UT-7/EPO细胞培养条件的优化及复壮

Optimization of culture condition and rejuvantion of UT-7/EPO cells

目的使复苏后处于衰亡状态的UT-7/EPO细胞恢复正常生长状态,并优化其生长密度。方法通过更换不同培养基(改良的RPMI1640、DMEM和IMDM培养基)、调整血清浓度(10%、15%和20%)、改变生长因子加量(1、3、5 U/ml)及将小鼠腹腔细胞作为饲养细胞与UT-7/EPO细胞共培养,对UT-7/EPO细胞进行复壮。对恢复正常生长状态后的UT-7/EPO细胞,利用CCK-8法绘制细胞生长曲线,并根据生长曲线通过梯度密度培养确定UT-7/EPO细胞在不同培养器皿(48、24、12、6孔板及25 cm2细胞培养瓶)中的最适培养密度。将传代后第2天处于对数生长期的UT-7/EPO细胞冻存及复苏传代3次后,连续培养9 d,检测复壮后UT-7/EPO细胞的初步稳定性。将复壮后UT-7/EPO细胞分别用人源STR试剂及小鼠细胞STR位点进行鉴定。结果更换培养基、改变血清和生长因子加量均不能使衰亡的UT-7/EPO细胞的生长状态有明显改善;饲养细胞与UT-7/EPO细胞共培养可使UT-7/EPO细胞恢复正常生长状态;获得了UT-7/EPO细胞在不同培养器皿中的最适培养密度。复苏后的细胞生长状况良好,与未冻存前的生长状态及活力相当。人源STR位点检测结果显示,复壮后的UT-7/EPO细胞STR图谱均为单个或2等位基因峰,无其他人源细胞的污染;小鼠STR位点检测结果显示,复壮后的细胞未发现饲养细胞的污染。结论饲养细胞能够有效地使UT-7/EPO细胞复壮;最适的细胞生长密度能有效地使细胞维持在稳定、良好的生长状态。

Objective To rejuvenate the dying UT-7/EPO cells after resuscitation and optimize their growth density. Methods UT-7/EPO cells were rejuvanted by changing the media （modified RPMI1640, DMEM and IMDM）, adjusting the serum concentration （10%, 15% and 20%）, adding various contents （1, 3 and 5 U/ml） of growth factors and co- culture with feeder cells derived from mouse peritoneal cavity. The growth curve of ceils recovered to normal status was plotted by CCK-8 method, based on which the culture densities of UT-7/EPO cells in various vessels （48-, 24-, 12- and 6-well plates as well as 25 em2 flask） were optimized by density gradient culture. The cells on the second day after subculture, in logarithmic growth phase, were stored in frozen, then resuscitated, subcuhured for three passages and cultured for 9 consecutive days to evaluate the stability. The cells after rejuvantion were identified with STR reagent from human origin and mouse cell STR site respectively. Results The growth status of UT-7 / EPO cells was not improved significantly by changing the medium, serum concentration and growth factor content. Co-culture with feeder cells recovered the UT-7 / EPO cells to a normal growth status. The culture densities of UT-7 / EPO cells in various vessels were optimized. The UT-7 / EPO cells after resuscitation grew well, of which the status and vigor were similar to those before storage in frozen. The test result for STR site from human origin showed a single or two allele peaks on the STR profile of UT-7/EPO cells after rejuvenation, while no contamination with other ceils from human origin was observed. However, the test resuh for murine STR site showed no contamination with feeder cells. Conclusion UT-7 / EPO cells were effectively rejuvenated after co-cuhure with feeder cells. However, the optimal growth density maintained the cells in a stable and normal growth status.