Investigating the impact of serum-free culture on HEK293T cells transfection efficiency for lentiviral vector production using fixed-bed minibioreactors

Details

Supervisors

Alsteens, David ; Drugmand, Jean-Christophe

Faculty

Faculté des bioingénieurs

Degree label

Master [120] : bioingénieur en chimie et bioindustries, à finalité spécialisée

Abstract

enLentiviral vectors (LV) have emerged as key tools for gene therapy applications, creating a growing demand for robust, scalable and regulatory-compliant manufacturing processes. In this context, serum-free culture media (SFM) are increasingly considered as alternatives to conventional serum-containing formulations to reduce process variability and facilitate regulatory compliance. The shift to suspension cells and stirred-tank bioreactors support SFM use but exposes cells and LV to shear stress throughout the entire process, lowering yield and quality. Fixed-bed bioreactors, such as the Scale-X from Univercells Technologies (UTEC), address these limitations by decreasing LV residence time and enabling (multiple) vector harvests or perfusion without additional cell retention devices. However, when evaluating SFM to produce LV in fixed-bed bioreactor platforms, UTEC observed a reduction in the transfection efficiency of HEK293T in some SFM conditions. As part of a collaborative project with UTEC, this work aimed to investigate the impact of HEK293T cells growth in serum-free media on this phenomenon. Because transient transfection constitutes a critical upstream step with a direct impact on subsequent vector production, understanding the origin of this performance loss has become essential to further optimise processes. To explain the reduced transfection efficiency observed following serum-free growth, some hypotheses were considered by UTEC. The first proposed that aggregate formation could limit the accessibility of PEI-DNA complexes to cells, while the second suggested that serum-free growth induces phenotypic modifications that reduce cellular permissiveness to transfection. HEK293T cells were therefore cultured under serum-containing and serum-free conditions in both TF25 and fixed-bed minibioreactors. PEI-mediated transfection using GFP-expressing lentiviral was compared with Respiratory Syncytial Virus-GFP (RSV) infection to distinguish between accessibility-related effects and changes in cellular permissiveness. Cell morphology was assessed by confocal microscopy, while transfection and infection efficiencies were quantified by flow cytometry. Some experiments indicated that these phenotypic modifications were mainly established during the growth phase. While aggregate formation may partially limit PEI-DNA complex accessibility, RSV infection experiments suggested that accessibility alone could not explain the reduced transfection performance. These results therefore support the hypothesis that serum-free growth induces phenotypic changes that reduce HEK293T permissiveness to PEI-mediated transfection. Overall, this study highlights the critical influence of upstream cell growth conditions on downstream transfection performance. By identifying phenotypic alterations acquired during serum-free growth as a major contributor to reduced transfection efficiency, this work provides valuable insights for the rational optimisation of serum-free culture strategies and supports the further development of robust serum-free processes for lentiviral vector production.