Knibbs Randall N, Dame Michael, Allen Melissa R, Ding Yunhong, Hillegas William J, Varani James, Stoolman Lloyd M
Department of Pathology, University of Michigan, Ann Arbor 48109-0602, USA.
Biotechnol Prog. 2003 Jan-Feb;19(1):9-13. doi: 10.1021/bp020092r.
The present study shows that COS-7 cells transiently transfected and maintained on positively charged (trimethylamine-coated) microcarrier beads synthesize recombinant protein at higher levels and for longer periods of time than cells transfected and maintained on polystyrene flasks in monolayer culture. Sustained, high-level synthesis was observed with secreted chimeric proteins (murine E-selectin- and P-selectin-human IgM chimeras) and a secreted hematopoietic growth factor (granulocyte-macrophage colony-stimulating factor). Studies with green fluorescent protein indicated that the transfected cells attached more firmly to the trimethylamine-coated microcarriers than to polystyrene flasks. After 10-14 days in culture, most of the transfected cells detached from the surface of the polystyrene flasks, whereas most transfected cells remained attached to the microcarriers. The transiently transfected microcarrier cultures produced higher levels of protein per transfected cell due to this prolonged attachment. The prolonged attachment and higher output of transfected cells on microcarriers resulted in a 5-fold increase in protein production from a single transfection over two weeks. Thus, microcarrier-based transient transfection yields quantities of recombinant proteins with a significant savings of time and reagents over monolayer culture.
本研究表明，与在单层培养的聚苯乙烯培养瓶中转染并维持培养的细胞相比，在带正电荷（三甲胺包被）的微载体珠上进行瞬时转染并维持培养的COS-7细胞能以更高的水平、更长的时间合成重组蛋白。对于分泌型嵌合蛋白（小鼠E-选择素和P-选择素-人IgM嵌合体）和一种分泌型造血生长因子（粒细胞-巨噬细胞集落刺激因子），均观察到持续的高水平合成。对绿色荧光蛋白的研究表明，转染细胞与三甲胺包被的微载体的附着比与聚苯乙烯培养瓶的附着更牢固。培养10 - 14天后，大多数转染细胞从聚苯乙烯培养瓶表面脱离，而大多数转染细胞仍附着在微载体上。由于这种延长的附着，瞬时转染的微载体培养物中每个转染细胞产生的蛋白水平更高。转染细胞在微载体上延长的附着和更高的产量导致单次转染在两周内产生的蛋白量增加了5倍。因此，基于微载体的瞬时转染与单层培养相比，能产生大量的重组蛋白，同时显著节省时间和试剂。
