Culture and Cryopreservation of Fibroblasts from Sheep Skin Tissue Stored at 4

This study was carried out to investigate whether the skin fibroblasts from the dying rare animals could be utilized as donor cells in somatic cell nuclear transfer for the production of cloned mammals.The skin tissues from auricle of young sheep were collected and preserved in mPBS at 4℃ for 0 hour(as control),24 hours,48 hours or 72 hours respectively,then processed primary culture in DMEM(Dulbecco's Modified Eagle's Medium) including glutamine and FBS(fetal bovine serum).After the primarily cultured cells reached to 85% confluence,they were subcultured directly or were frozen-thawed and then subcultured.The results showed that:(1) after stored at 4℃,the skin fibroblast in primary culture reached to 85% confluence requiring more times(8～15 d)than control group(4 d);(2) after subcultured 4 times,the fibroblasts from samples stored at 4 ℃ grew to 85% confluence requiring identical time to those from control samples;(3) although the frozen-thawed cells from different groups in the first subculture reached to 85% confluence in disparate time(11～13 d),all the cells in the second subculture grew to 85% confluence after 4 days.All these results indicated that if the skin tissues were soon collected and kept in mPBS at 4℃ for several hours once the animal died,the skin fibroblasts could have resumed growing after primary culture and subculture or primary culture and frozen-thawed,then subculture.