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目的优化高通量生物反应器Tubespin培养CHO细胞的条件,提高蛋白表达量。方法采用Tubespin生物反应器在不同转速(160、180和200 r/min)和培养体积(5、10、20和35 ml)条件下振荡悬浮培养CHO细胞,测定不同条件下的气体交换速率、细胞密度、细胞活力及蛋白相对表达量,优化细胞培养条件。在最佳培养条件下,向培养基中分别添加30、60和90 mmol/L氯化钠,观察不同浓度氯化钠对细胞密度、细胞活力、摄氧率、溶氧及重组蛋白表达的影响。结果 Tubespin具有良好的气体交换速率,可满足高密度细胞培养的需要;经优化,细胞培养最佳条件为:摇床转速180 r/min,培养体积10 ml,此时细胞生长密度高,较快进入稳定期进行重组蛋白的表达。细胞培养72 h时向培养基中添加60 mmol/L氯化钠,能使重组蛋白的表达量提高1倍。结论优化了Tubespin培养CHO细胞的条件和氯化钠诱导浓度,提高了重组蛋白的产量,为大规模发酵生产奠定了基础。
Objective To optimize the conditions of Tubespin high-throughput bioreactor to culture CHO cells and increase the protein expression. Methods The CHO cells were cultured by shaking with different rotating speed (160,180 and 200 r / min) and culture volume (5, 10, 20 and 35 ml) using Tubespin bioreactor. The gas exchange rate Density, cell viability and protein relative expression, optimize cell culture conditions. Under optimal culture conditions, 30, 60 and 90 mmol / L sodium chloride were added to the culture medium to observe the effect of different concentrations of sodium chloride on cell density, cell viability, oxygen uptake rate, dissolved oxygen and recombinant protein expression . Results Tubespin had a good gas exchange rate, which could meet the needs of high-density cell culture. Optimized conditions for cell culture were as follows: shaking speed 180 r / min, culture volume 10 ml, high cell density, faster Into the stable phase of recombinant protein expression. At 72 h after culture, 60 mmol / L sodium chloride was added to the medium to increase the expression of recombinant protein by a factor of two. Conclusion The conditions of Tubespin culture of CHO cells and the concentration of sodium chloride were optimized and the yield of recombinant protein was increased, which laid the foundation for large-scale fermentation production.