无血清培养基悬浮培养BHK21细胞驯化的研究

The Study on BHK21 Cells Domesticated by Serum-Free Medium Suspension Culture

本研究主要探索用常规BHK21贴壁细胞(F38)驯化为适应无血清培养基的全悬浮细胞的过程并使用生物反应器放大进行了相关研究。实验表明:常规BHK21细胞通过运用相应的商品化培养基进行低血清贴壁驯化、再到低血清全悬浮驯化、最后到无血清全悬浮驯化,使细胞达到全悬浮的状态。经过四十代的传代后,细胞在无血清悬浮培养基中生长细胞数量可达3.0×106/mL以上,且形态较好,倍增时间稳定,活力在90%以上。实验中采用速降和缓降两种方法进行过渡驯化,结果表明,在贴壁状态下,细胞对缓降方式比速降方式适应性要好,细胞的形态较好,活力在90%以上。细胞从低血清悬浮到无血清悬浮过程中,两种过渡方式无明显差异,均经过了5～10代驯化,细胞就完全适应了无血清悬浮培养基。无血清悬浮细胞从液氮中复苏后,采用直线放大的形式从1.5 mL到250 mL再到5 L反应器最后到650 L反应器中生长。在培养参数方面进行了摸索,最终验证用直线放大的方法是可行的。细胞数量可达4.5×106/mL左右,细胞活力在90%以上,且细胞在放大过程中,形态良好,倍增时间稳定在18 h。

In this research,BHK21 cells （F38） was domesticated with serum-free medium and could be in suspended state and at the same time, used biological reactor amplifier doing some experiments. The results showed that the usual BHK21 cells was slowly becoming suspended cell through low serum adherence cell culture, low serum suspended cell culture, and serum-free suspended cell culture. Cells number can reach over 3.0~ 106/ml in serum-free suspended medium and these ceils have good forms, stabilizing doubling time, and more than 90% cell vitality.The fast drop and slow drop was adopted to domesticate in this research.The results showed that in adherence state, the ceils adapted better to slow drop than fast drop. The cells showed good forms and high cell vitality. But in the suspended state, two methods have no obvious differences, and after 5-10 generations suspended cell culture, ceils can adapt to the suspension culture medium. Serum-free suspended cells were recovered from liquid nitrogen. The volume of cell culture was added from 1.Sml to 250ml to 5L to 650L using the linear amplification form. Through researching related researcher have found that this method was practicable.The cell mumble can reach 4.5 vitality was more than 90% and have good forms, stable doubling time（in 18h）. culture parameter, the ×10^6/ml and the cells