confidential, has complex components and high price, is not beneficial to the research of specific cell strain culture technology, and cannot be directly used for the research, development and optimization of cell culture processes. Also, as a partial serum replacement, a derivative of human serum, including human serum, platelet derivatives, umbilical serum, and the like, may be used. These products come from human sources, but the components are still unclear, the resources are few, the mass production is difficult to realize, and the large-scale culture of the MSCs in vitro cannot be guaranteed. Aiming at the defects that the performance of mainstream products in the market is not stable enough, the cell passage capacity and the amplification capacity are not enough, and high-density and large-scale amplification cannot be realized, a culture medium which is free of animal sources and has definite chemical components is continuously developed in the field, and the culture medium is particularly suitable for large-scale and clinical culture of stem cells.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a serum-free medium for mesenchymal stem cells, which is free of animal sources, has a definite chemical composition, and can realize the large-scale, clinical-grade and high-density culture of mesenchymal stem cells. The invention also provides a clinical-grade large-scale culture method of the mesenchymal stem cells.
The serum-free culture medium of the mesenchymal stem cells comprises:
amino acids, vitamins, inorganic salts, pH buffers, lipids, nucleic acids, and others;
the amino acids include: glycine, alanine, asparagine, aspartic acid, cystine, cysteine, leucine, isoleucine, glutamic acid, glutamine, arginine, histidine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine;
the vitamins comprise: vitamin C, biotin, choline chloride, pantalcin, folic acid, vitamin B2, vitamin B12, pyridoxine hydrochloride, thiamine hydrochloride, inositol, and nicotinamide;
the inorganic salts include: calcium chloride, copper sulfate pentahydrate, iron sulfate heptahydrate, magnesium sulfate, manganese sulfate, zinc sulfate heptahydrate, ferric citrate, sodium chloride, potassium chloride, lithium chloride, sodium butyrate, ammonium molybdate, nickel chloride hexahydrate, disodium hydrogen phosphate and sodium dihydrogen phosphate;
the pH buffers include: sodium bicarbonate and Hepes;
the lipids include: linoleic acid
The nucleic acids include: adenine and thymidine;
other species include: glucose, putrescine, lipoic acid and sodium pyruvate.
The serum-free culture medium provided by the invention is based on a classical DMEM/F12 basic culture medium, the components such as amino acid, inorganic salt, vitamins and the like and the addition concentration are adjusted through experimental optimization, transferrin is replaced by a composition of ferric citrate and sodium butyrate, insulin is replaced by zinc sulfate, the composition of other components is also quite reasonable, sufficient nutrients can be provided for the growth and proliferation of cells, and the serum-free culture medium is more suitable for the serum-free condition and high-density culture of MSCs, so that a good culture effect is obtained.
In some embodiments, the amino acid group comprises the following components in parts by mass:
15-20 parts of glycine, 1-10 parts of alanine, 1-10 parts of asparagine, 1-10 parts of aspartic acid, 10-50 parts of cystine, 10-20 parts of cysteine, 10-100 parts of leucine, 10-100 parts of isoleucine, 1-10 parts of glutamic acid, 100-600 parts of glutamine, 100-200 parts of arginine, 10-50 parts of histidine, 50-100 parts of lysine, 10-20 parts of methionine, 10-50 parts of phenylalanine, 10-50 parts of proline, 10-50 parts of serine, 10-100 parts of threonine, 2-20 parts of tryptophan, 10-100 parts of tyrosine and 10-100 parts of valine.
In some embodiments, the amino acid group comprises the following components in parts by mass:
18.75 parts of glycine, 4.45 parts of alanine, 7.5 parts of asparagine, 6.65 parts of aspartic acid, 31.29 parts of cystine, 17.56 parts of cysteine, 59.05 parts of leucine, 54.47 parts of isoleucine, 7.35 parts of glutamic acid, 292 parts of glutamine, 105 parts of arginine, 31.46 parts of histidine, 91.25 parts of lysine, 17.24 parts of methionine, 35.48 parts of phenylalanine, 30 parts of proline, 26.25 parts of serine, 53.45 parts of threonine, 10 parts of tryptophan, 55.79 parts of tyrosine and 52.85 parts of valine.
In some embodiments, the vitamins comprise the following components in parts by mass:
1-100 parts of vitamin C, 0.0005-0.005 part of biotin, 1-10 parts of choline chloride, 0.4-4.0 parts of calcitonin, 0.4-4.0 parts of folic acid, 20.4-4.0 parts of vitamin B, 120.4-4.0 parts of vitamin B, 0.4-4.0 parts of pyridoxine hydrochloride, 0.4-4.0 parts of thiamine hydrochloride, 2-20 parts of inositol and 0.4-4.0 parts of nicotinamide.
In some specific embodiments, the vitamins comprise the following components in parts by mass:
50 parts of vitamin C, 0.0035 parts of biotin, 8.98 parts of choline chloride, 2.24 parts of calcium pantothenate, 2.65 parts of folic acid, 20.219 parts of vitamin B, 120.68 parts of vitamin B, 2.25 parts of pyridoxine hydrochloride, 2.17 parts of thiamine hydrochloride, 12.60 parts of inositol and 2.02 parts of nicotinamide;
in some embodiments, the inorganic salt comprises the following components in parts by mass:
100 to 150 parts of calcium chloride, 0.0001 to 0.002 part of copper sulfate pentahydrate, 0.1 to 0.5 part of ferric sulfate heptahydrate, 10 to 100 parts of magnesium sulfate, 0.0003 to 0.003 part of manganese sulfate, 1.0 to 2.0 parts of zinc sulfate heptahydrate, 3000 to 5000 parts of ferric citrate, 5000 to 10000 parts of sodium chloride, 300 to 500 parts of potassium chloride, 100 to 500 parts of lithium chloride, 0.0003 to 0.003 part of sodium butyrate, 0.0003 to 0.003 part of ammonium molybdate, 0.0001 to 0.0005 part of nickel chloride hexahydrate, 10 to 100 parts of disodium hydrogen phosphate and 20 to 200 parts of sodium dihydrogen phosphate.
In some specific embodiments, the inorganic salt comprises the following components in parts by mass:
116.6 parts of calcium chloride, 0.0013 part of copper sulfate pentahydrate, 0.417 part of ferric sulfate heptahydrate, 97.67 parts of magnesium sulfate, 0.0025 part of manganese sulfate, 1.45 parts of zinc sulfate heptahydrate, 3920 parts of ferric citrate, 6995.5 parts of sodium chloride, 400 parts of potassium chloride, 423.9 parts of lithium chloride, 0.0025 part of sodium butyrate, 0.0025 part of ammonium molybdate, 0.00025 part of nickel chloride hexahydrate, 71.02 parts of disodium hydrogen phosphate and 140 parts of sodium dihydrogen phosphate.
In some embodiments, the pH buffer comprises the following components in parts by mass:
1000-2000 parts of sodium bicarbonate and 3000-5000 parts of Hepes;
in some embodiments, the pH buffer comprises the following components in parts by mass:
1200 parts of sodium bicarbonate and 3574.5 parts of Hepes;
the grease comprises the following components in parts by mass:
0.001-0.05 part of linoleic acid;
in some specific embodiments, the lipid comprises the following components in parts by mass:
0.042 part of linoleic acid;
in some embodiments, the nucleic acid comprises the following components in parts by mass:
2-20 parts of adenine and 0.1-0.5 part of thymidine;
in some embodiments, the nucleic acid comprises the following components in parts by mass:
adenine 10 parts and thymidine 0.365 parts;
in some embodiments, the other substances comprise the following components in parts by mass:
1000-4500 parts of glucose, 0.005-0.05 part of putrescine, 0.1-1.0 part of lipoic acid and 50-100 parts of sodium pyruvate.
In some specific embodiments, the mass parts of the components in other substances are as follows:
3151 parts of glucose, 0.015 part of putrescine, 0.105 part of lipoic acid and 55 parts of sodium pyruvate.
In some embodiments, the serum-free medium comprises water and:
Figure BDA0002388853690000051
Figure BDA0002388853690000061
Figure BDA0002388853690000071
in some embodiments, the serum-free medium comprises water and:
Figure BDA0002388853690000072
Figure BDA0002388853690000081
Figure BDA0002388853690000091
the serum-free culture medium is applied as a basic culture medium for stem cell culture.
The serum-free culture medium is an improved DMEM/F12 basal culture medium, and can replace a DMEM/F12 basal culture medium in animal cell culture. In the present invention, the serum-free medium is used as a basic medium for stem cell culture. The culture medium can make the cells adapt to a large-scale and high-density culture system and obtain good culture effect. The stem cell is a mesenchymal stem cell, in particular to a mesenchymal stem cell from fat, umbilical cord or bone marrow. In the embodiment of the invention, mesenchymal stem cells from umbilical cord are used as a test material to verify the function of the serum-free culture medium.
The present invention also provides a supplemental factor for stem cell culture, comprising:
hydrocortisone, human epidermal growth factor, human basic fibroblast growth factor, WNT-5a, poly-L-lysine, L-glutathione, β -mercaptoethanol, sodium selenite, linolenic acid, cholesterol, ethanolamine, Pluronic F-68, Tween80, soybean lecithin, soybean pancreatin inhibitor and soybean hydrolysate.
In the supplementary factor, the soybean hydrolysate is adopted to replace animal-derived components (such as bovine serum albumin and the like), and the soybean hydrolysate can also be replaced by wheat bran hydrolysate or cotton seed hydrolysate.
In some embodiments, the supplementary factor comprises the following components in parts by mass:
0.002-0.02 part of hydrocortisone, 0.01-0.05 part of human epidermal growth factor, 0.01-0.05 part of human basic fibroblast growth factor, 0.01-0.1 part of WNT-5a, 0.01-0.1 part of poly-L-lysine, 0.4-4 parts of L-glutathione, 0.1-1.0 part of β -mercaptoethanol, 0.005-0.01 part of sodium selenite, 0.01-0.05 part of linolenic acid, 0.4-4 parts of cholesterol, 0.04-0.4 part of ethanolamine, 0-68100-1000 parts of Pluronic F-68100, 804-40 parts of Tween, 5-50 parts of soybean lecithin, 20-200 parts of soybean pancreatin inhibitor and 2500-5000 parts of soybean hydrolysate.
In some specific embodiments, the supplementary factor comprises the following components in parts by mass:
0.018 portion of hydrocortisone, 0.02 portion of human epidermal growth factor, 0.02 portion of human basic fibroblast growth factor, 0.05 portion of WNT-5a, 0.05 portion of poly-L-lysine, 2 portions of L-glutathione, 0.39 portion of β -mercaptoethanol, 0.0067 portion of sodium selenite, 0.025 portion of linolenic acid, 2.2 portions of cholesterol, 0.2 portion of ethanol amine, 0.25 portion of Pluronic F-681700 portions, 8022 portions of Tween, 25 portions of soybean lecithin, 100 portions of soybean pancreatin inhibitor and 3000 portions of soybean hydrolysate.
In the supplementary factor, the hydrocortisone, the human epidermal growth factor, the human basic fibroblast growth factor and the WNT-5a are used as hormones or growth factor components, the poly-L-lysine is used as an adherence factor, the L-glutathione, the β -mercaptoethanol and the sodium selenite are used as antioxidants, lipid components and a soybean pancreatin inhibitor are also contained, and soybean hydrolysate is added to replace animal-derived components.
The invention also provides a culture medium for stem cell culture, which comprises the serum-free culture medium and the supplement factors.
The culture medium for stem cell culture comprises water and:
Figure BDA0002388853690000101
Figure BDA0002388853690000111
Figure BDA0002388853690000121
the culture medium is applied to clinical-grade and large-scale culture of stem cells.
The invention also provides a clinical-grade and large-scale culture method of the stem cells, which comprises the step of culturing the stem cells by using the culture medium.
The clinical-grade and large-scale stem cell culture method comprises the following steps: inoculating stem cells and microcarriers to the culture medium, culturing for 48-72 h with dissolved oxygen of 50% +/-0.5%, changing the solution by half, and culturing for 120-144 h with dissolved oxygen of 40% +/-0.5%.
In the culture method of the present invention:
the inoculation amount of the mesenchymal stem cells is 2.0 × 104cells/mL～4.0×104cells/m L, the inoculation amount of stem cells in the example of the present invention was 2.5 × 104The cell/m L, the inoculation amount of the microcarrier is 2-5 g/L, the using amount of the microcarrier is 3 g/L in the embodiment of the invention, the system of the culture system is 3-20L, and the system of the culture system is 3L in the embodiment of the invention.
The microcarrier is incubated before culture, and specifically comprises: soaking the microcarrier in PBS for 3h, replacing fresh PBS, sterilizing with high-pressure steam at 121 ℃ for 20min, removing PBS, adding complete culture medium, and incubating for 4h at 37 ℃.
The culture condition is 37 ℃ stirring culture, and the stirring procedure is as follows: stirring at 40-60 rpm for 5-10 min, stopping stirring for 10-20 min, and circulating for 3-6 h; after the intermittent stirring, continuously stirring at 80-120 rpm.
In the embodiment of the invention, the stirring procedure is as follows: and (3) intermittently stirring for 0-4 h: stirring at 60rpm for 5min, stopping for 15min, and circulating; continuously stirring for 4-120 h: 100 rpm.
The mesenchymal stem cell serum-free culture medium provided by the invention comprises amino acids, vitamins, inorganic salts, a pH buffering agent, lipids, nucleic acids and other substances, can provide sufficient nutrients for the growth and proliferation of cells, and is more suitable for MSCs serum-free conditions and high-density culture, so that a good culture effect is obtained. The invention also provides a supplementary factor which comprises hormone, growth factor, adherence factor, antioxidant ingredient, lipid, soybean pancreatin inhibitor and soybean hydrolysate. The supplement factor is matched with the serum-free culture medium for use, so that the stem cells can be rapidly proliferated and keep good dryness under the condition of no animal-derived component addition.
Drawings
FIG. 1 shows a morphology of groups of MSCs (100 ×);
FIG. 2 shows the expansion fold of UC-MSCs cells in each group;
FIG. 3 shows the cell viability of UC-MSCs in each group;
FIG. 4 is a graph showing the results of pH monitoring;
FIG. 5 shows the adipogenic differentiation effect of UC-MSCs in each group (400 ×);
FIG. 6 shows the osteogenic differentiation effect of UC-MSCs in each group (100 ×).
Detailed Description
The invention provides a serum-free culture medium of mesenchymal stem cells and a clinical-grade large-scale culture method of the mesenchymal stem cells, and a person skilled in the art can realize the culture by properly improving process parameters by referring to the content. It is expressly intended that all such alterations and modifications which are obvious to those skilled in the art are deemed to be incorporated herein by reference. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
The soybean or wheat bran or cottonseed hydrolysate refers to a soybean hydrolysate, a wheat bran hydrolysate or a cottonseed hydrolysate, and in the embodiment of the invention, the soybean hydrolysate is adopted.
The human epidermal growth factor of the invention is a recombinant human epidermal growth factor, the human basic fibroblast growth factor is a recombinant human basic fibroblast growth factor,
the soybean pancreatin inhibitor is extracted from soybean and purchased from sigma, and the product number is T6522.
The serum-free medium of the present invention may be in powder state or liquid state, and when it is in liquid state, its concentration may be equal to or several times that in the examples, for example, 1 ×, 2 ×, 10 ×.
The supplementary factor of the invention exists in the form of freeze-dried powder or liquid, and when the supplementary factor exists in the form of liquid, the concentration of the supplementary factor can be equal to or multiple times of that of the embodiment, such as 1 ×, 2 × and 10 ×.
The invention is further illustrated by the following examples:
example 1: preparation of MSCs basic culture medium
The MSCs basic culture medium is prepared by adjusting components such as amino acid, inorganic salt, vitamin and the like and addition concentration through experimental optimization on the basis of a classical DMEM/F12 basic culture medium, and is more suitable for MSCs serum-free condition and high-density culture. The concrete components are as follows:
TABLE 1 basal Medium formulation
Figure BDA0002388853690000151
Figure BDA0002388853690000161
Figure BDA0002388853690000171
Example 2 preparation of the complementary factor
According to the mixture ratio of table 2, the supplement factors are prepared:
TABLE 2 complement factor formulas
Name of ingredient Mass portion (portion)
Hydrocortisone 0.018
Recombinant human epidermal growth factor 0.02
Recombinant human basic fibroblast growth factor 0.02
WNT-5a 0.05
Poly-L-lysine 0.05
L glutathione 2.00
β -mercaptoethanol 0.39
Sodium selenite 0.0067
Linolenic acid 0.025
Cholesterol 2.20
Ethanolamine 0.20
Pluronic F-68 1700
Tween 80 22.00
Soybean lecithin 25.00
Soybean pancreatin inhibitor 100.00
Soybean hydrolysate 3000
EXAMPLE 3 complete Medium preparation
1L complete medium preparation:
the basal medium of example 1 of 1L was supplemented with the combination of factors of example 2 to give the concentrations of the components in the medium shown in Table 3:
TABLE 3 complete Medium
Figure BDA0002388853690000181
Figure BDA0002388853690000191
Figure BDA0002388853690000201
Figure BDA0002388853690000211
The components are fully stirred and uniformly mixed until being completely dissolved, and the pH value is adjusted to 7.0-7.2.
Example 4 preparation of complete Medium for Experimental group 2
1L complete medium preparation:
1L example 1 basal medium was supplemented with various combinations of supplements to achieve the concentrations of the components in the medium as shown in Table 4:
TABLE 4 complete Medium
Figure BDA0002388853690000212
Figure BDA0002388853690000221
Figure BDA0002388853690000231
The components are fully stirred and uniformly mixed until being completely dissolved, and the pH value is adjusted to 7.0-7.2.
Example 5 Large-Scale culture Process for MSCs
Cell type including MSCs derived from umbilical cord, selecting P3 generation cells, and performing experiment according to 1 × 104/cm2Inoculating into T175 culture flask at a density of 5% CO2The culture was carried out in an incubator at 37 ℃. After culturing for 3-4 days, the confluency of the cells reaches more than 90%, and enough cells are collected for later use;
microcarrier type: cytodex-1;
bioreactor type Applikon bioreactor (5L);
TABLE 5 Experimental group design
Figure BDA0002388853690000241
The culture process comprises the following steps:
1. cell inoculation amount of 2.5 × 104cells/m L/m L, 1L complete medium resuspension 7.5 × 107MSCs are ready for use;
2. culture volume 3L;
3. weighing 9g of microcarrier, pouring the microcarrier into a 1L bottling bottle, soaking the microcarrier in PBS for 3h, removing the PBS, adding new PBS, sterilizing the mixture for 20min by high-pressure steam at 121 ℃, removing the PBS, adding 1L complete culture solution, and incubating the mixture for 4h at 37 ℃ for later use;
4. cell inoculation: transferring the cell suspension and the microcarrier suspension into a sample loading bottle, and slightly shaking and uniformly mixing; slowly adding the mixed solution into the bioreactor by adopting a peristaltic pump;
5. intermittent stirring: stirring at 60rpm for 5min, stopping stirring for 15min, and circulating for 4 h;
6. and (3) continuous stirring: after the intermittent stirring, the stirring was continued at 100 rpm;
7. the pH value is maintained between 7.0 and 7.2 48 hours before the cell culture; the dissolved oxygen is maintained at 50% +/-0.5;
8. liquid changing time: culturing for 60 h; changing liquid by half amount;
9. after the liquid is changed, the pH value is maintained between 7.0 and 7.4; the dissolved oxygen is maintained between 40 percent and 50 percent;
10. culturing for 120 h.
Example 6 morphological Observation and Activity detection
The cells of the three groups of example 3 were sampled at 48h and 120h, and the morphological results are shown in FIG. 1 (100 ×).
After 120h of incubation, samples were taken, each group of UC-MSCs was digested with 0.25% trypsin solution and the number of cells, fold expansion (Table 6, FIG. 2) and cell viability (Table 6, FIG. 3) of each group were calculated. The result shows that the amplification fold of the experimental group 1 is obviously higher than that of other groups (p is less than 0.01); the cell viability rate was significantly higher than that of the other groups (p < 0.01).
TABLE 6 detection results of UC-MSCs cell number and activity in each group
Experimental groups Number of viable cells (× 10)4/mL) Amplification factor Cell viability (%)
Control group 1 25.23±0.94 10.09 91.01±0.68
Control group 2 18.23±0.62 7.29## 84.57±1.15#
Experimental group 1 33.03±1.14 13.21** 95.38±0.74**
Experimental group 2 20.79±0.69 8.32ΔΔ 90.52±1.14
Note: experimental group 1 showed very significant differences compared to the other groups, p < 0.01;
# shows that control 2 has significant difference compared with control 1, p is less than 0.05;
# indicates that the control group 2 has a very significant difference compared with the control group 1, and p is less than 0.01;
delta delta shows the significant difference between the experimental group 2 and the control group 1, and p is less than 0.01.
Example 7 pH monitoring results
During the cell culture, samples were taken every 12h to determine the pH, and the results are shown in FIG. 4. The experimental result shows that the pH value of the experimental group is more stable relative to the pH values of the two control groups when half of the culture medium is replaced within 60 hours, which proves that the culture medium can better maintain the stability of the pH value of the culture medium, is beneficial to high-density culture of cells, and maintains vigorous proliferation capacity and good activity.
Example 8 UC-MSCs surface marker detection
After 120H of culture, sampling the control group 1 and the experimental group respectively, digesting with 0.25% trypsin solution to collect UC-MSCs of each group, detecting the expression conditions of surface markers such as CD105, CD73, CD90, CD34, CD45, H L A-DR and the like by a flow cytometer, wherein the results are shown in Table 7.
Table 7 detection results of UC-MSCs surface marker of each group
Experimental groups CD105 CD90 CD73 CD34 CD45 HLA-DR
Control group 1 100.0％ 98.50％ 96.35％ 0.02％ 0.15％ 0.01％
Control group 2 96.35％ 91.75％ 92.35％ 0.12％ 0.45％ 0.02％
Experimental group 1 100.0％ 97.46％ 99.80％ 0.01％ 0.00％ 0.01％
Experimental group 2 99.98％ 97.23％ 96.25％ 0.08％ 0.12％ 0.01％
Example 9 UC-MSCs multidirectional differentiation potential assay
After 120h of culture, samples were taken from control 1 and experimental 1, and UC-MSCs were collected by digestion with 0.25% trypsin solution as 1 × 105The seeds were seeded at a density of/m L in 6-well plates, charged with 5% CO2Culturing in a culture box at 37 ℃. When the fusion degree of UC-MSCs in each group reaches more than 80%, a control hole and an induction hole are respectively arranged to induce the UC-MSCs to form bone and fat differentiation. And (3) carrying out oil red O staining on the cells in the adipogenic differentiation experiment group after 14 days, and carrying out alizarin red staining on the cells in the osteogenic differentiation experiment group after 21 days. The experimental result shows that the UC-MSCs cultured by the serum-free culture medium does not influence the osteogenic differentiation potential of the adipogenic bone marrow and maintains the dryness (figure 5 and figure 6).
The foregoing is only a preferred embodiment of the present invention, and it should be noted that it is obvious to those skilled in the art that various modifications and improvements can be made without departing from the principle of the present invention, and these modifications and improvements should also be considered as the protection scope of the present invention.

Claims (10)

1. A serum-free medium of mesenchymal stem cells, comprising:
hydrogen acids, vitamins, inorganic salts, pH buffers, lipids, nucleic acids and other substances;
the amino acids include: glycine, alanine, asparagine, aspartic acid, cystine, cysteine, leucine, isoleucine, glutamic acid, glutamine, arginine, histidine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine;
the vitamins comprise: vitamin C, biotin, choline chloride, pantalcin, folic acid, vitamin B2, vitamin B12, pyridoxine hydrochloride, thiamine hydrochloride, inositol, and nicotinamide;
the inorganic salts include: calcium chloride, copper sulfate pentahydrate, iron sulfate heptahydrate, magnesium sulfate, manganese sulfate, zinc sulfate heptahydrate, ferric citrate, sodium chloride, potassium chloride, lithium chloride, sodium butyrate, ammonium molybdate, nickel chloride hexahydrate, disodium hydrogen phosphate and sodium dihydrogen phosphate;
the pH buffers include: sodium bicarbonate and Hepes;
the lipids include: linoleic acid
The nucleic acids include: adenine and thymidine;
other species include: glucose, putrescine, lipoic acid and sodium pyruvate.
2. The serum-free medium according to claim 1,
the amino acid comprises the following components in parts by mass:
15-20 parts of glycine, 1-10 parts of alanine, 1-10 parts of asparagine, 1-10 parts of aspartic acid, 10-50 parts of cystine, 10-20 parts of cysteine, 10-100 parts of leucine, 10-100 parts of isoleucine, 1-10 parts of glutamic acid, 100-600 parts of glutamine, 100-200 parts of arginine, 10-50 parts of histidine, 50-100 parts of lysine, 10-20 parts of methionine, 10-50 parts of phenylalanine, 10-50 parts of proline, 10-50 parts of serine, 10-100 parts of threonine, 2-20 parts of tryptophan, 10-100 parts of tyrosine and 10-100 parts of valine;
the vitamins comprise the following components in parts by mass:
1-100 parts of vitamin C, 0.0005-0.005 part of biotin, 1-10 parts of choline chloride, 0.4-4.0 parts of calcium pantothenate, 0.4-4.0 parts of folic acid, 20.4-4.0 parts of vitamin B, 120.4-4.0 parts of vitamin B, 0.4-4.0 parts of pyridoxine hydrochloride, 0.4-4.0 parts of thiamine hydrochloride, 2-20 parts of inositol and 0.4-4.0 parts of nicotinamide;
the inorganic salt comprises the following components in parts by mass:
100-150 parts of calcium chloride, 0.0001-0.002 part of copper sulfate pentahydrate, 0.1-0.5 part of ferric sulfate heptahydrate, 10-100 parts of magnesium sulfate, 0.0003-0.003 part of manganese sulfate, 1.0-2.0 parts of zinc sulfate heptahydrate, 3000-5000 parts of ferric citrate, 5000-10000 parts of sodium chloride, 300-500 parts of potassium chloride, 100-500 parts of lithium chloride, 0.0003-0.003 part of sodium butyrate, 0.0003-0.003 part of ammonium molybdate, 0.0001-0.0005 part of nickel chloride hexahydrate, 10-100 parts of disodium hydrogen phosphate and 20-200 parts of sodium dihydrogen phosphate;
the pH buffering agent comprises the following components in parts by mass:
1000-2000 parts of sodium bicarbonate and 3000-5000 parts of Hepes;
the grease comprises the following components in parts by mass:
0.001-0.05 part of linoleic acid;
the nucleic acid comprises the following components in parts by mass:
2-20 parts of adenine and 0.1-0.5 part of thymidine;
the mass parts of the components in other substances are as follows:
1000-4500 parts of glucose, 0.005-0.05 part of putrescine, 0.1-1.0 part of lipoic acid and 50-100 parts of sodium pyruvate.
3. The serum-free medium according to claim 1 or 2, comprising water and:
Figure FDA0002388853680000021
Figure FDA0002388853680000031
Figure FDA0002388853680000041
4. use of the serum-free medium according to any one of claims 1 to 3 as a basal medium for stem cell culture.
5. A supplemental factor for stem cell culture, comprising:
hydrocortisone, human epidermal growth factor, human basic fibroblast growth factor, WNT-5a, poly-L-lysine, L-glutathione, β -mercaptoethanol, sodium selenite, linolenic acid, cholesterol, ethanolamine, Pluronic F-68, Tween80, soybean lecithin, soybean pancreatin inhibitor and soybean hydrolysate.
6. The supplement factor according to claim 5, wherein the mass parts of the components are as follows:
0.002-0.02 part of hydrocortisone, 0.01-0.05 part of human epidermal growth factor, 0.01-0.05 part of human basic fibroblast growth factor, 0.01-0.1 part of WNT-5a, 0.01-0.1 part of poly-L-lysine, 0.4-4 parts of L-glutathione, 0.1-1.0 part of β -mercaptoethanol, 0.005-0.01 part of sodium selenite, 0.01-0.05 part of linolenic acid, 0.4-4 parts of cholesterol, 0.04-0.4 part of ethanolamine, 0-68100-1000 parts of Pluronic F-68100, 804-40 parts of Tween, 5-50 parts of soybean lecithin, 20-200 parts of soybean pancreatin inhibitor and 2500-5000 parts of soybean hydrolysate.
7. A culture medium for stem cell culture, comprising the serum-free medium according to any one of claims 1 to 3 and the supplement factor according to any one of claims 5 to 6.
8. The culture medium according to claim 7, comprising water and:
Figure FDA0002388853680000042
Figure FDA0002388853680000051
Figure FDA0002388853680000061
Figure FDA0002388853680000071
9. use of the culture medium according to claim 7 or 8 for clinical-grade, large-scale culture of stem cells.
10. A method for clinical-scale culture of stem cells, comprising culturing the stem cells in the medium of claim 7 or 8.
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