玻璃化冷冻预处理方法对牛卵巢颗粒细胞的影响

Effects of the pretreatments of vitrification cryopreservation on bovine ovarian granulosa cells

为了研究不同玻璃化冷冻预处理对牛卵巢颗粒细胞培养的影响,试验采用4种不同的玻璃化冷冻预处理方法(①将冷冻保存管直接投入液氮罐中;②将冷冻保存管先置于4℃冰箱15 min,然后投入液氮罐中;③将冷冻保存管先置于-20℃冰箱10 min,然后投入液氮罐中;④将冷冻保存管先置于4℃冰箱15 min,再置于-20℃冰箱10 min,然后投入液氮罐中)处理牛卵巢颗粒细胞,解冻后检测细胞活力和传代时间。结果表明:解冻后4种方法的细胞活力分别为72%、74%、79%和86%;传代时间分别为76 h、73 h、67 h和62 h;方法④的细胞活力和传代时间与其他组比较差异显著(P<0.05)。说明玻璃化冷冻预处理方法有利于复苏后的牛卵巢颗粒细胞的进一步培养。

To study the effects of different pretreatments of vitrification cryopreservation on culture of bovine granulose cells. The following four different pretreatments were used to detect the viability and generation time of bovine ovarian granulosa cells after thawing： （1） putting directly the frozen pipes into a liquid nitrogen tank. （2） keeping the frozen pipes in a refrigerator at 4 degrees C for 15 min and then putting them into the liquid nitrogen tank. （3） keeping the frozen pipes in the refrigerator at 20 degrees C below zero for 10 min and then putting them into the liquid nitrogen tank. （4） keeping the frozen pipes in the refrigerator at 4 degrees C for 15 rain and at 20 degrees C below zero for 10 min, then putting them into liquid nitrogen tank. The results showed that the cell viability of the four pretreatments were 72.8%, 74.2%, 79.3% and 86.6% respectively, and the generation time were 76 h, 73 h, 67 h and 62 h respectively. There were significant differences （P 〈 0.05 ）in cell viability and generation time between the fourth pretreatment and other treatments. It indicates that the pretreatments of vitrification cryopr- eservation is conducive to further culture of bovine ovarian granulosa cells after resuscitation.