无血清悬浮培养CHO-K1-SFS细胞系的建立及其生物学特性

Establishment and biological characteristics of serum-free suspension culture of CHO-K1-SFS cell line

目的建立无血清悬浮培养CHO-K1-SFS细胞系,并检测其生物学特性。方法将CHO-K1贴壁细胞通过逐步降低血清浓度,获得低血清CHO-K1贴壁细胞,以3×105个/ml接种于含无血清培养基SFM4CHO的150 ml三角瓶中,摇床无血清适应培养,获得CHO-K1-SFS细胞。对该细胞进行生长曲线绘制、微生物污染检测、代谢分析及外源基因表达检测。结果所获得的CHO-K1-SFS细胞系可无血清悬浮培养,以3×105个/ml的初始密度接种,最大增殖浓度可达3.8×106个/ml,平均倍增时间为29.7 h;无细菌、真菌、病毒和支原体污染;对葡萄糖利用率较高,乳酸产生较多;能较好地表达外源基因。结论成功建立了无血清悬浮培养CHO-K1-SFS细胞系,为建立高效的外源基因表达平台奠定了基础。

Objective To establish the serum-free suspension culture of CHO-K1-SFS cell line and determine its biological characteristics. Methods Adherent CHO-K1 cells were cultured by gradually decreasing serum concentration,and the obtained CHO-K1 cells in low serum medium were inoculated at a density of 3 × 10^5cells / ml to 150 ml Erlenmeyer flask containing serum-free SFM4CHO medium and cultured in shaker. The growth curve of the obtained CHO-K1-SFS cells was plotted,the microbial contamination was tested,while the metabolism was analyzed,and the exogenous gene expression was determined. Results The obtained CHO-K1-SFS cell line was suitable for serum-free suspension culture,of which the maximum density reached 3. 8 × 10^6cells / ml when inoculated at an initial density of 3 × 10^5cells / ml,while the mean doubling time was 29. 7 h. No contamination with bacteria,fungi,viruses or mycoplasma was observed. The utilization rate of glucose was high,and a quantity of lactic acid was produced. Exogenous gene was effectively expressed. Conclusion The serum-free suspension culture of CHO-K1-SFS cell line was successfully established,which laid a foundation of high expression of exogenous gene.