Method for separating and amplifying mesenchymal stem cell from fresh tissue of umbilical cord
A technique for stem cells and umbilical cord tissue, applied in the field of isolation and expansion of stem cells
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[0064] Example 1. Method for separating and expanding mesenchymal stem cells from umbilical cord
[0065] (1) Disinfection and cleaning: In the biological safety cabinet, disinfect the surface of the umbilical cord tissue with alcohol, cut the umbilical cord from the middle, spread it flat on a sterile 10cm cell culture dish, and wash the tissue with PBS buffer to minimize Red blood cells on the tissue;
[0066] (2) Digestion treatment: add 0.1g of type I collagenase to 100ml of DMEM-F12, then filter with a 20μm filter to obtain the digestion solution, transfer the umbilical cord tissue obtained in step (1) to another 10cm cell culture plate, Cut the umbilical cord tissue into 1cm 3 Put the tissue block in the prepared digestion solution and digest it in a constant temperature shaker at 37°C for 1.5 hours. Use a 100μm filter to remove the remaining tissue block. Add the interfill at a volume ratio of 1:1. Stem cell culture medium (15%FBS+1%L-Glutamine+0.05%Gentamicin+84%DMEM-F12) ...
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[0073] Example 2. Method for separating and expanding mesenchymal stem cells from umbilical cord
[0074] Refer to the method of Example 1. The whole cells of the umbilical cord tissue obtained after digestion began to have adherent cells crawl out on the 8th day of culture, and the fusion rate reached 90% on the 20th day. After 3 passages, the cell purity is greater than 95%.
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[0075] Example 3. Method of separating and expanding mesenchymal stem cells from umbilical cord
[0076] Refer to the method of Example 1. The whole cells of the umbilical cord tissue obtained after digestion began to have adherent cells crawl out on the 7th day of culture, and the fusion rate reached 90% on the 18th day. After 3 passages, the cell purity is greater than 90%.
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