Contents lists available at ScienceDirect Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec Research Article Real-time imaging reveals unique heterogeneous population features in insect cell cultures David Hidalgo, Enrique Paz, Laura A. Palomares ⁎ , Octavio T. Ramírez ⁎ Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Ave. Universidad 2001, Col. Chamilpa, Cuernavaca, Morelos, 62210, Mexico ARTICLE INFO Keywords: Baculovirus Insect cells Live cell imaging Population heterogeneity Single cell monitoring Phagocytosis ABSTRACT 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 ob- served. 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. 1. Introduction Animal cell lines have been shown to be heterogeneous, even after cloning (Altschuler and Wu, 2010). Traditional bioprocess monitoring yield parameters with values that represent the average behavior of a population, which can have a very broad distribution, follow an ab- normal distribution or is constituted by two or more populations co- habiting in a culture (Slack et al., 2008). Therefore, the information obtained from bulk observations of a culture may not be representative of the behavior of the various populations present. To overcome this limitation, monitoring of individual cells has been reported (Fritzch et al., 2012). Using this approach, a number of cells (from tens to hundreds) can be individually monitored, and the individual popula- tions present can be identified. This approach has been utilized to characterize inherently heterogeneous populations, such as hemato- poietic cells (Lecault et al., 2011) or T cells (Varadarajan et al., 2012). The use of fluorescent reporter proteins has allowed monitoring in real- time the expression driven by specific promoters (Coutu and Schroeder, 2013; George et al., 2015), enabling the determination of the effect of a specific condition in each cell using noninvasive methods (Coutu and Schroeder, 2013). The characterization of specific populations of cells with particular phenotypes that are advantageous for a process allows either the identification of factors critical for productivity, which can be engineered into a cell line, or the possibility of isolating and/or en- riching a particular population of cells. Live cell imaging is especially relevant for the insect cell-baculo- virus expression system (IC-BES), as it is an inherently heterogeneous system. Factors introducing heterogeneities are the polyploidy of insect cell lines (Meneses-Acosta et al., 2001) and the progression of infection by the baculovirus vector, which is a stochastic process (Roldao et al., 2008). Wild-type baculovirus has a four-stage infection cycle and two phenotypes (Palomares et al., 2015). In the immediate phase (0–4h post-infection, hpi), virions are transported and viral DNA is released to the nucleus. In the early phase (4–7 hpi), baculovirus DNA replication begins. From 7 to 24 hpi (late phase), budded baculovirus, the pheno- type that spreads infection through the insect, are released and sec- ondary infection occurs. At 24 hpi and until cell death, very late pro- moters are active and occluded viruses, covered with polyhedrin, http://dx.doi.org/10.1016/j.jbiotec.2017.08.019 Received 8 June 2017; Received in revised form 11 August 2017; Accepted 18 August 2017 ⁎ Corresponding authors. E-mail addresses: laura@ibt.unam.mx (L.A. Palomares), tonatiuh@ibt.unam.mx (O.T. Ramírez). Abbreviations: DOT, dissolved oxygen tension; eGFP, enhanced green fluorescent protein; hpi, hours postinfection; IC-BES, insect cell-baculovirus expression system; MOI, multiplicity of infection; pfu, plaque forming units; Ppolh, promoter of the polh gene; Pvp39, promoter of the vp39 gene; RFP, red fluorescent protein; RFU, relative fluorescence units; w.r.s.a., with respect to saturation with air Journal of Biotechnology 259 (2017) 56–62 Available online 24 August 2017 0168-1656/ © 2017 Elsevier B.V. All rights reserved. MARK