Xiao C, Zhang Y, Kong W, Wang H
Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences, Beijing.
Chin Med Sci J. 1992 Jun;7(2):67-71.
We have successfully cultured four cell lines--L929, BHK-13, BHK-21 and CHO-K1 using an MC-1 type microcarrier made in our academy. With the microcarrier in a concentration of 5 mg/ml, the cell density was about 30 x 10(4) cells/ml; after 3 days in suspension culture, the cells could proliferate to 1 x 10(6) cells/ml. At this time, when L929 cells were primed with 25 IU/ml MuIFN for 14-24 h and then superinduced with NDV, cycloheximide (20 micrograms/ml) and actinomycin D (2 micrograms/ml), the titer of IFN reached approximately 10(5) IU/ml (10(5) IU/mg of protein, expressed in specific activity). When VSV was inoculated into the other three cell line cultures, the viral titer reached 6 Log TCID50/ml or much higher. The yield with the CHO-K1 cell line was the highest, reaching titers of 7-8 Log TCID50/ml. These titers were similar to those seen in stationary culture. With trypsin-citrate solution and a more rapid stirring speed, the cells could be satisfactorily released from the microcarriers and reattached on fresh ones. These experiments show that the microcarrier suspension culture system is suitable for producing large scale, high-titer, low-cost vaccines and IFNs, both natural and recombinant, using genetically-engineered CHO cell lines.
我们使用本校制造的MC-1型微载体成功培养了四种细胞系——L929、BHK-13、BHK-21和CHO-K1。当微载体浓度为5 mg/ml时，细胞密度约为30×10⁴个细胞/ml；在悬浮培养3天后，细胞可增殖至1×10⁶个细胞/ml。此时，用25 IU/ml的鼠干扰素（MuIFN）预处理L929细胞14 - 24小时，然后用新城疫病毒（NDV）、放线菌酮（20微克/ml）和放线菌素D（2微克/ml）进行超诱导，干扰素的滴度可达约10⁵IU/ml（以比活性表示为10⁵IU/mg蛋白质）。当将水泡性口炎病毒（VSV）接种到其他三种细胞系培养物中时，病毒滴度达到6 Log TCID₅₀/ml或更高。其中CHO-K1细胞系的产量最高，滴度达到7 - 8 Log TCID₅₀/ml。这些滴度与在静止培养中观察到的滴度相似。使用胰蛋白酶 - 柠檬酸盐溶液并以更快的搅拌速度，细胞能够令人满意地从微载体上释放出来并重新附着在新鲜的微载体上。这些实验表明，微载体悬浮培养系统适用于使用基因工程CHO细胞系大规模生产高滴度、低成本的天然和重组疫苗及干扰素。
