猪滋养层细胞囊泡的冷冻保存

Cryopreservation of Pig Trophoblastic Vesicles(TRV)

冲洗和轻微抖动子宫角收集猪１４日龄的伸展胚胎，剪成０.５ｍｍ左右节段，培养２小时，将体外形成的滋养层细胞囊泡（ＴｒｏｐｈｏｂｌａｓｔｉｃＶｅｓｉｃｌｅｓＴＲＶ）在含１０％甘油（处理１）或１０％甘油和０.２Ｍ海藻糖（处理２）的Ｈｅｐｅｓ－Ｍ１９９（ＢＳＡ）冷冻液中平衡并装入０.２５ｍｌ的塑料细管。处理１和处理２的细管降温至－７℃后植冻并保持１０分钟，再以－０.５℃／分钟的速率分别降至－２５℃和－３６℃后直接浸入液氮保存。细管在室温下解冻，用含０.５Ｍ蔗糖的Ｈｅｐｅｓ－Ｍ１９９（ＢＳＡ）除去抗冻剂，洗涤５次后进行培养。上述不同方法冷冻的猪ＴＲＶ在解冻后２４－４８小时以生长囊泡计算成活率，分别为８４.８％和７９.３％，即可为有关研究提供稳定而可靠的猪ＴＲＶ来源。该研究还对猪ＴＲＶ体外培养和冷冻过程中的形态学变化及其分类进行了光镜观察和讨论。

Pig trophoblastic vesicles were by cutting into sections 20.5 mm in length from enlongated embryos collected onday-14 after Artificial Insemination by flashing the uterus.The sections were cultured in vitro for 2 h before equilibrated in freezing medium containing 10%(v/v)glycerol(treatment A)or 10%glycerol and 0.2 M trehalose(treatment B),and loaded into 0.25 ml plastic straws.The straws were seeded at-7℃and cooled at a rate of 0.5℃/min to- 36℃(treatpoent A)and- 25℃(treatment B)respectively.Then the straws were directly insert into Liquid Nitrogen.After thawing,the TRV were cultured for at least 48 h in Hapes-TCM199 (BSA)medium.The post-thaw viabilities of the growing vesicles were 84.8%and 79.3%for treatment A and B respectively.The results show that this experiment may provide a reliable method to achieve TRV used in embryoco-culture and co-transfer as well as the early embryo model for relative research.The morphologicchanges and sorting standard under the light microscope were also observed and discussed.