Citation
- Authors: Vaz TA. et al.
- Year: 2023
- Journal: Biotechnol J
- Applications: in vitro / DNA / PEIpro
- Cell type: HEK-293T
Description: Human embryonic kidney Fibroblast
Known as: HEK293T, 293T
Abstract
The use of lentiviral vectors (LV) in gene therapy has been growing in recent years.
To meet the increasing clinical demand, LV production platforms will benefit from
improved productivity and scalability to enable cost-effective manufacture of LVbased therapies. Here we report the adaptation of 293T cells to serum-free suspension
cultures and the improvement of LV yields through transfection parameters optimization, process intensification and medium supplementation with nutrient boosters.
Cells were sequentially adapted to different serum-free culture media, transfection
parameters were optimized and the two best-performing conditions were selected to
explore process intensification by increasing cell density at the time of transfection.
LV production at higher cell densities increased volumetric titers up to 12-fold and
lipid supplementation was the most efficient metabolic optimization strategy further
enhancing LV productivity by 3-fold. Furthermore, cell concentration was identified
and validated as an important source of transfection variability impairing cellular
uptake of DNA polyplexes, impacting transfection efficiency and reducing LV titers
down to 6-fold. This work contributes to improving LV-based gene therapy by establishing new scalable manufacturing platforms and providing key metabolic insights,
unveiling important bioreaction parameters to improve vector yields.