Supplement for mesenchymal stem cell culture and culture method
A stem cell and cell culture technology, applied in the field of stem cell culture, can solve problems such as the inability to achieve high density, large-scale culture, loss, and reduced ability of cells to express products
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Embodiment 1
[0043] Embodiment 1 MSCs large-scale culture technique
[0044] Cell type: MSCs derived from umbilical cord; choose P3 generation cells to carry out the experiment, press 1×10 4 / cm 2The density was inoculated in T175 culture flasks, and cultured at 37°C in a 5% CO2 incubator. After 3-4 days of culture, the confluence of the cells reaches more than 90%, and enough cells are collected for use;
[0045] Microcarrier type: Cytodex-1;
[0046] Bioreactor type: Applikon bioreactor (5L);
[0047] Feeding 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; 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:
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[0051] Cultivation process:
[0052] 1. Culture system volume: 3L; cell inoculation volume: 2.5×10 4 cells / mL; microca...
Embodiment 2
[0060] Example 2 Morphological observation and activity detection
[0061] The cells of each group in Example 1 were sampled, and the sampling time points were: 48h, 120h. Morphological results such as figure 1 Shown (100×).
[0062] After culturing for 120 h, samples were taken and digested with 0.25% trypsin solution to collect UC-MSCs in each group, and the number of cells, amplification factor and cell viability in each group were calculated. The results are shown in Table 2, Figure 2~3 , indicating that the amplification factor 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).
[0063] Table 2 UC-MSCs cell number and activity detection results in each group (** means p<0.01)
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Embodiment 3
[0066] Example 3 pH value monitoring results
[0067] During the cell culture process, samples were taken every 12 hours to detect the pH value, and the results were as follows: Figure four shown. The experimental results show that fresh culture medium is supplemented between 48-60h, and the pH value of each experimental group is more stable relative to the matched group, indicating that the present invention's culture medium feed can better maintain the stability of the culture medium pH value, which is beneficial to Cells are cultured at high density to maintain strong proliferative ability and good activity ( Figure 4 ).
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