Research ArticleReal-time imaging reveals unique heterogeneous population features in insect cell cultures

- Live cell imaging revealed heterogeneous populations in Sf9 insect cell cultures.
- Live cell imaging revealed the presence of motile elongated cells that were resistant to baculovirus infection.
- The migration speed of motile elongated cells was measured, and was comparable to that of melanoma cells.
- Low or high dissolved oxygen induced the appearance of motile cells, suggesting that stress triggers their appearance.
- A dual recombinant baculovirus with fluorescent proteins under promoters with different temporalities in the infection cycle allowed the identification of conditions that altered the infection cycle.

Heterogeneity of cellular populations has been frequently observed. We used live cell imaging to follow Sf9 insect cells before and after infection with baculovirus, to understand population dynamics. It was possible to identify in real time cells with distinctive phenotypes. Mobile cells with an elongated bipolar shape were observed. They extended pseudopods and actively moved about the culture surface. The presence of actively moving elongated cells increased when cultures were subjected to oxygen limiting or excessive conditions, suggesting that stress triggered differentiation of cells to the mobile phenotype. A dual reporter baculovirus (DRBac), coding for two fluorescent proteins under promoters with different temporality, was designed to follow sequential phenomena through infection. Oxygen limitation reduced the number of cells that expressed the reporter proteins, possibly because it reduced the efficiency of baculovirus infection. Elongated cells did not show signs of infection. To our knowledge, this is the first time that actively moving cells are observed in real time in Sf9 cultures, which had distinctive responses towards infection. Anoxia was identified as a factor that modulates baculovirus infection. Results open a new approach for understanding the insect-cell baculovirus system. Particular cellular phenotypes with unique traits can be isolated for specific applications.