Cryopreservation protective fluid and method for umbilical cord mesenchymal stem cells
A technology of stem cells and protective solution, applied in the field of stem cells, can solve the problems of low cell recovery rate and cell viability rate, unsatisfactory effect of cryopreservation of umbilical cord mesenchymal stem cells, etc., and achieve the effect of excellent cell proliferation ability.
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Embodiment 1
[0050] Preparation of umbilical cord mesenchymal stem cell conditioned medium: Take umbilical cord mesenchymal stem cells of P1 generation with a confluence of 90%, wash them once with PBS, and add 0.015mL / cm2 to the cells 2 Digest with 0.05% trypsin + 0.01% EDTA for 1min, stop the enzymolysis with complete medium 5 times the size of the digestion solution, centrifuge at 200g for 5min, reselect with complete medium, inoculate in a petri dish, the passage density is 8000 cells per cm 2 . The cells continued to grow for 72 hours, and the culture supernatant was collected, centrifuged at 200g for 5 minutes, and the supernatant was collected, aliquoted and stored at -80°C for later use.
[0051] Before cell cryopreservation, prepare umbilical cord mesenchymal stem cell cryopreservation protection solution: mix 80% umbilical cord mesenchymal stem cell conditioned medium and 10% fetal bovine serum, then add 10% DMSO, place in ice water bath until umbilical cord mesenchymal stem cel...
Embodiment 2
[0055] Preparation of umbilical cord mesenchymal stem cell conditioned medium: Take umbilical cord mesenchymal stem cells of P5 generation with a confluence of 80%, wash them with PBS three times, and add 0.04mL / cm2 to the cells 2 Digest with 0.05% trypsin + 0.04% EDTA for 1min, stop the enzymolysis with complete medium 10 times the size of the digestion solution, centrifuge at 400g for 5min, reselect with complete medium, inoculate in a culture bottle, and the passage density is 15,000 cells per cm 2 . The cells continued to grow for 24 hours, and the culture supernatant was collected, centrifuged at 400g for 5 minutes, and the supernatant was collected, aliquoted and stored at -80°C for later use.
[0056] Before cell cryopreservation, prepare umbilical cord mesenchymal stem cell cryopreservation protection solution: mix 30% umbilical cord mesenchymal stem cell conditioned medium and 50% fetal bovine serum, then add 20% DMSO, place in an ice water bath until cryopreservatio...
Embodiment 3
[0060] Preparation of umbilical cord mesenchymal stem cell conditioned medium: take umbilical cord mesenchymal stem cells of P3 generation with a confluence of 80%, wash them twice with PBS, and add 0.04 mL / cm2 to the cells 2 Digest with 0.05% trypsin + 0.04% EDTA for 1min, stop the enzymolysis with complete medium 10 times of the digestion solution, centrifuge at 400g for 5min, reselect with complete medium, inoculate in culture dish or culture bottle, passage density 10,000 cells per cm 2 . The cells continued to grow for 48 hours, and the culture supernatant was collected, centrifuged at 400g for 5 minutes, and the supernatant was collected, aliquoted and stored at -80°C for later use.
[0061] Before cryopreservation of cells, prepare umbilical cord mesenchymal stem cell cryopreservation protection solution: mix 55% umbilical cord mesenchymal stem cell conditioned medium and 30% fetal bovine serum, then add 15% DMSO, place in an ice water bath until cryopreservation solut...
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