Establishment Of Bovine Oviduct And Endometrial Epithelial Cell Lines

There is "developmental block" phenomenon widely in the course of in vitro production of various animal embryos. Lots of researches showed one of effective ways to overcome this block stage was to let the embryos co-culture with somatic cells. In the process of co-culture, the somatic cells could improve the efficiency of embryo production by means of secreting certain factors which were conducive embryos developing and some nutrients which contributed to maintain metabolic balance by autocrine and paracrine, reducing oxygen free radicals in cultured micro-environment and keep the stability of physical and chemical conditions of the culture system, clearing metabolites in the medium which did not conduce embryos developing. However, in the numerous somatic cells of animal, the oviduct epithelial cells and endometrial epithelial cells which belong to reproductive tract cells are closer to the environment of embryonic development physiologically in vivo. Thus they could be cultured in vitro and then used for embryo co-culture, which may have a positive significance. Moreover, because of the limitation of research in vivo, the two types of cells cultured in vitro could also provide an ideal model for studying microenvironments of early embryo development and physiological functions and physical disease of oviduct and uterus.This investagation mainly studied culture condition of the bovine oviduct epithelial cells (BOEC) and the bovine endometrial epithelial cells (BEEC) in vitro. The BOEC and BEEC were isolated and obtained separately by using the mechanical scraping+ centrifugation method and mechanical stripping+centrifugation method, and then cultured by explanting culture. Followed by morphology observation under the inverted and scanning microscope, the growth and proliferation condition were measured by MTT. In addition, when the cells were stable through passage and purification, the cultured cells type and purity were detected with the immunocytochemical examinations. Afterwards, the number of living cells was also examined by trypan blue exclusion test after cells freezing and thawing. By detecting the above biological properties, the effect of cells cultured in vitro could be assessed as a whole.The results were as follow: 1. The bovine oviduct epithelial cells (BOEC) were isolated by mechanical scraping +centrifugation method, and then they were identified by immunocytochemical examinations after passage and purification, the result suggested that its positive rate was more than 93%. The morphology of the cells was typically cobblestone in the inverted microscope. And cell proliferation curve was drawed by MTT method. The cells were frozen and thawed, and the cell viability over 75% with trypan blue exclusion test. However after passage, the living cells were more than 90%. By detecting the biological characteristics, it confirmed that this cell line was to be bovine oviduct epithelial cells.2. The bovine endometrial epithelial cells (BEEC) were isolated by mechanical stripping+centrifugation method, and they were identified by immunocytochemical examinations after passage and purification, the result indicated that the positive rate of BEEC was more than 96%. Under the inverted microscope, the morphology of the cells was typically pavement. The cell proliferation curve was also drawed by MTT method. The living cells were more than 65% using trypan blue exclusion test after frozen and thawed, whereas the cell viability over 85% after passage. Through detecting the biological characteristics, we confirmed that a bovine endometrial epithelial cell line was established.