Preparation method and application of human placenta stem cell extract freeze-dried powder
A technology of placental stem cells and extracts, which is applied in freeze-dried transportation, medical preparations of non-active ingredients, powder transportation, etc., can solve the problems of application limitations of embryonic stem cells, and achieve industrial utilization, good solubility, and safety high effect
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[0030] The embodiment of the present invention relates to a preparation method of freeze-dried powder of human placental stem cell extract, the method comprising the following steps:
[0031] (1) collecting healthy placenta, extracting and separating to obtain placental stem cells;
[0032] (2) placental stem cells are subcultured, and placental stem cell extracts are obtained by isolation;
[0033] (3) After mixing the placental stem cell extract with a lyoprotectant, freeze-drying to obtain a freeze-dried powder of the placental stem cell extract.
[0034] In one embodiment of the present invention, in step (1), under sterile conditions, shred healthy human placenta to 1-3mm 3 The blocks were washed with phosphate buffer solution containing double antibodies, then inoculated in serum-free medium containing double antibodies for culture, and when the cell density was 80% to 90%, primary placental stem cells were obtained.
[0035] In one embodiment of the present invention,...
Embodiment 1
[0052] Prepare the freeze-dried powder of human placental stem cell extract by the following method:
[0053] (1) Under sterile conditions, cut the healthy placenta to 1~3mm 3 The block was washed repeatedly 5 times with phosphate buffer solution containing double antibody; then the placenta was picked up with sterile ophthalmic forceps and inoculated in serum-free medium containing double antibody at 37°C, 5% CO 2 cultured under conditions. The fresh medium was replaced every 2-3 days, and the primary placental stem cells were obtained when the cell density reached 90%.
[0054] (2) The obtained primary placental stem cells were mixed with 0.1×10 5 ~1.0×10 5 cells / cm 2 Inoculate the cell culture dish at a density of 37°C, 5% CO 2 cultured under conditions. When the cells of passage 10 are obtained, the culture medium containing placental stem cells is added with water to form a suspension, transferred to a centrifuge tube, and centrifuged at a speed of 1000 rpm for 5 mi...
Embodiment 2
[0062] Prepare the freeze-dried powder of human placental stem cell extract by the following method:
[0063] (1) collecting healthy placenta, extracting and separating to obtain placental stem cells;
[0064] (i) adding the placenta to a solution containing 0.1% to 0.3% trypsin and 0.01% to 0.03% EDTA, and treating it at 37° C. for 0.5 to 1.5 hours;
[0065] (ii) After taking out the placenta, cut it into 1~3mm 3 block, and washed with phosphate buffer solution containing double antibody;
[0066] (iii) Add the placenta block to a solution containing 1-2g / L collagenase II, 0.04-0.05g / L DNase I and 8%-10% fetal bovine serum, and treat at 37°C for 1-3 hours;
[0067] (iii) After centrifuging the liquid obtained in step (iii), discard the supernatant to obtain the culture, inoculate it in serum-free medium containing double antibody, at 37 ° C, 5% CO 2 cultured under conditions. The fresh medium was replaced every 2-3 days, and the primary placental stem cells were obtained ...
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