Supplement and culture method for culturing mesenchymal stem cells
A technology for cell culture and qualitative stem cells, applied in the field of stem cell culture, can solve the problems of reduced ability of cells to express products, inability to achieve high density, large-scale culture, loss, etc.
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Embodiment 1
[0043] Example 1 MSCs large-scale culture process
[0044] Cell type: MSCs derived from umbilical cord; select P3 generation cells for experiments, press 1×10 4 / cm 2 The density was inoculated in T175 culture flasks and cultured at 37°C in a 5% CO2 incubator. After 3-4 days of culture, the cell confluency reaches more than 90%, and enough cells are collected for future use;
[0045] Microcarrier type: Cytodex-1;
[0046] Bioreactor Type: Applikon Bioreactor (5L);
[0047] Feed formula: glucose 20g / L; sodium bicarbonate 2g / L; soybean=hydrolyzate 2.5g / L; 100× non-essential amino acid mixture 1vol%; glutamine 365mg / L; sodium pyruvate 55mg / L; reconstituted Human epidermal growth factor 0.02mg / L; recombinant human basic fibroblast growth factor 0.02mg / L; linoleic acid 0.042mg / L.
[0048] Table 1 Experimental group design:
[0049]
[0050] Cultivation process:
[0051] 1. Culture system volume: 3L; cell inoculation volume: 2.5×10 4 cells / mL; microcarrier usage: 3g / L. Spe...
Embodiment 2
[0059] Embodiment 2 Morphological observation and activity detection
[0060] The cells of each group in Example 1 were sampled, and the sampling time points were: 48h and 120h. morphological results such as figure 1 shown (100×).
[0061] After culturing for 120 h, samples were taken, and 0.25% trypsin solution was digested to collect UC-MSCs in each group, and the number of cells, expansion fold and cell viability in each group were calculated. The results are shown in Table 2. Figures 2 to 3 , indicating that the amplification fold of each experimental group was significantly higher than that of the two control groups (p<0.01); the cell viability was significantly higher than that of the two control groups (p<0.01).
[0062] Table 2 The results of the number and activity of UC-MSCs in each group (** means p<0.01)
[0063] Experimental group Number of viable cells (×10 4 / mL)
Embodiment 3
[0064] Example 3 pH monitoring results
[0065] During the cell culture process, samples were taken every 12 hours to detect the pH value, and the results were as follows: Figure 4 shown. The experimental results show that when fresh medium is supplemented between 48-60h, the pH value of each experimental group is more stable than that of the control group, indicating that the medium supplement of the present invention can better maintain the stability of the medium pH value, which is beneficial to Cells are cultured at high density to maintain vigorous proliferation and good activity ( Figure 4 ).
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