Mammalian cell culture is an important and essential technology in pharmaceutical production and regenerative medicine. Cell adhesion and growth in culture generally require the addition of fetal calf serum (FCS) to a medium, because FCS can supply trace amounts of essential elements, such as hormones, vitamins, and growth factors. However, the use of FCS should be avoided owing to possible contamination by pathogens, such as prions derived from infected calves. Thus, the development of new effective substitutes for FCS is necessary. Such substitutes are required to have high medical safety, that is, no containing virulent factors for humans, and an effect on cell growth comparable to that of FCS.
To date, no DNA or RNA viruses infecting fish have been reported to infect humans (Yoshimizu and Kasai 2005
). Thus, employing fish serum (FS) in mammalian cell culture for medically related use should be safer than employing FCS. However, the stimulating activities of FS for the adhesion and proliferation of mammalian cells are unknown.
Insulin-like growth factor-I (IGF-I), insulin, growth hormone (GH), and thyroxine were detected in the plasma of fish such as gilthead seabream (Sparus aurata
) (Funkenstein et al. 1989
), coho salmon (Oncorhynchus kisutch
) (Larsen et al. 2001
), Atlantic salmon (Nordgarden et al. 2005
), and channel catfish (Ictalurus punctatus
) (Small and Peterson 2005
). A cDNA encoding fibroblast growth factor-2 (FGF-2) has been isolated from the cDNA library of rainbow trout (Hata et al. 1997
). A probable fibroblast growth factor was obtained from the swim bladder of red seabream (Pagrus major
) (Suzuki et al. 1994
). Furthermore, primary cultures of cells from fish gills and kidneys were developed using serum from the North African catfish (Clarias gariepinus
) (Rathore et al. 2001
). Therefore, FS is expected to have some growth-stimulating effects on mammalian cells.
The Chinese hamster ovary (CHO) cell line is industrially important in the production of pharmaceutical proteins, such as human granulocyte-macrophage colony-stimulating factor (hGM-CSF). In our previous study, the stimulatory effects of FS from a red seabream (P. major
) on the adhesion and proliferation of, and protein production by CHO cells were investigated (Fujiwara et al. 2007
). It was concluded that the use of some FS lots resulted in a cell density of only 75% that in the FCS-containing medium (FCS medium), whereas the use of other FS lots resulted in very low cell density (data not shown). Thus, cell culture using FS-containing medium (FS medium) needs to be optimized to become as suitable as that with FCS medium.
Animal serum generally contains complement system proteins. A complement system cascade may be activated during cell culture and lead to the death of cultured cells. Thus, FCS is often heat treated at 56 °C for 30 min to inactivate the complement system before addition to the cell culture. Therefore, heat-treated FS was expected to be effective in improving cell growth.
FCS is generally added to a culture medium at a concentration of 10%. However, to date, the concentration of FS has not yet been optimized to obtain maximum cell growth. Therefore, optimization of the concentration of FS in a culture medium may also be effective in improving cell growth.
In this study, the effects of heat treatment and concentration of FS on cell growth and hGM-CSF production in an adhesion culture of recombinant CHO cells were investigated and compared with those of FCS.