Nucleus pulposus cell separating and purifying method
A technology for separation and purification of nucleus pulposus cells, applied in cell dissociation methods, biochemical equipment and methods, animal cells, etc., can solve the problems of small cell volume, low survival rate, and large differences, and achieve short digestion time, The effect of high survival rate and low cost
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Embodiment 1
[0036] The surgically removed nucleus pulposus tissue was placed in PBS containing double-antibody (penicillin-streptomycin), and a small amount of surrounding fibrous ring tissue was carefully separated, and then washed three times with PBS until there was no obvious blood. Cut the nucleus pulposus tissue into a diameter of about 1 mm with ophthalmic scissors, add 2 times the volume of 0.5% type II collagenase, and shake and digest overnight in a 37°C constant temperature water bath. Stop the digestion with DMEM medium containing 10% fetal bovine serum, centrifuge at 1000r / min for 5min; discard the supernatant (mainly to remove residual digestive enzymes), and resuspend the cells in DMEM containing 10% fetal bovine serum . Use trypan blue staining to detect cell viability, calculate the cell concentration with a hemocytometer, and adjust the cell concentration to 2×10 5 / ml, inoculated in T25 culture flasks, placed at 37°C, 5% CO 2 Cultivate in an incubator, change the medi...
Embodiment 2
[0038] The surgically removed nucleus pulposus tissue was placed in PBS containing double-antibody (penicillin-streptomycin), and a small amount of surrounding fibrous ring tissue was carefully separated, and then washed three times with PBS until there was no obvious blood. Use ophthalmic scissors to cut the nucleus pulposus tissue into a size less than 1 mm in diameter, add 2 times the volume of 0.5% type II collagenase, and shake and digest in a constant temperature water bath at 37°C overnight. Stop the digestion with DMEM medium containing 10% fetal bovine serum, centrifuge at 1500r / min for 10min; discard the supernatant (mainly to remove residual digestive enzymes), and resuspend the cells in DMEM containing 10% fetal bovine serum . Use trypan blue staining to detect cell viability, calculate the cell concentration with a hemocytometer, and adjust the cell concentration to 2×10 5 / ml, inoculated in T25 culture flasks, placed at 37°C, 5% CO 2 Cultivate in an incubator, ...
Embodiment 3
[0040] The surgically removed nucleus pulposus tissue was placed in PBS containing double-antibody (penicillin-streptomycin), and a small amount of surrounding fibrous ring tissue was carefully separated, and then washed three times with PBS until there was no obvious blood. Use ophthalmic scissors to cut the nucleus pulposus tissue into a size smaller than 1 mm in diameter, add 2 times the volume of 0.25% trypsin, and shake and digest in a constant temperature water bath at 37°C for 2 hours. Stop the digestion with DMEM medium containing 10% fetal bovine serum, centrifuge at 1500r / min for 10min; discard the supernatant (mainly to remove residual digestive enzymes), and resuspend the cells in DMEM containing 10% fetal bovine serum . Use trypan blue staining to detect cell viability, calculate the cell concentration with a hemocytometer, and adjust the cell concentration to 2×10 5 / ml, inoculated in T25 culture flasks, placed at 37°C, 5% CO 2 Cultivate in an incubator, change...
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