Cryopreservation And Culture Of Bovine Mammary Epithelial Cells

In this experiment, mammary epithelial cells were cultured at the level of the two-dimensional by taking Holstein cow's breast tissue as the material. On the base of it, the effect of different cryoprotectants, different cryopreservation methods and different cryopreservation density on the cryopreservation quality of bovine mammary epithelial cells were studied.1. Bovine mammary epithelial cells were collected by ways of digestion with collagenase typeⅡ, and the casein gene expression and protein secretion were detected. The results showed thatαs1-casein andβ-casein gene expression were detected at the gene level,β-casein wasn't detected in purified cells or cell culture medium except primary cells, which indicated that the translation ofβ-casein was restricted.2. The effect of cryoprotectants, cryopreservation methods and cryopreservation density on the cryopreservation quality of the fifth generation of bovine mammary epithelial cells were studied respectively, group A: 85% DMEM +10% FBS +5% DMSO; group B: 80% DMEM +10% FBS +10% DMSO; group C: 75% DMEM +10% FBS +15% DMSO; group D: 70% DMEM +10% FBS +20% DMSO; group E: 85% DMEM +5% FBS +10% DMSO; group F: 75% DMEM +15% FBS +10% DMSO; group G: 70% DMEM +20% FBS +10% DMSO; group H: 80% DMEM +10% FBS +10% glycerol; group I: 70% DMEM +10% FBS +20 % glycerol; group J: 60% DMEM +10% FBS +30% glycerol, group K: gradient freezing, group L: put the cryopreservation tubes in -20℃freezer for an hour , and then -80℃freezer for 12 hour, stored in liquid nitrogen, group M: put the cryopreservation tubes in -80℃freezer directly for 12 hour and then stored in liquid nitrogen, group N: put the cryopreservation tubes into the self-made frozen foam box in -80℃freezer directly for 12 hour and then stored in liquid nitrogen, group O: cryopreservation tubes were wrapped by 15g cotton in -80℃freezer directly for 12 hour and then stored in liquid nitrogen, group P: cryopreservation tubes were wrapped by 200 layer of medical gauze in -80℃freezer directly for 12 hour and then stored in liquid nitrogen, group Q: 1×10~4/ mL, group R: 1×10~5/ mL, group S: 1×10~6/ mL, group T: 1×10~7/ mL. Measure the survival rate, 24h adhering rate and apoptosis rate of bovine mammary epithelial cells, so as to determine the optimal conditions for cell cryopreservation. The results showed that the best cryopreservation conditions of cryopreserved bovine mammary epithelial cells were 80% DMEM +10% FBS +10% DMSO, self-made frozen foam box in -80℃freezer directly then stored in liquid nitrogen and 1×10~6 / mL of cryopreservation density.