Cell therapy agents are used to alleviate or improve the method of vascular lesions
A technology of cell therapy and vascular disease, applied in cardiovascular system diseases, animal cells, vertebrate cells, etc., can solve problems such as difficult to be suitable for clinical use, difficult to maintain, and decreased cell viability
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Embodiment 1
[0179] Embodiment 1, the pretreatment of human umbilical cord sample
[0180] Under sterile conditions, take the near-fetal umbilical cord of normal full-term production, disinfect the surface of the umbilical cord tissue with alcohol, and cut the umbilical cord from the middle;
[0181] Wash thoroughly with PBS containing 100U / mL penicillin and 100U / mL streptomycin to remove residual blood stains;
[0182] Divide the umbilical cord into 0.5cm umbilical cord segments, carefully remove the arteries and veins, take 50mL centrifuge tubes to hold the umbilical cord segments, 1 segment / centrifuge tube;
[0183] In a centrifuge tube, cut the umbilical cord segment into pieces with a size of about 1mm×1mm×1mm, and add LG-DMEM culture solution dropwise during the cutting process to keep the tissue moist;
[0184] Wash twice with PBS buffer solution containing 100 U / mL penicillin and 100 U / mL streptomycin (pH7.2, sodium phosphate salt, phosphate root concentration 0.025M, if not spec...
Embodiment 2
[0185] Embodiment 2, enzymolysis
[0186] The umbilical cord fragments obtained in Example 1 were mixed with enzyme solution (0.1% type I collagenase, 0.1% trypsin, 0.1% hyaluronidase, 0.1% DNase, 0.02% EDTA; % represents the percentage of mass concentration), Digest at 37°C for 2h;
[0187] After digestion, centrifuge for 15 minutes with a centrifugal force of 400g and a temperature of 4±2°C;
[0188] Discard the mixed enzyme solution to obtain single cells derived from umbilical cord.
Embodiment 3
[0189] Embodiment 3, primary culture of cells
[0190] The cells obtained from the treatment in Example 2 were divided into 1×10 6 The density of cells / ml was inoculated into T-25 culture flasks, placed in 20ml of RPMI1640 culture medium supplemented with 10% (v / v) fetal bovine serum, placed at 37°C, 5% CO 2 (v / v), incubating and cultivating for 3 days in an incubator with 95% humidity;
[0191] Change the medium for the first time after 3 days to remove unattached cells;
[0192] The culture medium was replaced every 48 h thereafter.
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