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).