Lentiviral Vector Production in HEK293T Cells

HEK293T cells (ATCC^® CRL-3216™) obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) were adapted to grow in suspension as described in [70 ]. The cells were cultivated in BalanCD^® HEK293 medium (FUJIFILM IrvineScientific, Santa Ana, CA, USA) supplemented with 4 mM GlutaMAX™ (Thermo Fisher Scientific™, Waltham, MA, USA) and maintained at 37 °C in an incubator with a humidified atmosphere of 8% CO₂ in the air. Cell concentration and viability were assessed by the trypan blue exclusion method. LVs were produced by transient transfection using pALD-Lenti System (Aldevron^®, Fargo, ND, USA) including the pALD-VSV-G-K, pALD-GagPol-K, pALD-Rev-K, and pALD-Lenti-EGFP-K plasmids. Briefly, the cells were transfected at 2.5 × 10⁶ cells/mL in a shake flask using linear 25 kDa polyethyleneimine, PEIpro^® Transfection Reagent (Polyplus-transfection^®, Illkirch-Graffenstaden, France) at a mass ratio of 1:3 (DNA:PEI) and 1 µg of total DNA per 1 × 10⁶ cells. At 48 h-post-transfection, VSVG-pseudotyped LVs (VSVG-LVs) were harvested at a cell density ranging from 4–6 × 10⁶ cells/mL.