Serum-free culture medium for mesenchymal stem cells
A serum-free medium and necessary technology, applied in the biological field, can solve the problems of not supporting MSC primary culture, increasing workload and pollution, and expensive medium, avoiding instability, clear properties, and ensuring consistency Effect
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Embodiment 1
[0023] Embodiment 1: the preparation of culture medium
[0024]The medium SFM composition of the present invention is as follows:
[0025]
[0026]
[0027] More preferably, the medium SFM composition of the present invention is as follows:
[0028]
[0029]
[0030] The adhesive amine in the culture medium of the present invention can be replaced by 1 mg / L recombinant human fibronectin.
[0031] The amino acids mentioned in the table above include L-arginine, L-cystine, L-histidine, L-isoleucine, L-leucine, L-lysine, L-methionine , L-Phenylalanine, L-Threonine, L-Tryptophan, L-Tyrosine, L-Valine, L-Alanine, L-Aspartic Acid, L-Asparagine , L-glutamic acid, glycine, L-proline, L-serine;
[0032] The vitamins include calcium pantothenate, choline chloride, folic acid, meso-inositol, nicotinamide, pyridoxal hydrochloride, riboflavin, thiamine hydrochloride;
[0033] The lipids comprise arachidonic acid, cholesterol, DL-α-tocopheryl acetate, linoleic acid, linoleni...
Embodiment 2
[0043] Example 2: Isolation and culture of adherent cells in umbilical cord
[0044] Aseptically take 2 cm of the umbilical cord, cut open the blood vessel, rinse with DPBS to remove residual blood, and then digest it with 1 mg / mL type II collagenase DPB S 35 mL at 37 °C for 2 h, centrifuge at 2500 r / min for 10 min (centrifugal radius 22 cm), Remove the sediment from the lower layer and digest it with 0.25% trypsin in PBS for 20min, centrifuge at 2500r / min for 10min (centrifugal radius 22cm), take the precipitate and use PBS to pipette the cells to suspend, filter with a 100μm filter, and centrifuge at 2000r / min for 10min (centrifugal radius 22cm) to obtain single cells , followed by centrifugation with PBS 800r / min for 10min (centrifugal radius 22cm), washing the cells twice, with 1×10 6 Individual / mL density suspended in serum-free medium and serum-free medium of the control group, placed at 37°C, 5% CO 2 Cultivate in an incubator, and then change the medium every 3 to 4 da...
Embodiment 3
[0046] Example 3: Detection of cell phenotype by flow cytometry
[0047] Take the cells passed to the fifth generation, remove the culture medium, wash twice with PBS, digest with 1:1 0.25% trypsin solution, centrifuge at 1000r / min for 5min, wash once with PBS, adjust the cell concentration, and make the concentration for 10 9 L -1 Add 10 μL of antibody to the single cell suspension, incubate at 4°C for 30 min, wash once with PBS, centrifuge at 1000 r / min for 5 min, resuspend the cells in 500 μL of PBS, and then detect on a flow cytometer for comparison. The results are shown in Table 2. The results of comparison of the flow cytometry phenotypes of MSC cultured in the serum-free medium of the present invention and the medium of the control group showed that the MSCs in the serum-free medium provided by the present invention and the medium of the control group positively expressed CD13, CD29, CD44, CD73, CD90, CD105, CD166 and HLA-ABC, negative expression of CD14, CD19, CD45...
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