冷冻保护剂和平衡方法对鸡胚原始生殖细胞冷冻保存效果的影响

Cryopreservation of the Chicken PGCs Using Different Freezing Media and Equilibrium Methods

采用Ficoll密度梯度离心,提取第19期(孵化72h)和第28期(孵化132h)性腺中的原始生殖细胞(PGCs),应用不同的冷冻保护剂和平衡方法进行冷冻保存。PGCs解冻后用DMEM培养液进行体外培养,并采用苔盼蓝染色法鉴定复苏后PGCs的存活情况。结果表明:1从第19期或第28期性腺中获取的PGCs在同一种冷冻保护剂下,平衡方法对PGCs的存活率有显著或极显著影响,平衡方法1的冷冻保护效果优于平衡方法2。2采用相同的平衡方法,冷冻保护剂C的冻存效果与其他保护剂之间存在显著或极显著差异,冷冻保护剂E和F对PGCs的冻存效果次之,冷冻保护剂A、B和D对PGCs的冻存效果最差。复苏后PGCs进行体外培养,其存活时间与分离后PGCs直接培养相比未出现显著缩短现象。

Primordial germ cells (PGCs) were isolated from gonads of development stage 19 and 28 by Ficoll density gradient centrifugation. The PGCs were frozen by different cryopreservation media and equilibrium steps. Thawed PGCs were cultured in vitro in DMEM medium, and the vitality of the PGCs were identified by the Trypan Blue exclusion method. The main results showed below: ① Vitality of the PGCs from stage 19 and 28 which were frozen in a same cryopreservation medium showed significant difference or among the different equilibrium steps. Cryopreservation effects of the PGCs frozen by the step 1 were better than those by the step 2. ② Among different cryopreservation media by the same step, media C showed more effective, followed by medium E and F while medium A, B and D was less effective. Vitality time of PGCs showed no significant differences between the frozen-thawed treatment and the control treatment during culture of the tested PGCs in vitro.