title: Effects of conditioned medium factors and passage number on Sf9 cell physiology and productivity.
abstract: 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.
authors.affiliation: Karo Bio AB, Huddinge, SE-141 57 Huddinge, Sweden.
authors.forename: Karin
authors.initials: K
authors.lastname: Calles
authors.forename: Ingrid
authors.initials: I
authors.lastname: Svensson
authors.forename: Eva
authors.initials: E
authors.lastname: Lindskog
authors.forename: Lena
authors.initials: L
authors.lastname: Häggström
mesh_terms: 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
chemical_list: D017077:Culture Media, Conditioned