Optimised protocols to generate high titre lentiviral vectors using a novel transfection agent enabling extended HEK293T culture following transient transfection and suspension culture

In this study, we sought to improve lentiviral titre over time by testing two alternative commercially available transfection reagents Fugene® 6 and Genejuice® with the commonly used polycation, polyethyleneimine. Our aim was to identify less cytotoxic transfection reagents that could be used to generate LV particles at high titre past the often used 72 h period when vector is usually collected before producer cell death is caused due to post transfection cytotoxicity. We show that LV could be produced in extended culture using Genejuice® and even by transfected cells in glass flasks in suspension. Because this delivery agent is less toxic to 293 T producer cells, following optimisation of transfection we found that LV can be harvested for more than 10 days at high titre. Using our protocol, titres of 109 TU/ml and 108 TU/ml were routinely reached via traditional monolayer conditions or suspension cultures, respectively. We propose, this simple change in vector production enables large volumes of high titre vector to be produced, cost effectively for non-clinical in vivo and in vitro applications or for more stringent downstream clinical grade vector purification.PMID:38218417 | DOI:10.1016/j.jviromet.2024.114884
Source: Journal of Virological Methods - Category: Virology Authors: Source Type: research