Abstract: At present,it has become a common method to study bovine endometritis by using the in vitro culture technology of bovine endometrial epithelial cells to establish a model of bovine endometritis so as to reduce uncontrollable factors.Thus obtaining high purity and identical endometrial epithelial cells are the key link in in vitro research.The methods used at home and abroad are mainly enzymatic digestion,tissue block adherence and tissue block digestion and adherence.In this paper,the previous method was modified to establish a simple and easy-to-use technique for in vitro culture of primary bovine endometrial epithelial cells with short cell culture period and good cell activity and morphology.The healthy non-pregnant cow uterus was used as the test material.The endometrial tissue was removed,and 5 mL of DMEM/F12 medium containing 2% double antibody and 40% trypsin was added,and digested at 4 ℃ for 12 h.The tissue was transferred to a 25 cm² cell culture flask for adherent culture.After obtaining the primary cells,the cells were subjected to immunohistochemical fluorescence identification using keratin-18 antibody,and the CCK-8 method was used for the third generation cells.The detection wavelength was 450 nm,and the cell growth was drawn according to the D_{450 nm} values at different time points curve.The results showed that the primary cells were basically covered with the bottom of the cell culture flask on the 8th day,which effectively shortened the cycle of the conventional tissue block adherence method.By keratin-18 immunohistochemical fluorescence identification,the positive rate of primary epithelial cells was more than 98%.Compared with the conventional tissue block adherence method,the purity of primary epithelial cells was significantly improved,and the steps of cell purification were omitted.The proliferation of the cells was in line with the normal characteristics of division and growth.The results showed that the conventional tissue block attachment method was optimized and improved,which not only shortened the culture period,but also improved the purity and maintained the cell activity,which provided a certain reference for the improvement of the in vitro culture technique of the primary bovine endometrial epithelial cells.

Key words: bovine endometrial epithelial cells; cell culture; cell identification