Effects of conditioned medium factors and passage number on Sf9 cell physiology and productivity.
The effects of conditioned medium (CM) and passage number on Spodoptera frugiperda Sf9 cell physiology and productivity have been studied. Low passage (LP) cells at passages 20-45 were compared to high passage (HP) cells at passages >100. Addition of 20% CM or 10 kDa filtrated CM to LP cells promoted growth. LP cells passed a switch in growth kinetics, characterized by a shorter lag phase and a higher growth rate, after 30-40 passages. After this point, CM lost its stimulating effect on proliferation. HP cells displayed a still shorter lag phase and reached the maximum cell density 24-48 earlier than LP cells. HP cells also exhibited higher specific productivity of recombinant protein compared to LP cells, when infected with baculovirus during the initial 48 h of culture. The specific productivity of LP cells was decreased by 30-50% by addition of 20% CM or 10 kDa filtrated CM, whereas addition of CM to cells having passed the switch in growth kinetics had no negative effect on productivity. Cell cycle analysis showed that a large proportion of HP cells, >60%, was transiently arrested in G2/M after inoculation. In LP cultures this proportion was lower, 40-45%, and addition of CM decreased the arrested population further. This correlated to the cell size, the HP cells being the largest: HP cells > LP > LP + 20% CM > LP + 20% 10 kDa filtrated CM. Since the degree of synchronization in G2/M correlated to the productivity, yeastolate limitation was used to achieve 85% G2/M synchronized cells. In this culture the specific productivity was maintained during a prolonged production phase and a 69% higher volumetric yield was obtained. The results suggest that a decreasing degree of synchronization during the course of culture partly explains the cell-density-dependent drop in productivity in Sf9 cells.
Calles
Karin
K
Karo Bio AB, Huddinge, SE-141 57 Huddinge, Sweden.
Svensson
Ingrid
I
Lindskog
Eva
E
Häggström
Lena
L
D000818:Animals; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D016922:Cellular Senescence / Q000187:drug effects* / Q000502:physiology*; D017077:Culture Media, Conditioned / Q000737:chemistry* / Q000494:pharmacology*; D008970:Molecular Weight; D018411:Spodoptera / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism
D017077:Culture Media, Conditioned