1株悬浮培养的Vero细胞驯化及其生物学特性研究

Domestication and Biological Characteristics of a Vero Cell Line in Suspension Culture

目的 将贴壁培养的Vero细胞驯化成悬浮培养细胞,并对其生物学特性进行研究。方法 采用无血清培养和“摇床适应法”对细胞进行培养优化,对其进行成瘤性、短串联重复序列(short tandem repeat,STR)分析、透射电子显微镜形态,以及对流感病毒敏感性检测。结果 成功建立悬浮细胞系Vero-S,Vero-S细胞生长期在24~72 h,平台期在108~144h之间,衰退期在144h以后,细胞PDT为36 h,细胞密度在14.01~14.88×10^(8)个/L之间,细胞平均圆度在0.73~0.75之间,细胞直径在12.12~14.44μm之间。Vero-S细胞在裸鼠皮下形成肿瘤,而悬浮培养前的Vero细胞不具有成瘤性;STR显示为猴源细胞系;电镜下细胞结构完整;Vero-S细胞对流感病毒感染不敏感。结论 悬浮细胞系Vero-S细胞的生长经历与贴壁细胞类似的生长期、平台期、衰退期,且细胞大小均一,建立的悬浮培养基能够支持Vero-S细胞高密度生长。STR结果表明悬浮Vero细胞具有完全的遗传稳定性,透射电镜下与贴壁Vero细胞无明显区别。为细胞悬浮驯化培养技术提供参考价值,并可用于细胞致肿瘤的机理研究。

Objective To domesticate adherently cultured Vero cells into suspension-cultured cells and investigate their biological characteristics.Methods Cell culture was optimized using serum-free media and the "shaker adaptation method." Additionally,tumorigenicity,short tandem repeat( STR) profiles,morphological characters under transmission electron microscope(TEM),and influenza virus sensitivity were analyzed in these cells.Results Suspension cell line Vero-S was successfully established.The growth period of Vero-S cells ranged from 24 to 72 hours,with a plateau phase between 108 and 144 hours,and a decline phase after 144 hours.The population doubling time(PDT) was 36 hours,and cell density ranged from 14.01 to 14.88 × 10^(8) cells/L.Average cell roundness ranged from 0.73 to 0.75,and cell diameter ranged from 12.12 to 14.44 μm.Vero-S cells formed t umors subcutaneously in nude mice,while Vero cells before suspension culture did not exhibit tumorigenicity.STR analysis indicated a primate origin of the cell line.Under electron microscopy,cell structure appeared intact.VeroS cells showed insensitivity to influenza virus infection.Conclusion The established suspension cell line Vero-S exhibiting growth patterns similar to adherent cells,with uniform cell size and high-density growth supported by the established suspension culture medium.STR results demonstrated complete genetic stability of suspended Vero cells,with no apparent differences observed under transmission electron microscopy compared to adherent Vero cells.This study provides reference for suspension culture domestication techniques and can be applied in mechanistic studies of cell tumorigenicity.