【摘要】 主要阐述了在以二甲亚砜为抗冻剂,不同的4℃平衡时间对PCK细胞低温保存的影响。把第24代PCK细胞悬浮于含10％二甲亚砜(DMSO)的冻存保护液,置于4℃冰箱,按不同的时间间隔取出细胞,重新培养,用来研究不同的平衡时间对细胞的贴壁率及生长的影响。将19代PCK细胞,按40min、1h、2h三种不同的4℃平衡时间进行-700℃冻存,再以较低的细胞密度(2×10~4个/mL)复苏细胞,用以研究不同的平衡时间对细胞冻存的复苏率及形态学的影响。结果表明,较长时间放置在4℃,DMSO的毒副作用明显,不利于细胞的生长。同直接传代的细胞相比,放置24h以上明显影响细胞的贴壁率及单层形成时间。在PCK细胞的-70℃保存中,4℃下平衡40min,DMSO不能对细胞形成充分的保护,细胞碎片较多;平衡2h以上,DMSO有影响了细胞对低温伤害的抵御能力,同样不利于细胞的冻存;平衡1h,细胞复苏及生长情况最佳。更多还原

【Abstract】 This paper mainly described the effect of the different equilibrium time on cryopreservation of PCK cells when dimethylsulphoxide (DMSO) was used as the cryoprotectant. PCK cells at passage 24 were suspended in M199 with 20% newborn bovine serum(NBS) and 10% DMSO, then they were placed at 4t. The cells were taken out by different time intervals. After centrifugation and the discard of cryopreservative solution, the cells were cultured again. This was used to study the effect of different equilibrium time at 4℃ on the seeding efficiency and growth. PCK cells at passage 19 were divided into three series, respectively, passed by three kinds of equilibrium time and cryopreserved at - 70℃. Then, those cells were thawed every 5 days in low density(2× 104cells/mL). This was used to study the effect of the different equilibrium time at 4℃ on the thawing rates of the cryopreserved cells. The result showed that when the cells were placed too long at 4℃, the toxicity of DMSO became obviously strong and showed disadvantage to the growth. Compared with the directly subcultured cells, those cells which were placed at 4t more than 24 hours had lower seeding efficiency and longer monolayer-formed time. In the process of cryopreservation, when the equilibrium time is within 40min, DMSO could not protect cells thoroughly; when beyond 2h, DMSO decreased the ability of cells to resist the detriment of low temperature; the thawing rates and the growth were best in 1h equilibrium at 4c℃.更多还原