3.1. Cell line generation and FACS-aided enrichment of stable cell pools
We used suspension-adapted HEK293 cells originating from an animal component-free GMP cell bank (from NRC, Canada) to express VLPs comprising the structural proteins prM and E of the yellow fever virus. The HEK293 cell line was chosen due to its ability to produce VLPs of different viruses and to grow well in continuous suspension cultivation processes (Fontana et al., 2015; Fuenmayor et al., 2018; Alvim et al., 2019; Venereo-Sanchez et al., 2017).
The prM-E gene construct was inserted into a plasmid containing a selection marker cassette based on the neomycin phosphotransferase gene in order to allow selection of stable transfectants by including G418 antibiotics in the medium. Selection pressure by G418 addition was started on day 2 post-transfection, and cell culture showed low viability and enhanced cell death for the following 2 weeks, due to death of cells where the heterologous DNA had not been integrated into the cell genome. After that period, cell viability recovered, indicating that stable producer cells had been successfully selected. A research cell bank of the original cell pool was cryopreserved 4 weeks after transfection, and then FACS-aided cell sorts were carried out to select subpopulations of high-producer cells.
Our focus is on VLPs that are secreted to the extracellular medium, but since secreted VLPs can be transiently found on the cell membrane on their way to the extracellular medium, it is possible to stain the cells and sort for high producers. The first round of cell sorting was carried out 40 days after transfection by collecting 0.3% of cells presenting the highest levels of 4G2-PE fluorescence. These cells were named cell pool 1 and a cell bank was cryopreserved. The second round of cell enrichment was carried out starting from cell pool 1 by again collecting the cells with highest fluorescent intensity and then expanding them. This double sorted cell pool was named cell pool 2 and a cell bank was cryopreserved. Passage history of the original stable cell pool and of cell pools 1 and 2 is shown in Figure 1A.
With the purpose of evaluating VLP production after each round of cell sorting, an immunoblot assay was performed (Figure 1B). The results show that the concentration of VLPs in the supernatant increased after each round of cell sorting, confirming that cell enrichment was successful and that FACS is a powerful high-throughput technique also for selection of cells secreting a recombinant product (Sutermaster & Darling, 2019).