成年小鼠雄性生殖细胞的冷冻保存

在含10％小牛血清（NBS）的DMEM培养基中，分别各添加5％，10％，15％，20％和25％的二甲基亚砜（DMSO）,丙二醇（PG），乙二醇（EG）和甘油（G），对成年小鼠睾丸生殖细胞冷冻保存；复苏后台盼蓝染色测定细胞复苏率。结果显示，5％－25％DMSO冻存液组的细胞复苏率分别为88.5%,88.0%,65.6%及51.3%;5%-25%PG冻存液组的细胞复苏率分别为87.2%,86.4%,79.0%,73.4%及40.1%.;5%-25%EG冻存液组的细胞复苏率分别为6.6%,80.9%,60.8%,51.3%及30.0%;5%-25%G冻存液组的细胞复苏率分别为86．5％，86．3％，65．3％，36．0％及31．4％。其中各抗冻剂5％和10％组的细胞复苏率最高，与15％组相比均存在显著或极显著差异。4种抗冻剂的最高细胞复苏率之间无显著差异。DMSO，PG，EG和G分别冷冻保存成年小鼠睾丸生殖细胞对的最小损失率分别为4．8％，6．1％，6．7％，6．8％。结果表明，采用慢速冷冻时，DMSO，PG，EG及G均适宜用作成年小鼠睾丸生殖细胞的抗冷冻剂，最佳使用含量均为5％－10％。成年小鼠睾丸生殖细胞分别在含5％－10％的DMSO，PG，EG和G的DMEM（含10％NBS）冻存液中，2步慢速降温，液氮储存，37℃水浴复苏，是一种具有较高复苏率的冷冻保存方法。

Cryopreservation of Adult Mouse Testicular Germ Cells

Adult mouse testicular germ cells were cryopreserved,and the cell recovery rates were examined by trypan blue exclusion staining after thawing.The freezing solutions were DMEM/10% NBS supplemented with 5%,10%,15%,20% and 25% cryoprotectants dimethyl sulphoxide(DMSO),propanediol(PG),ethylene glycol(EG) or glycerol(G),respectively.The cell recoveries of DMSO at concentrations of 5%,10%,15%,20%,and 25% were 88 5%,88 0%,80 0%,65 6%,and 51 3%,respectively.The cell recoveries of PG at concentrations of 5%,10%,15%,20%,and 25% were 87 2%,86 4%,79 0%,73 4%,and 40 1%,respectively.The cell recoveries of EG at concentrations of 5%,10%,15%,20%,and 25% were 86 6%,80 9%,60 8%,51 3%,and 30 0%,respectively.The cell recoveries of G at concentrations of 5%,10%,15%,20%,and 25% were 86 5%,86 3%,65 3%,36 0%,and 31 4%,respectively.When the concentrations of four cryoprotectants were 5% 10%,the cell recoveries all were highest,and showed significant difference as compared with those at concentration of 15%.There were no significant differences among the highest recoveries of four cryoprotectants.The minimum cell losses of four cryoprotectants after freezing/thawing were 4 8%,6 1%,6 7%,and 6 8%,respectively.The results suggest that DMSO,PG,EG and G all are suitable for cryopreservation of adult mouse germ cells and their favorable concentration all are at 5% 10%.The two step freezing procedure is one with high cell recovery rates for cryopreservation of adult mouse germ cells.