RESUMO
When grown under hypothermic conditions, CHO (Chinese-hamster ovary) cells become growth-arrested in the G0/G1 phase of the cell cycle and also often exhibit increased recombinant-protein production. We have shown in the accompanying paper [Fox, Tan, Tan, Wong, Yap and Wang (2005) Biotechnol. Appl. Biochem. 41, 255-264] that the positive effect of low temperature on recombinant-protein production is due to elevated mRNA levels and not due to G0/G1-phase growth arrest and that a cell line can still show growth-associated productivity at low temperature. This finding led to the hypothesis that improved total production of recombinant protein would be achieved by stimulating cells to actively grow at low temperature, a culture condition previously unreported in the literature. In the present study we have validated this hypothesis by stimulating hypothermic (32 degrees C) growth through the use of different growth factors. Hypothermic growth was stimulated in fetal-bovine-serum-supplemented adherent cultures using basic fibroblast growth factor or insulin. Hypothermic growth was also stimulated in suspension cultures normally grown in protein-free medium by using supplementation with fetal bovine serum. These methods resulted in up to 7.7- and 4.9-fold increases in total recombinant-protein production compared with the 37 and 32 degrees C control cultures respectively. This proof-of-concept study will motivate the creation of cell lines capable of growing at low temperatures for use in industrial processes.
