The platform based on suspension-adapted HEK293FT-DS cells enables high-density transfection and provides a cost-effective, scalable alternative to commercial LV production systems, particularly suited for academic CAR-T cell manufacturing.
Abstract
Lentiviral vector (LV) production for CAR-T therapy remains challenging due to the limited scalability of adherent HEK293 cell cultures. To address this, we adapted HEK293FT cells directly to serum-free FreeStyle 293 Expression Medium, generating a novel suspension cell line, HEK293FT-DS. We characterized growth kinetics, stability over 35 passages, and transient transfection parameters (PEI:DNA ratio, cell density). The suspension-adapted cells grew with >93% viability and a specific growth rate of 0.54 day−1, and maintained stable viable cell density (1.51 ± 0.03 × 106 cells/mL) over 35 passages. The optimal PEI:DNA ratio was 2.5:1, and increasing the transfection cell density to 16 × 106 cells/mL boosted functional titers to approximately 9 × 106 TU/mL in clarified supernatant. Scaled production (1 L culture supernatant per batch) yielded up to 5 × 109 TU. The resulting vectors efficiently transduced primary human T cells (57% CAR-positive cells) in a CliniMACS Prodigy-based process. The platform based on suspension-adapted HEK293FT-DS cells enables high-density transfection and provides a cost-effective, scalable alternative to commercial LV production systems, particularly suited for academic CAR-T cell manufacturing.
Purpose Extracellular vesicles (EVs) are increasingly explored as natural vehicles for drug delivery and gene therapy approaches. However, reproducible yield and scalability of EV production still pose major challenges in the clinical translation of EV-based therapies. In this study, we sought to quantify and characterize EVs released by suspension-cultured HEK293 cells (Expi293F cells) grown in shaker flasks or small-scale bioreactors, to investigate how the culturing environment affects EV production yield. Methods Expi293F cells were cultivated (N=3) in either shaker flasks or a bioreactor system, and total cell density, viability, and size were monitored. Supernatants were drawn daily post-cell seeding and were analyzed for EV quantity, size, morphology, and CD63 expression. Results No significant differences were observed in terms of total cell density, viability, and cell size between both cultivation settings. However, cultivation of Expi293F cells in the bioreactor environment significantly increased EV yield by 3-fold compared to shaker flask cultivation (p < 0.01). Other parameters such as average nanoparticle size, EV morphology, and CD63 expression remained comparable between both cultivation methods. Conclusion These results demonstrate that Expi293F-derived EV yield can be increased by culturing cells in a scalable bioreactor system. These findings pave the way towards the production of therapeutic-based EVs in a scalable and reproducible manner suitable for future (pre-)clinical applications.
W. Woud, E. Dilla, N. Dits et al.· bioRxiv· 0 citations
Stable lentiviral vector producer cell lines represent a promising platform for scalable and cost-efficient vector manufacturing, yet their productivity is often limited by intrinsic host-cell constraints. In this study, we aimed to identify cellular factors restricting LVV production in stable doxycycline-inducible GPRTG producer cell lines and to evaluate whether targeted host-cell engineering can improve vector yield. Comparative transcriptomic analysis of high- and low-producing clones revealed distinct differences in cellular pathways related to transcription, translation, energy metabolism, lipid homeostasis, stress response, and chromatin regulation. Based on differential gene expression, candidate genes were modified by CRISPR/Cas9-mediated knockout or overexpression and functionally analyzed in a low-producing clone. Single-gene KO screening identified H1-2, ADAMTS1, INSIG1, GADD45B, and HSPA1B to increase cell-specific LVV productivity up to 2.6-fold, with H1-2 showing the strongest effect. In addition, combinatorial disruption further improved productivity. In contrast, overexpression of selected candidates did not enhance LVV production. Overall, our results demonstrate that transcriptomics-guided host-cell engineering is an effective strategy to identify and relieve intrinsic bottlenecks in stable LVV producer cell lines.
Jona Röscheise, Lena-Marie Eberle, Holger Laux et al.· Biotechnology and Bioenginee...· 0 citations
The growing demand for high-quality plasmid DNA (pDNA) in cell and gene therapy, including chimeric antigen receptor T-cell therapy (CAR-T) manufacturing, is constrained by the limited availability of transfection-grade plasmids. This study describes a practical, scalable, and cost-effective acidic phenol extraction method based on a modified alkaline lysis protocol. This approach efficiently removes endotoxins and residual genomic DNA without requiring chromatography or ultracentrifugation. When benchmarked against CsCl density gradient ultracentrifugation and a commercial midiprep kit, the acidic phenol method delivered comparable plasmid purity and functional performance while providing superior scalability, yielding 5–10 mg of pDNA per 500 mL culture. Notably, residual endotoxin levels were negligible for downstream applications such as transfection and lentiviral vector packaging. Lentiviral vectors produced with these plasmids reached titers exceeding 5 × 106 TU/mL, with transduction efficiencies statistically indistinguishable from those obtained with CsCl-purified DNA. The acidic phenol extraction method is a robust, scalable, and cost-effective alternative to industry-standard methods, matching them in yield and purity while being readily scalable, making it a practical tool for both academic research and preparative laboratory production.
Gaziza Nigmatulla, Aitolkyn Kydyrbayeva, T. Kulatay et al.· Bioengineering· 0 citations
An industrial-grade platform based on monoclonal producer cell lines that enables the continuous and scalable generation of engineered virus-like particles (eVLPs) co-packaging Cas9–gRNA ribonucleoproteins (RNPs) and provides a GMP-compliant and broadly adaptable strategy for the streamlined manufacturing of next-generation autologous and allogeneic gene-edited CAR-T/NK therapies.
Wei Lin, Jiaru Shi, Hanyi Chen et al.· Frontiers in Immunology· 0 citations
This proof-of-concept study underlines the utility of the Env variant BaEV-TR for the establishment of stable pseudotype VPCLs and the utilization of HT1080 cells as an alternative to frequently used 293T host cells.
Angelina Hallik, Mona Pißarreck, Mats Jürgen Grummel et al.· Frontiers in Molecular Medic...· 0 citations
TcBuster-M-mediated cell engineering achieved high CD19-CAR expression in both T and NK cells while preserving cell viability and growth, and demonstrated potent, target-specific cytotoxicity and favorable cytokine secretion.
Jessica K. Fiege, R. Haugen, Ellie A. Mews et al.· Journal of Immunology· 0 citations
