CN117363566A - Human umbilical cord mesenchymal stem cell culture medium - Google Patents
Human umbilical cord mesenchymal stem cell culture medium Download PDFInfo
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- CN117363566A CN117363566A CN202311307836.1A CN202311307836A CN117363566A CN 117363566 A CN117363566 A CN 117363566A CN 202311307836 A CN202311307836 A CN 202311307836A CN 117363566 A CN117363566 A CN 117363566A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/70—Undefined extracts
- C12N2500/76—Undefined extracts from plants
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2500/00—Specific components of cell culture medium
- C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/999—Small molecules not provided for elsewhere
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Abstract
The invention belongs to the technical field of stem cell culture medium preparation, and particularly relates to a human umbilical cord mesenchymal stem cell culture medium. The human umbilical cord mesenchymal stem cell culture medium comprises the following components: IMDM basal medium, F12 basal medium, ruxolitinib, seaweed extract, small molecule compound Thiazovivin and SD-208, normal human AB serum. The invention creatively introduces the ruxolitinib, thiazovivin, SD-208 and other small molecular compounds, and can prevent the stem cells from differentiating, aging and apoptosis in the in-vitro culture process, thereby maintaining the stem cells' dryness; and creatively introduces seaweed extract to promote cell proliferation. The culture medium prepared by the invention can be used for culturing human umbilical mesenchymal stem cells, has the advantages of high cell proliferation speed, large harvesting quantity, stable cell phenotype, capability of maintaining multipotential differentiation capacity and high expression of mesenchymal stem cell surface antigen after multiple passages, no in-vitro tumorigenicity and good safety.
Description
Technical Field
The invention belongs to the technical field of stem cell culture medium preparation, and particularly relates to a human umbilical cord mesenchymal stem cell culture medium.
Background
Mesenchymal stem cell definition: mesenchymal stem cells [ mesenchymal stem cells, MSC ] are important members of the stem cell family, derived from early stages of development mesoderm and ectoderm. MSCs were originally found in bone marrow and have been increasingly attracting attention due to their multipotent differentiation potential, hematopoietic support, and promotion of stem cell engraftment, immune regulation, and self-replication. For example, the mesenchymal stem cells can be differentiated into various tissue cells such as fat, bone, cartilage, muscle, tendon, ligament, nerve, liver, cardiac muscle, endothelial and the like under specific induction conditions in vivo or in vitro, and still have multidirectional differentiation potential after continuous subculture and cryopreservation, and can be used as ideal seed cells for repairing tissue organ injury caused by aging and pathological changes.
Currently, we can isolate and prepare mesenchymal stem cells from bone marrow, fat, synovial membrane, bone, muscle, lung, liver, pancreas, etc., amniotic fluid, umbilical cord blood, and the most used are bone marrow-derived mesenchymal stem cells. However, bone marrow-derived mesenchymal stem cells have the following problems: with aging, the number of stem cells is obviously reduced, and the proliferation and differentiation capability is greatly reduced; the preparation process is not easy to control quality; transplantation of the variant may cause an immune response; the patient is injured when the materials are obtained, and the patient cannot collect the bone marrow when the patient has bone marrow diseases, so that even a healthy donor cannot extract too much bone marrow. This limits the clinical application of bone marrow mesenchymal stem cells, making searching for alternative mesenchymal stem cell sources other than bone marrow an important issue.
Mesenchymal stem cells have been successfully isolated from human umbilical cord tissue, and the tissue-derived mesenchymal stem cells maintain the biological properties of the mesenchymal stem cells. The research to date shows that the mesenchymal stem cells of human umbilical cord source can not only become ideal substitutes of bone marrow mesenchymal stem cells, but also have greater application potential. The human umbilical cord mesenchymal stem cells express the special molecular markers of various embryonic stem cells, and have the characteristics of large differentiation potential, strong proliferation capability, low immunogenicity, convenient material acquisition, no limitation of ethical issues, easy industrial preparation and the like, so the human umbilical cord mesenchymal stem cells are likely to become the pluripotent stem cells with the most clinical application prospect.
The serum-free culture medium of the human umbilical cord mesenchymal stem cells in the current market has potential nutrition deficiency because of the fact that nutrition is not as comprehensive as serum, and when the serum-free culture medium is used for culturing the human umbilical cord mesenchymal stem cells, the problems that proliferation and adhesion capacity are reduced, cell aging or apoptosis are caused along with the extension of passage and culture time, spontaneous differentiation is easy to occur, and multipotential differentiation capacity is weakened or multipotential differentiation is lost are solved. MSC is very sensitive to factors such as chemical components of a culture medium, mechanical and hydrodynamic factors of a culture environment and the like, and long-term culture of MSC in vitro is easy to cause DNA damage accumulation, slow proliferation, weak differentiation capability, cell phenotype change and function decline of MSC. In the in vitro amplification process, the intervention measures can reduce the accumulation of DNA damage in MSC, and are of great clinical significance for obtaining MSC with good functions.
Disclosure of Invention
In view of the above, the invention aims to overcome the defects of the existing serum-free culture medium technology and provide a preparation method of a culture medium required by preparing human umbilical cord mesenchymal stem cells, which comprises the following steps: the preparation of the serum-free culture medium of the stem cells is suitable for a stable large-scale mesenchymal stem cell culture system; the invention can establish a stable stem cell preparation flow; can maintain and promote the proliferation and multipotent differentiation capacity of mesenchymal stem cells, and has good safety. The seaweed extract is rich in mineral nutrient elements such as calcium, iron, magnesium, zinc and the like, seaweed polysaccharide such as brown seaweed starch, fucoidin, xylan, glucan and the like, and natural substances such as sugar alcohol, amino acid, vitamins, cytochromes, betaine, phenolic compounds, auxin, cytokinin and the like, and is a good additive for culturing MSCs. Ruxolitinib is a tyrosine kinase inhibitor, namely a small molecule inhibitor of protein kinases JAK1 and JAK2, can inhibit mesenchymal stem cell aging, and keeps stem cell activity and dryness. The small molecular compounds SD-208 and Thiazovivin can promote cell proliferation, alleviate apoptosis and inhibit spontaneous differentiation of MSCs in vitro culture. The AB serum of normal people is rich in various growth factors, and the concentration and the proportion of the various growth factors meet the physiological requirement, so that the proliferation and differentiation of MSCs and the formation of local new blood vessels can be obviously promoted.
The invention provides a stable, high-yield and safe human umbilical cord mesenchymal stem cell culture medium, which can be used for rapidly preparing human umbilical cord mesenchymal stem cells, has the advantages of high cell proliferation speed, large cell harvest, stable phenotype and capability of maintaining multipotent differentiation capacity after multiple passages, and does not generate in-vitro tumorigenicity.
In order to achieve the above object, the technical scheme provided by the invention is as follows: a human umbilical cord mesenchymal stem cell culture medium, comprising the following components: IMDM basal medium (Dulbecco's Modified Eagle Medi. Mu.M), F12 basal medium (Ham's F nutrient medium. Mu.M), ruxolitinib, seaweed extract, small molecule compound ROCK inhibitor Thiazovivin, TGF-beta pathway inhibitor SD-208, normal human AB serum.
Preferably, the volume ratio of IMDM basal medium to F12 basal medium in the culture medium is 1:1-3, preferably 1:1.
Preferably, the concentration of ruxolitinib in the mesenchymal stem cell medium is 1-100nM.
Preferably, the concentration of the seaweed extract in the mesenchymal stem cell medium is 1-100g/L.
Preferably, the concentration of Thiazovivin in the mesenchymal stem cell medium is 1-100nM.
Preferably, the concentration of SD-208 in the mesenchymal stem cell medium is 1-100nM.
Preferably, the normal human AB serum is 5-20% of the total volume of the culture medium.
The invention also provides a preparation method of the culture medium, which comprises the following steps:
respectively dissolving and preparing concentrated solutions of ruxolitinib, thiazovivin and SD-208 by using DMSO (dimethyl sulfoxide) to ensure that the concentration of each concentrated solution is 1-100 mu M; mixing IMDM basal medium and F12 basal medium in proportion, sequentially adding ruxolitinib, seaweed extract, small molecular compound Thiazovivin and SD-208 concentrated solution, and finally adding normal human AB serum.
The invention also aims to provide the application of the human umbilical cord mesenchymal stem cell culture medium, wherein the application is to apply the culture medium to culture human umbilical cord mesenchymal stem cells.
The human umbilical cord mesenchymal stem cell culture medium prepared by the invention is used for culturing human umbilical cord mesenchymal stem cells, and the method comprises a conventional tissue adherence method for primary culture and an enzyme digestion method for subculture, and comprises the following steps:
(1) Washing, sterilizing, separating and shearing human umbilical cord, and adding into the culture medium for culturing;
(2) Culturing until the human umbilical cord mesenchymal stem cells grow to 80-90% and are fused, and passaging the cells.
(3) Subculturing and collecting the P2, P8 and P16 generation cells.
Preferably, in (1), the washing is carried out by sterilizing with iodophor and then washing with normal saline, the soaking is carried out by soaking with iodophor for 0.5-3min, and the shearing is carried out by shearing human umbilical cords into tissue blocks with the length of 1mm multiplied by 1 mm.
Preferably, in (1) to (3), the culture is performed in an incubator containing 5% CO2 by volume at 37 ℃.
Preferably, in (3), the specific process of subculturing is as follows: digesting the cells into single cells by using 0.25% trypsin solution, inoculating the single cells into a culture flask according to the density of 2000-8000 cells/square centimeter, and adding 10-30ml of prepared culture medium according to the size of the culture flask; the flask was horizontally placed in an incubator for cultivation.
Compared with the prior art, the invention has the technical advantages that:
(1) The invention creatively introduces the ruxolitinib, thiazovivin, SD-208 and other small molecular compounds, and can prevent the stem cells from differentiating, aging and apoptosis in the in-vitro culture process, thereby maintaining the stem cells' dryness; and creatively introduces seaweed extract to promote cell proliferation. The culture medium of the human umbilical cord mesenchymal stem cells prepared by the invention can stably prepare the human umbilical cord mesenchymal stem cells with high yield, has high cell proliferation speed and cell yield, is stable in phenotype, and can still maintain the multi-directional differentiation capability after multiple passages. And after multiple passages, the cells still do not generate in-vitro tumorigenicity, thus showing good safety.
(2) The serum-free culture medium provided by the invention is used by compounding low-concentration normal human AB serum with ruxolitinib, seaweed extract, thiazovivin and SD-208, so that continuous in-vitro passage culture of mesenchymal stem cells can be satisfied; the test result shows that the human umbilical cord mesenchymal stem cells cultured by adopting the serum-free culture medium provided by the invention are fast in proliferation, good in state and capable of being induced to differentiate into osteoblasts, adipocytes and chondrocytes, the expression and expression of cell surface antigens accord with the mesenchymal stem cell standard, and the culture effect is not obviously different from that of a culture medium containing 10% Fetal Bovine Serum (FBS).
(3) In the culture medium provided by the invention, ruxolitinib, seaweed extract, thiazovivin and SD-208 have better synergistic effect, and the culture medium is promoted to continuously culture in vitro umbilical cord mesenchymal stem cells.
Drawings
FIG. 1 is a graph showing comparison of cell harvest amounts of each of examples and comparative examples P2, P8 and P16.
FIG. 2 is a graph comparing cell proliferation curves of examples and comparative examples.
FIG. 3 is a graph showing comparison of the stained areas of alizing differentiation alizarin red of cells of each example and comparative example.
FIG. 4 is a graph showing comparison of areas of adipogenic induced differentiation of cells stained with oil red O.
FIG. 5 is a graph of contrast in areas stained with Alxin blue for chondrogenic induced differentiation of cells of each of the examples and comparative examples.
Detailed Description
The present invention is described in conjunction with the following specific embodiments, which are given as examples of the present invention and are not to be construed as limiting the invention in any way, and any person skilled in the art can, with reference to the present invention, suitably modify the process parameters. Any simple modification of the following embodiments according to the technical substance of the present invention falls within the scope of the present invention, without departing from the scope of the present invention.
The reagents applied to the human umbilical cord mesenchymal stem cell culture medium provided by the invention can be obtained from commercial approaches; and the different sources have no significant effect on the product performance.
Example 1
A human umbilical cord mesenchymal stem cell culture medium, comprising the following components: IMDM basal medium, F12 basal medium, ruxolitinib, seaweed extract, thiazovivin and SD-208 normal human AB serum.
The volume ratio of the IMDM basal medium to the F12 basal medium in the culture medium is 1:1.
The concentration of ruxolitinib in the mesenchymal stem cell medium was 2nM.
The mass concentration of the seaweed extract in the mesenchymal stem cell culture medium is 5g/L.
The concentration of Thiazovivin in the mesenchymal stem cell medium was 10nM.
The concentration of SD-208 in the mesenchymal stem cell medium was 10nM.
The normal human AB serum is 10% of the total volume of the IMDM basal medium and the F12 basal medium.
The preparation method of the culture medium comprises the following steps: respectively dissolving and preparing concentrated solutions of ruxolitinib, thiazovivin and SD-208 by using DMSO (dimethyl sulfoxide) to ensure that the concentrations of the concentrated solutions are 100 mu M; mixing IMDM basal medium and F12 basal medium in proportion, sequentially adding ruxolitinib, seaweed extract, thiazovivin and SD-208, and finally adding normal human AB serum.
Example 2
A human umbilical cord mesenchymal stem cell culture medium, comprising the following components: IMDM basal medium, F12 basal medium, ruxolitinib, seaweed extract, thiazovivin and SD-208 normal human AB serum.
The volume ratio of the IMDM basal medium to the F12 basal medium in the culture medium is 1:2.
The concentration of ruxolitinib in the mesenchymal stem cell medium is 5nM.
The mass concentration of the seaweed extract in the mesenchymal stem cell culture medium is 2g/L.
The concentration of Thiazovivin in the mesenchymal stem cell medium was 5nM.
The concentration of SD-208 in the mesenchymal stem cell medium was 5nM.
The normal human AB serum is 5% of the total volume of the IMDM basal medium and the F12 basal medium.
The preparation method of the culture medium comprises the following steps: respectively dissolving and preparing concentrated solutions of ruxolitinib, thiazovivin and SD-208 by using DMSO (dimethyl sulfoxide) to ensure that the concentrations of the concentrated solutions are 100 mu M; mixing IMDM basal medium and F12 basal medium in proportion, sequentially adding ruxolitinib, seaweed extract, thiazovivin and SD-208, and finally adding normal human AB serum.
Example 3
A human umbilical cord mesenchymal stem cell culture medium, comprising the following components: IMDM basal medium, F12 basal medium, ruxolitinib, seaweed extract, thiazovivin and SD-208 normal human AB serum.
The volume ratio of the IMDM basal medium to the F12 basal medium in the culture medium is 1:3.
The concentration of ruxolitinib in the mesenchymal stem cell medium is 10nM.
The mass concentration of the seaweed extract in the mesenchymal stem cell culture medium is 10g/L.
The concentration of Thiazovivin in the mesenchymal stem cell medium was 20nM.
The concentration of SD-208 in the mesenchymal stem cell medium was 20nM.
The normal human AB serum is 20% of the total volume of the IMDM basal medium and the F12 basal medium.
The preparation method of the culture medium comprises the following steps: respectively dissolving and preparing concentrated solutions of ruxolitinib, thiazovivin and SD-208 by using DMSO (dimethyl sulfoxide) to ensure that the concentrations of the concentrated solutions are 100 mu M; mixing IMDM basal medium and F12 basal medium in proportion, sequentially adding ruxolitinib, seaweed extract, thiazovivin and SD-208, and finally adding normal human AB serum.
Comparative example 1
The difference from example 1 is that normal human AB serum is replaced with Fetal Bovine Serum (FBS).
A human umbilical cord mesenchymal stem cell culture medium, comprising the following components: IMDM basal medium, F12 basal medium, ruxolitinib, seaweed extract, thiazovivin and SD-208, FBS.
The volume ratio of the IMDM basal medium to the F12 basal medium in the culture medium is 1:1.
The concentration of ruxolitinib in the mesenchymal stem cell medium was 2nM.
The mass concentration of the seaweed extract in the mesenchymal stem cell culture medium is 5g/L.
The concentration of Thiazovivin in the mesenchymal stem cell medium was 10nM.
The concentration of SD-208 in the mesenchymal stem cell medium was 10nM.
The FBS was 10% of the total volume of IMDM basal medium and F12 basal medium.
The preparation method of the culture medium comprises the following steps: respectively dissolving and preparing concentrated solutions of ruxolitinib, thiazovivin and SD-208 by using DMSO (dimethyl sulfoxide) to ensure that the concentrations of the concentrated solutions are 100 mu M; mixing IMDM basal medium and F12 basal medium in proportion, sequentially adding ruxolitinib, seaweed extract, thiazovivin and SD-208 concentrated solution, and finally adding FBS.
Comparative example 2
The difference from example 1 is that vitamin C is used instead of ruxolitinib.
A human umbilical cord mesenchymal stem cell culture medium, comprising the following components: IMDM basal medium, F12 basal medium, vitamin C, seaweed extract, thiazovivin and SD-208 normal human AB serum.
The volume ratio of the IMDM basal medium to the F12 basal medium in the culture medium is 1:1.
The concentration of vitamin C in the mesenchymal stem cell medium is 200nM.
The mass concentration of the seaweed extract in the mesenchymal stem cell culture medium is 5g/L.
The concentration of Thiazovivin in the mesenchymal stem cell medium was 10nM.
The concentration of SD-208 in the mesenchymal stem cell medium was 10nM.
The normal human AB serum is 10% of the total volume of the IMDM basal medium and the F12 basal medium.
The preparation method of the culture medium comprises the following steps: firstly, respectively dissolving and preparing concentrated solutions of Thiazovivin and SD-208 by using DMSO (dimethyl sulfoxide) to ensure that the concentration of each concentrated solution is 100 mu M; mixing IMDM basal medium and F12 basal medium at a certain proportion, sequentially adding vitamin C, seaweed extract, thiazovivin and SD-208 concentrated solution, and finally adding normal human AB serum.
Comparative example 3
The difference from example 1 is that algal polysaccharide is used instead of algal extract.
A human umbilical cord mesenchymal stem cell culture medium, comprising the following components: IMDM basal medium, F12 basal medium, ruxolitinib, algal polysaccharide, thiazovivin and SD-208, normal human AB serum.
The volume ratio of the IMDM basal medium to the F12 basal medium in the culture medium is 1:1.
The concentration of ruxolitinib in the mesenchymal stem cell medium was 2nM.
The mass concentration of the seaweed polysaccharide in the mesenchymal stem cell culture medium is 5g/L.
The concentration of Thiazovivin in the mesenchymal stem cell medium was 10nM.
The concentration of SD-208 in the mesenchymal stem cell medium was 10nM.
The normal human AB serum is 10% of the total volume of the IMDM basal medium and the F12 basal medium.
The preparation method of the culture medium comprises the following steps: respectively dissolving and preparing concentrated solutions of ruxolitinib, thiazovivin and SD-208 by using DMSO (dimethyl sulfoxide) to ensure that the concentrations of the concentrated solutions are 100 mu M; mixing IMDM basal medium and F12 basal medium in proportion, sequentially adding ruxolitinib, algal polysaccharide, thiazovivin and SD-208, and finally adding normal human AB serum.
Comparative example 4
The difference from example 1 is the removal of Thiazovivin.
A human umbilical cord mesenchymal stem cell culture medium, comprising the following components: IMDM basal medium, F12 basal medium, ruxolitinib, seaweed extract, SD-208, normal human AB serum.
The volume ratio of the IMDM basal medium to the F12 basal medium in the culture medium is 1:1.
The concentration of ruxolitinib in the mesenchymal stem cell medium was 2nM.
The mass concentration of the seaweed extract in the mesenchymal stem cell culture medium is 5g/L.
The concentration of SD-208 in the mesenchymal stem cell medium was 10nM.
The normal human AB serum is 10% of the total volume of the IMDM basal medium and the F12 basal medium.
The preparation method of the culture medium comprises the following steps: dissolving and preparing concentrated solutions of ruxolitinib and SD-208 by using DMSO to ensure that the concentration of the concentrated solutions is 100 mu M; mixing IMDM basal medium and F12 basal medium in proportion, sequentially adding ruxolitinib, algal polysaccharide and SD-208, and finally adding normal human AB serum.
Comparative example 5
The difference from example 1 is that the removal SD-208 is used.
A human umbilical cord mesenchymal stem cell culture medium, comprising the following components: IMDM basal medium, F12 basal medium, ruxolitinib, seaweed extract, thiazovivin, normal human AB serum.
The volume ratio of the IMDM basal medium to the F12 basal medium in the culture medium is 1:1.
The concentration of ruxolitinib in the mesenchymal stem cell medium was 2nM.
The mass concentration of the seaweed extract in the mesenchymal stem cell culture medium is 5g/L.
The concentration of Thiazovivin in the mesenchymal stem cell medium was 10nM.
The normal human AB serum is 10% of the total volume of the IMDM basal medium and the F12 basal medium.
The preparation method of the culture medium comprises the following steps: dissolving and preparing concentrated solutions of ruxolitinib and Thiazovivin by using DMSO to ensure that the concentration of the concentrated solutions is 100 mu M; mixing IMDM basal medium and F12 basal medium in proportion, sequentially adding ruxolitinib, algal polysaccharide and Thiazovivin, and finally adding normal human AB serum.
1. Isolated culture of human umbilical cord mesenchymal stem cells
After wiping the human umbilical cord with iodophor, placing the umbilical cord in a10 cm sterile culture dish, adding 10ml of sodium chloride injection for washing, repeating for 4-8 times, and removing blood stains. Then adding iodophor to soak for 0.5min, and then cleaning with sodium chloride injection. The umbilical cord is sheared into sections of about 2-3 cm by the sterile surgical scissors, and then 10ml of sodium chloride injection is added for washing again. Removing umbilical cord blood vessel, separating Wharton's jelly, washing the jelly, and shearing the tissue into 1-4 mm3 tissue homogenate blocks. After a proper amount of human umbilical cord mesenchymal stem cell culture medium is added, homogenizing is carried out uniformly by blowing, 1ml of tissue homogenate is inoculated in a T75 culture flask, 10ml of human umbilical cord mesenchymal stem cell culture medium is added, and the culture flask is placed in a carbon dioxide constant temperature and humidity incubator. Culture conditions: 37 plus or minus 0.5 ℃ and the volume fraction of the carbon dioxide is 5 plus or minus 0.2 percent. Changing liquid once every 3-4 days, when the area percentage of the cell clone groups reaches 70% -80%, digesting and harvesting cells by using 0.25% trypsin solution, and using 5000-10000 cells/cm 2 Is a density of cells.
2. Comparison of human umbilical cord mesenchymal Stem cell harvesting
The generation of P2, P8 and P16 cells is respectively carried out at 5000/cm 2 Is planted in a T75 culture flask, is cultured by the culture mediums prepared in each example and comparative example, and after 3 days of culture, the harvested cells are digested by 0.25% trypsin solution, counted by a Countstar cell counter, and the cell harvest count is performed; the cell harvest comparison is shown in FIG. 1.
The results show that: cells cultured in the culture protocol of examples 1-3 were passaged to P16, were still strong in cell proliferation capacity, were larger in cell harvest, and were similar to comparative example 1 containing 10% FBS, and were superior to other comparative examples.
3. Human umbilical cord mesenchymal stem cell proliferation curve
Proliferation curve comparison was performed on human umbilical cord mesenchymal stem cells cultured in each example and comparative example.
The detection method comprises the following steps: taking P2, P8 and P16 generation human umbilical cord mesenchymal stem cells respectively, inoculating the cells into a 96-well plate at the density of 1000 cells per well, adding 100 mu L of human umbilical cord mesenchymal stem cell serum-free culture medium per well, replacing 1 culture solution per group of 3 compound wells, adding 10 mu L of CCK8 reagent into a culture system at 24, 48, 72, 96 and 120 hours respectively, incubating at 37 ℃ in a dark place for 2.5 hours, reading an absorbance value of 450nm by using an enzyme-labeled instrument, and drawing a cell proliferation curve according to the absorbance value, wherein the result is shown in figure 2.
The results show that the cell proliferation curves of each group are in an S shape and comprise a latent period, a logarithmic growth period and a plateau period, the cell proliferation curves of examples 1-3 are similar to those of comparative example 1 containing 10% of fetal bovine serum, the proliferation speeds are similar, the proliferation speed is faster than that of other comparative example groups, and the cells enter the logarithmic growth period from the next day, the proliferation is accelerated, and the cells enter the plateau period on the 5 th day. While comparative examples 2-5 had slower proliferation of cells, and also entered plateau at day 5, indicating less harvest.
4. Human umbilical cord mesenchymal stem cell surface marker detection
Collecting P2, P8 and P16 generation human umbilical cord mesenchymal stem cells, washing with sodium chloride injection, centrifuging for 2 times, and mixing to obtain powder with density of 2×10 6 Per ml, adding fluorescent labeled antibodies (CD 73, CD90, CD105, CD34, CD45, HLA-DR), incubating at room temperature for 30min, washing with sodium chloride injection again, centrifuging for 2 times, incubating with sodium chloride injection instead of antibody in blank control, and detecting with flow cytometry. The results are shown in Table 1.
TABLE 1 identification of human umbilical cord mesenchymal Stem cell surface antigen
| Group of | CD73(％) | CD90(％) | CD105(％) | CD34(％) | CD45(％) | HLA-DR |
| Example 1 | 99.89 | 99.79 | 98.38 | 0.12 | 0.78 | 0.63 |
| Example 2 | 99.78 | 99.68 | 98.21 | 0.29 | 0.98 | 0.79 |
| Example 3 | 99.79 | 99.67 | 98.10 | 0.26 | 0.95 | 0.94 |
| Comparative example 1 | 99.80 | 99.60 | 98.12 | 0.18 | 0.89 | 0.95 |
| Comparative example 2 | 97.78 | 97.40 | 96.11 | 1.90 | 1.89 | 1.70 |
| Comparative example 3 | 96.86 | 96.12 | 95.19 | 1.89 | 1.75 | 1.82 |
| Comparative example 4 | 95.43 | 95.28 | 94.27 | 1.67 | 1.97 | 1.93 |
| Comparative example 5 | 94.59 | 94.32 | 93.41 | 1.98 | 1.86 | 1.75 |
According to the flow detection results (table 1), each group of cell surface antigens CD73, CD90 and CD105 are positively expressed, and CD34, CD45 and HLA-DR are negatively expressed, wherein the positive rates of CD73, CD90 and CD105 obtained by culturing in examples 1-3 are all above 95%, the positive rates of CD45 and CD34 are lower than 2%, the cell surface antigens conform to the phenotypic characteristics of human umbilical cord mesenchymal stem cells, and the cell surface antigens have no obvious difference from the expression of the surface antigens of the human umbilical cord mesenchymal stem cells cultured by 10% fbs, which are superior to comparative examples 2, 3, 4 and 5. The detection result shows that different compositions of the culture medium have a certain influence on the phenotypic characteristics of cells, and the serum-free culture medium provided by the invention is suitable for culturing human umbilical cord mesenchymal stem cells.
5. Identification of human umbilical cord mesenchymal stem cell multipotential
The media prepared in each of examples and comparative examples were evaluated for the multipotent differentiation capacity (osteogenic, adipogenic and chondrogenic differentiation-inducing capacity) of mesenchymal stem cells obtained by isolated culture of human umbilical cord mesenchymal stem cells according to the following method.
(1) Osteogenesis induction: taking P2, P8 and P16 generation human umbilical cord mesenchymal stem cells respectively according to 1×10 4 Planting in six-hole plate at density of/cm 2, adding corresponding culture medium, culturing, discarding old culture medium on day 4, adding osteogenesis inducing medium (Gibco StemPro) TM Osteogenesis differentiation kit a 1007201), the liquid was changed every 3 days, and the negative control group replaced the induction differentiation medium with human umbilical cord mesenchymal stem cell medium. After 3 weeks of culture, alizarin red staining observation is carried out after fixation by using 4% paraformaldehyde solution, and the staining area is counted after observation under a microscope.
(2) Lipid formation induction: taking P2, P8 and P16 generation human umbilical cord mesenchymal stem cells respectively according to 5×10 3 Planting in six-hole plate at density of/cm 2, adding corresponding culture medium, culturing, discarding old culture medium on day 4, adding lipid formation induction medium (Gibco Stempro TM Adipogenic differentiation kit a 1007001), the liquid was changed every 3 days, and the negative control group replaced the induced differentiation medium with human umbilical cord mesenchymal stem cell medium. After 2 weeks of culture, the sample was fixed with a 4% paraformaldehyde solution, and then subjected to oil red O staining observation, microscopic observation, and statistics of the staining area.
(3) Cartilage induction: collecting P2, P8 and P16 generation human umbilical cord mesenchymal stem cells, concentrating the cells to a density of 1.6X10 7 Cell suspension of/cm 2, 5uL was takenPlanting in six-hole plate, culturing in carbon dioxide incubator at 37deg.C for 2 hr, adding into cartilage inducing medium (Gibco StemPro) TM Chondrogenic differentiation kit a 1007101), the liquid was changed every 3 days, and the negative control group replaced the induction differentiation medium with human umbilical cord mesenchymal stem cell medium. After 2 weeks of culture, the cells were fixed with 4% paraformaldehyde solution, paraffin-embedded, sectioned, stained with alcian blue, observed under a microscope, and the stained areas were counted.
The detection results show that the human umbilical cord mesenchymal stem cells prepared in examples 1-3 have no obvious difference from the human umbilical cord mesenchymal stem cells prepared in comparative example 1 in terms of osteogenesis, adipogenesis and chondrogenic induced differentiation potential after passage of each group of cells to the generation P16; however, the osteogenic, adipogenic, chondrogenic differentiation potential of examples 1-3 was significantly better than comparative examples 2-5. This shows that the human umbilical cord mesenchymal stem cell culture medium provided by the invention can better maintain the in-vitro differentiation potential of the human umbilical cord mesenchymal stem cells, and is suitable for culturing the human umbilical cord mesenchymal stem cells.
6. Human umbilical cord mesenchymal stem cell in-vitro tumorigenicity detection
The in vitro tumorigenicity of the P2, P8, P16 generation human umbilical cord mesenchymal stem cells prepared in examples 1, 2, 3 was examined by the following method to evaluate the safety of the culture medium.
In vitro tumorigenesis experiments: the P2, P8 and P16 generation human umbilical cord mesenchymal stem cells prepared in examples 1, 2 and 3 are regulated to have the density of 4 multiplied by 10 by adopting a low-melting-point agar clone formation test method and using a human umbilical cord mesenchymal stem cell culture medium 3 /ml; positive control Hela cell densities were adjusted to 2X 10 with IMDM/F12+10% FBS medium 3 Per ml, negative control MRC-5 cells 2X 10 3 /ml. After mixing with the prepared agarose with low melting point and 0.7% concentration, the final cell concentration of the positive control and the negative control was 1×10 3 Each ml, and umbilical cord mesenchymal stem cells are 0.5X10 3 Ml and 2X 10 3 Two densities per ml, 3 wells per group. 1ml of human umbilical cord mesenchymal stem cells were spread uniformly in a 6-well plate with underlying agar and cultured in a 5% CO2 incubator at 37 ℃. Every week, checking whether clone is formed, and continuously checking for 3 weeks untilPositive controls grew until obvious clones were grown.
Results: positive control Hela cells were cultured for 3 weeks, mulberry-like clusters were visible in the upper agar layer, clear clone round clusters were visible under the microscope, and at weeks 1, 2 and 3, the clusters were (315.7±19.8), (623.7 ±29.3) and (768.7 ±21.9), respectively; no clonal pellet was detected under both the human umbilical cord mesenchymal stem cell group and the negative control MRC-5 cell group.
The result shows that the human umbilical cord mesenchymal stem cells prepared by the human umbilical cord mesenchymal stem cell culture medium provided by the invention still have no in-vitro tumorigenicity after being cultured in vitro to P16 generation, and the culture medium has good safety.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (9)
1. A human umbilical cord mesenchymal stem cell culture medium, which is characterized by comprising the following components: IMDM basal medium, F12 basal medium, ruxolitinib, seaweed extract, small molecule compound Thiazovivin and SD-208, normal human AB serum.
2. The human umbilical cord mesenchymal stem cell medium of claim 1, wherein the volume ratio of IMDM basal medium to F12 basal medium in the medium is 1:1-3.
3. The human umbilical cord mesenchymal stem cell medium of claim 1, wherein the concentration of ruxolitinib in the mesenchymal stem cell medium is 1-100nM.
4. The human umbilical cord mesenchymal stem cell medium of claim 1, wherein the concentration of the seaweed extract in the mesenchymal stem cell medium is 1-100g/L.
5. The human umbilical cord mesenchymal stem cell medium of claim 1, wherein the concentration of Thiazovivin in the mesenchymal stem cell medium is 1-100nM.
6. The human umbilical mesenchymal stem cell medium of claim 1, wherein the concentration of SD-208 in the mesenchymal stem cell medium is 1-100nM.
7. The human umbilical cord mesenchymal stem cell medium of claim 1, wherein the normal human AB serum is 5-20% of the total volume of the medium.
8. A method for preparing a human umbilical cord mesenchymal stem cell culture medium, using the human umbilical cord mesenchymal stem cell culture medium of any one of claims 1-7, comprising the steps of: respectively dissolving and preparing concentrated solutions of ruxolitinib, thiazovivin and SD-208 by using DMSO (dimethyl sulfoxide) to ensure that the concentration of each concentrated solution is 1-100 mu M; mixing IMDM basal medium and F12 basal medium in proportion, sequentially adding ruxolitinib, seaweed extract, small molecular compound Thiazovivin and SD-208 concentrated solution, and finally adding normal human AB serum.
9. Use of a human umbilical cord mesenchymal stem cell medium, a method for preparing a human umbilical cord mesenchymal stem cell medium according to claim 8, for culturing human umbilical cord mesenchymal stem cells.
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2023
- 2023-10-10 CN CN202311307836.1A patent/CN117363566A/en active Pending
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