Method for hypoxia culture of endometrial stem cell from menstrual blood

[0033] Example 1 Collection of menstrual blood samples from adult women

[0034] (1) Preparation of specimen preservation solution: PBS buffer, respectively adding final concentration of 200U/ml gentamicin, 0.2mg/ml amphotericin, and 1.0mg/ml EDTA-NA2. Mix well.

[0035] (2) Recruit 8 healthy adult female volunteers 3 days before the menstrual cycle, collect about 10ml of their menstrual blood samples, and then transfer the menstrual blood samples to a 50ml centrifuge tube containing 10ml specimen preservation solution and mix gently. The menstrual blood sample preservation solution is stored at 4°C, and the endometrial stem cells are separated and extracted within 24 hours.

[0036] Example 2 Preparation of hypoxic endometrial stem cells

[0037] Take 4 copies of the menstrual blood sample preservation solution of Example 1, and centrifuge them at 300 g for 5 min. Collect some of the supernatant samples for microbiological testing, and discard the rest, leaving the precipitate to be further operated in a biological safety cabinet.

[0038] For further precipitation, add 20ml of normal saline to mix well, slowly add to the upper layer of 10ml of Ficoll lymphocyte separation solution, add the solution carefully to keep a clear interface between the blood sediment and Ficoll lymphocyte separation solution. Centrifuge at 400g for 20 minutes, and carefully aspirate the uppermost layer of liquid, leaving the buffy coat cells and endometrial fragments. Wash twice with PBS buffer, and then add 5ml complete medium (low-sugar DMEM and F12 are mixed in proportion and then add replacement plasma with a final concentration of 10%, gentamicin 100U/ml, amphotericin 0.1mg/ml) . 8 specimens are operated in the same way, 4 specimens are cultured in a normal oxygen carbon dioxide incubator, CO 2 The concentration is controlled at 5%, O 2 The concentration is controlled to 21% and the temperature is 37°C. The other 4 samples were cultured in a hypoxic carbon dioxide incubator, CO 2 The concentration is controlled at 5%, O 2 The concentration is controlled to 1-10%, preferably 5%, and the temperature is 37°C. When the fusion rate of the cells reaches 80% or more, trypsinization is used to collect the cells, and the cells are subcultured to multiple T75 culture flasks at an appropriate density for continued cultivation. The culture conditions are the same as the above.

[0039] The cells were collected by trypsinization and centrifuged at 300g for 5 min. The cells were counted and frozen; the cells in the hypoxic incubator were endometrial stem cells (hypoxic MenESCs) derived from hypoxic menstrual blood, and normal oxygen CO 2 The cells in the incubator are endometrial stem cells derived from normoxic menstrual blood (normoxic MenESCs).

[0040] Example 3 Growth curve of endometrial stem cells from menstrual blood

[0041] Menstrual blood endometrial stem cell growth curve detection: culture the endometrial stem cells to the third generation in a hypoxic carbon dioxide incubator and a normoxic carbon dioxide incubator respectively, digest the cells with 0.25% trypsin, and resuspend in an appropriate volume of medium Cells, take 100μl single cell suspension (1×10 3 ) Inoculated in 96-well plates. The cells were placed in a hypoxic incubator and a normoxic incubator for 8 days, each day was used as a time point and 3 re-wells were set up; 10 μl of CCK-8 solution was added to the 96-well plate 4 hours before the measurement; The calibrator detects the OD value of each hole at a wavelength of 490nm and records the result. Draw a growth curve, see the results figure 1.

[0042] From figure 1 It can be seen that MenESCs cultured in normoxia and hypoxia both showed good proliferation status. There was no significant difference in cell growth status between the two groups.