Feeder cells using in immature ovocyte in-vitro cultivating maturation
A technology for in vitro culture and feeding of cells, applied in the fields of reproductive medicine and cell biology, to achieve stable results, high maturation rate, and enhanced interaction effects
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Embodiment 1
[0030] Using MSCs as feeder cells for the culture of immature oocytes separated by stimulation
[0031] 8-10 weeks old female Kunming mice, intraperitoneal injection of 10IU PMSG, 48 hours later intraperitoneal injection of 10IU HCG, 16-20 hours later cut the oviduct, at 37 ℃, volume ratio of 5% CO 2 Equilibrate overnight in the BSS to pick eggs under a stereomicroscope. Oocytes below MII stage were incubated at 37°C in 5% CO by volume. 2 Under the conditions, a part was cultured with second-generation MSCs, and a part was cultured with conditioned medium for 40 minutes to obtain mature oocytes ( figure 2 shown).
[0032] Experiments have shown that oocytes below the MII stage were cultured with second-generation MSCs or conditioned medium at 37°C with a volume ratio of 5% CO 2 The maturation rate reaches 95% after 40 minutes of culture under the condition. In order to avoid the further division of mature oocytes in MSC or conditioned medium, the mature oocytes are then di...
Embodiment 2
[0035] Using MSCs as feeder cells for the culture of immature oocytes obtained by aspirating from ovarian tissue
[0036] Under aseptic conditions, remove both ovaries of 8-10 week-old female Kunming mice, and place them in 37°C, 5% CO by volume. 2 Equilibrate overnight in DMEM containing 10% fetal bovine serum by volume, and obtain free antral follicles and preantral follicles by aspirating with a 1ml 25G syringe needle under a stereomicroscope. 2 Under conditions, a part is cultured with the second-generation MSC, and a part is cultured with the conditioned medium for 24 hours to 7 days to obtain mature oocytes ( image 3 shown).
[0037] Experiments have shown that, depending on the developmental stages of the follicles, in order to obtain mature oocytes, the culture time varies from 24 hours to 7 days. The maturation rate of mother cells is 93%; after being cultured with conditioned medium for 7 days, the maturation rate of oocytes is 85%. In order to prevent mature ooc...
Embodiment 3
[0041] Using MSCs as feeder cells for the cultivation of ovarian tissue blocks
[0042] Prepare agar scaffolds with a weight-to-volume ratio of 0.4%: second-generation MSCs in 1×10 5 Inoculate a 6-well plate at a cell density of / ml, pour off the culture medium after the cells adhere to the wall; prepare 0.4% agar with the conditioned culture medium, wait until it is cooled to 37°C-42°C, suck 3ml and spread it on the MSC, at 37°C, the volume ratio is 5 %CO 2 Equilibrate overnight for ovarian tissue block culture.
[0043] Under aseptic conditions, take out both ovaries of 8-10 weeks old female Kunming mice, place them in 4°C pre-cooled DMEM containing 10% fetal bovine serum by volume, and quickly make 1 -2mm action sheet, attached to 37°C, 5% CO by volume 2 On overnight MSC-containing agar with a weight-to-volume ratio of 0.4%, lightly press the tissue block to embed in the agar, and at the same time add HCG at 3 international units per milliliter; some ovarian tissue slices ...
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