Marc-145细胞低血清适应培养传代稳定性和无血清驯化研究

通过驯化建立无血清悬浮培养型Marc-145细胞系,分析驯化传代对细胞特性的影响,为大规模生产疫苗用的宿主细胞提供基础。采用缓降培养液中血清浓度的方法,连续传代培养50代,并用无血清培养基进行悬浮培养驯化,对传代和驯化过程中的P10、P20、P30、P40和P50代细胞的形态、倍增时间、生长曲线、染色体和对蓝耳病病毒敏感性等特性进行对比分析。研究发现,传代过程中Marc-145细胞均呈扁平多边形上皮样生长,P10代细胞倍增时间为34.15 h,P50代细胞倍增时间为36.16 h,P50代细胞倍增时间稍有延长但差异不显著。P10、P20、P30、P40和P50代的Marc-145细胞生长曲线均呈“S”形;P10代和P50代细胞染色体众数都集中在88~90条。接种病毒后,P10、P20、P30、P40和P50细胞TCID₅₀间无显著差异,表明细胞传50代后,细胞特性没有发生明显变化。P50细胞进行无血清悬浮培养48 h后,细胞密度可达到4.14×10⁶ mL^-1。

Study on subculture stability and serum-free acclimation of Marc-145 cells in low serum adaptation culture

A serum-free suspension culture Marc-145 cell line was established through domestication, and the effects of domestication on cell characteristics were analyzed to provide basis for mass production of host cells for vaccines. The serum concentration in the slow drop culture medium was used to subculture for 50 generations, and the serum-free medium was used for suspension culture and domestication. The morphology, doubling time, growth curve and sensitivity to PRRSV of P10, P20, P30, P40 and P50 passage cells during passage and domestication were compared and analyzed. Results showed that Marc-145 cells grew like flat polygons. The doubling time of P10 passage cells was 34.15 h, while the doubling time of P50 passage cells was 36.16 h, and the doubling time of P50 passage cells was slightly prolonged, but the difference was not significant. The growth curves of P10, P20, P30, P40 and P50 Marc-145 cells were “S” type, and the chromosome numbers of P10 and P50 passage cells were between 88 and 90. After inoculating the virus, there was no significant difference in TCID₅₀ values between P10, P20, P30, P40 and P50 cells, indicating that there were no significant change in cell characteristics after 50 generations of cell passage. Therefore, P50 cells were cultured and domesticated in serum-free suspension culture. After 48 h of serum-free suspension culture, the cell density of P50 cells reached 4.14×10⁶ mL^-1.