Propagation and Maintenance of H1299-E3 and VeroE6 Cell Lines

The H1299-E3 (H1299-ACE2, clone E3, H1299 originally from ATCC as CRL-5803) cell line was derived from H1299 as described in our previous work (Cele et al. 2021a (link), 2021b (link)). The H1299-E3 cells were propagated in growth medium consisting of complete Roswell Park Memorial Institute (RPMI) 1640 medium with 10 per cent fetal bovine serum (Hyclone) containing 10 mM of hydroxyethylpiperazine ethanesulfonic acid (HEPES), 1 mM sodium pyruvate, 2 mM l-glutamine and 0.1 mM nonessential amino acids (all Sigma-Aldrich). Cells were passaged every second day. For virus isolation, Vero E6 cells (originally ATCC CRL-1586, obtained from Cellonex in South Africa) were propagated in complete growth medium consisting of Dulbecco’s Modified Eagle Medium (DMEM) with 10 per cent fetal bovine serum (Hyclone) containing 10 mM of HEPES, 1 mM sodium pyruvate, 2 mM l-glutamine and 0.1 mM nonessential amino acids (all Sigma-Aldrich). Vero E6 cells were passaged every 3–4 days. The VeroE6 cells expressing TMPRSS2 and ACE2 (VeroE6-TMPRSS2), originally BEI Resources, NR-54,970 were used for virus re-expansion of the virus from the day 6 swab and fusion assay. The Vero-TMPRSS2 cell line was propagated in the same way as VeroE6 cells.