Effects of heat treatment and concentration of fish serum on cell growth in adhesion culture of Chinese hamster ovary cells

Abstract

The effects of heat treatment and concentration of fish serum (FS) on cell growth and human granulocyte-macrophage colony-stimulating factor (hGM-CSF) production in an adhesion culture of recombinant Chinese hamster ovary (CHO) cells, DR1000L4N, were investigated. The addition of heat treated FS instead of non-heat-treated FS improved cell growth in terms of cell density, which reached 60% that in 10% fetal calf serum (FCS)-containing medium (FCS medium). A decrease in FS concentration from 10 to 1.25% markedly increased cell density, which was 79% that in 10% FCS medium. The combination of heat treatment at 56 degrees C and the addition of FS at a low concentration (1.25%) showed an additive effect on cell growth and resulted in the same cell density as that in 10% FCS medium, whereas the hGM-CSF concentration in the culture using FS-containing medium (FS medium) was approximately 50% that in 10% FCS medium. The total lipid concentration in FS was more than three fold that in FCS. The effect of decreasing FS concentration on cell growth may be due to the low lipid concentration in FS medium, because addition of the lipids extracted from FS to 10% FCS and 1.25% FS media markedly decreased cell density. Consequently, the addition of heat-treated FS at low concentrations to medium may be useful for the growth of CHO cells without FCS.

Fig. 1

Effect of heat treatment of fish serum on adhesive growth of CHO cells. CHO DR1000L4N cells were cultivated in adhesion for 120 h in media containing 10% FCS, heat-treated 10% FS (Seriola quinqueradiata) (46, 51, 56, or 61 °C), non-heat-treated 10% FS (NT), or no serum (0%) after initial adhesion for 24 h in 10% FCS medium. Mean ± SD (n = 3)

Fig. 2

Effect of concentration of fish serum on adhesive growth of CHO cells. CHO DR1000L4N cells were cultivated in adhesion for 120 h in media containing 10% FCS, FS (Seriola quinqueradiata) (1.25, 2.5, 5, 10, or 20%) or no serum (0%) after initial adhesion for 24 h in 10% FCS medium. Mean ± SD (n = 3)

Fig. 3

Effects of heat treatment and concentration of FS and FCS on growth of CHO cells. CHO DR1000L4N cells were cultivated in adhesion for 120 h in medium containing FS (Seriola quinqueradiata) (circles) or FCS (squares) with (closed symbols) or without (open symbols) heat treatment (56 °C) at various concentrations after initial adhesion for 24 h in 10% FCS medium. Mean ± SD (n = 3)

Fig. 4

Effects of heat treatment and concentration of fish serum from red seabream (Pagrus major) on growth of CHO cells. CHO DR1000L4N cells were cultivated for 120 h in medium containing FS (Pagrus major) with (closed bars) or without (open bars) heat treatment (56 °C) at various concentrations after initial adhesion for 24 h in 10% FCS medium. Mean ± SD (n = 3)

Fig. 5

Influences of lipids extracted from fish serum on growth of CHO cells. CHO DR1000L4N cells were cultivated in adhesion for 120 h in media containing 10% FCS supplemented with lipids extracted from FS (Seriola quinqueradiata) (open circles), heat-treated 1.25% FS (Seriola quinqueradiata) supplemented with lipids (closed squares), and no serum supplemented with lipids (open triangles), after initial adhesion for 24 h in 10% FCS medium. For comparison, the same cultivations were performed in media with various concentrations of lipids originally contained in heat-treated FS (Seriola quinqueradiata) (open squares). Data represent the means of triplicate cultures

Fig. 6

Time course of cell density and hGM-CSF concentration in adhesion culture of CHO cells using fish serum. a Cell density, b hGM-CSF concentration. CHO DR1000L4N cells were cultivated in adhesion for 168 h in media containing 10% FCS (open circles), 1.25% fish serum (Seriola quinqueradiata) with (closed squares) or without (open squares) heat treatment, and no serum (0%) (open triangles) after initial adhesion for 24 h in 10% FCS medium. Data represent the means of triplicate cultures