In-vitro cultivating matural process of immatural ovocyte in ovarium organized block
A technology of ovarian tissue and in vitro culture, applied in the fields of reproductive medicine and cell biology, can solve the problems of not reaching 90% oocyte maturation rate, limiting the advantages and application of IVM technology, and inconsistency of nucleoplasmic maturation of oocytes , to achieve stable results, easy operation, and maintain tissue activity
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[0027] The inventive method operates as follows:
[0028] 1. Isolation and culture of MSC: Obtain the bone marrow cavity flushing solution of 8-10 weeks old female mice, add equal proportions to the top of the sterile Percol solution with a density of 1.077 in the centrifuge tube, centrifuge at 2000 rpm for 20 minutes, and collect the two liquid layers The mononuclear cell layer in between was washed twice by centrifugation at 1200 rpm for 10 minutes with 10 times the volume of 1×PBS. Cells resuspended in DMEM containing 10% fetal bovine serum by volume, 2×10 6 / ml cell density inoculate culture flasks at 37°C in 5% CO by volume 2 to cultivate. After 3 days, gently shake the culture flask, pour off the non-adherent cells, wash 2 times with 1×PBS to obtain the purified primary MSC, add DMEM containing 10% fetal bovine serum by volume and continue to culture until 80% of the bottom of the flask is covered ( Shown in Figure 1), the weight volume ratio is 0.25% trypsin digestio...
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- IPC
- C12N5/06; C12N5/075
- Inventors
- 冯定庆; 凌斌