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.

Protein production using the baculovirus‐insect cell expression system

“…A total replacement of the culture medium pre‐ or postinfection in a large scale operation is impractical and adversely impacts the economics of protein production . A partial replacement of the medium allows the retention of the autocrine growth‐promoting factors and is less expensive . Alternatively, a part of the spent medium may be reused to provide autocrine growth factors.…”

Effects of Fetal Bovine Serum deprivation in cell cultures on the production of Anticarsia gemmatalis Multinucleopolyhedrovirus

“…However, there is little information about cycle synchronization in insect cell lines. It was described that Spodoptera frugiperda (Sf9) cells are affected when treated with conditioned medium; and it was observed that they are arrested in G 2 /M cell stage by AcMNPV infection generating implications in cell proliferation and recombinant protein production [20-24]. The same authors were also evaluated the effects of synchronization in Trichoplusia ni (TN-368) insect cell using similar tests [20-24].…”

“…It was described that Spodoptera frugiperda (Sf9) cells are affected when treated with conditioned medium; and it was observed that they are arrested in G 2 /M cell stage by AcMNPV infection generating implications in cell proliferation and recombinant protein production [20-24]. The same authors were also evaluated the effects of synchronization in Trichoplusia ni (TN-368) insect cell using similar tests [20-24]. On the other hand, there are standardized protocols for synchronization in mammalian cells, such as those based on serum deprivation, contact inhibition or chemical treatments [23,25-28].…”

Highly passage of Spodoptera litura cell line causes its permissiveness to baculovirus infection

“…The characteristics of cell lines possibly alter when cell lines are at high passage number because of the environmental selection (Briske-Anderson et al 1997;Calles et al 2006). A general experience is that long-term passaging increases the growth rate of cells (Peiser et al 1993;Park et al 2004).…”