Protease composition for separating primary cells of human amniotic mesenchymal stem cells and separation culture method
A technology for the isolation and cultivation of mesenchymal stem cells, which is applied in the field of protease composition and isolation and cultivation for the primary cell separation of human amniotic mesenchymal stem cells, and can solve the problems of difficulty in uniformly quantifying the digestion time of tissue blocks, poor proliferation ability, and destruction
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Embodiment 1
[0046] Isolation and culture of primary cells
[0047] 1) Preparation before separation: turn on the shaker and preheat it to 37°C; wipe the surface of the ultra-clean workbench, wipe the reagent and sterilized instrument box with 75% alcohol and then transfer it to the ultra-clean workbench;
[0048] 2) Prepare five 15cm petri dishes, pour 50mL 0.9% sodium chloride injection into them respectively, and set aside;
[0049] 3) After wiping the amnion tissue with 75% alcohol, transfer it to two 15cm petri dishes in the ultra-clean workbench;
[0050] 4) Remove the blood stains on the amnion tissue, transfer to a 15cm petri dish, and wash the amnion tissue until there is no obvious blood stain;
[0051] 5) Clamp the drained amniotic tissue into two 15cm petri dishes, and cut the amniotic tissue into about 1×1cm with 10cm ophthalmic curved scissors 2 The tissue pieces were evenly transferred to two 50mL centrifuge tubes;
[0052] 6) Add 37°C preheated trypsin (purchased from In...
Embodiment 2
[0062] Screening experiment of adding amount of trypsin
[0063] Wash the amniotic membrane with pre-cooled normal saline and cut it to a size of 1cm×1cm, add 0.5 times, 1 times, 1.5 times, and 2 times the volume of amniotic membrane tissue to digest the tissue for 1 hour (37°C, 150rpm), after digestion Transfer the tissue block to a new culture dish, wash it with normal saline, drain the water, and repeat the digestion again. After the tissue block is drained, transfer it to a new centrifuge tube and add the same volume as the digested tissue block. Digestion with compound collagenase until the tissue block is transparent, and the digested mesenchymal stem cells are collected. By investigating the number of cells, cell shape, cell purity and other indicators, the effect of the amount of trypsin on the quality of amnion mesenchymal stem cells was determined, and the results are as follows.
[0064] 1. Cell number
[0065] Table 1 shows the results of the number of primary ce...
Embodiment 3
[0083] The amount of collagenase added
[0084] After the amniotic membrane tissue digested with trypsin of 1 times the volume of amniotic membrane tissue was washed with pre-cooled normal saline and coagulated, it was cut to a size of 1cm×1cm, and compounded with 0.5 times, 1 time, 1.5 times, and 2 times the volume of amniotic membrane tissue were added. Type collagenase (enzyme activity 0.4U / ml) was used to digest the tissue until it was transparent. By examining the cell number, cell shape, and cell purity indicators, the effect of collagenase addition on the quality of amniotic mesenchymal stem cells was determined.
[0085] 1. Cell number
[0086] See Table 1 for the results of digestion time and the number of primary cells of different added volumes of compound collagenase treatment groups.
[0087] Table 1 Digestion time and quantity of primary cells
[0088] group 0.5 times 1 times 1.5 times 2 times tissue volume 3ml 3ml 3ml 3ml Collag...
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