1. Surface epithelial cells from normal ovaries from surgical residual specimen were isolated using standard and Institutional Review (References).
2. The surgical samples of ovarian tissue were immediately placed in plastic bags and kept on ice.
3. Tissues were gently washed twice (5 min for each wash) with phosphate-buffered saline (PBS) containing primary cell culture system with 10% penicillin and streptomycin.
4. Afterward, the outer surface of the ovarian tissues was scraped gently with a scalpel blade or more firmly with the blunt side of the blade.
5. The scraped cells were gently transferred to a tissue culture dish containing the growth medium of primary cells, and grown for 7–10 days at 37°C in 5% CO^₂ without changing the medium.
6. Trypsinization was used to remove any stromal fibroblasts, after which the cells were usually split in a 1:3 ratio (33.33%, passage 1) when they reached 85% confluence in average.
7. In this way, one passage is ≅1.3 population doublings (PD), which was calculated by referring to the method reported in literature (Reference).
8. Three primary cell cultures were maintained this way and used for additional experimentations.

References

1. Kruk P.A., Maines-Bandiera S.L., Auersperg N. A simplified method to culture human ovarian surface epithelium. Lab. Invest. 1990;63:132-136.

2. Auersperg N., Siemens C.H, Myrdal S.E. Human ovarian surface epithelium in primary culture. In Vitro 1984;20:743-755.

3. Hayflick L. Subculturing human diploid fibroblast cultures. In: Kruse P.F. Jr, Patterson M.K. Jr, editors. Tissue Culture Methods and Applications. New York: Academic Press; 1973. p. 220-223.