Suspension-Adapted HEK293FT Cells Enable High-Density Transfection for Efficient Lentiviral Vector Production in CAR-T Therapy

Shevtsov, Alexandr; Kydyrbayeva, Aitolkyn; Nigmatulla, Gaziza; Keyer, Viktoriya; Kulatay, Tolganay; Zauatbayeva, Gulzat; Ingirbay, Bakytkali; Zhumabekova, Maral; Zharlyganova, Dinara; Shustov, Alexandr V.

doi:10.3390/bioengineering13080856

Open AccessArticle

Suspension-Adapted HEK293FT Cells Enable High-Density Transfection for Efficient Lentiviral Vector Production in CAR-T Therapy

by

Alexandr Shevtsov

,

Aitolkyn Kydyrbayeva

,

Gaziza Nigmatulla

,

Viktoriya Keyer

,

Tolganay Kulatay

,

Gulzat Zauatbayeva

,

Bakytkali Ingirbay

,

Maral Zhumabekova

,

Dinara Zharlyganova

and

Alexandr V. Shustov

^*

National Center for Biotechnology, Korgalzhin hwy 13/5, 010000 Astana, Kazakhstan

^*

Author to whom correspondence should be addressed.

Bioengineering 2026, 13(8), 856; https://doi.org/10.3390/bioengineering13080856

Submission received: 9 June 2026 / Revised: 18 July 2026 / Accepted: 22 July 2026 / Published: 24 July 2026

(This article belongs to the Section Cellular and Molecular Bioengineering)

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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.

Keywords: lentiviral vectors; HEK293FT cells; suspension adaptation; high-density transfection; PEI transfection; serum-free medium; CAR-T therapy; transient transfection; cell line development; viral vector manufacturing

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MDPI and ACS Style

Shevtsov, A.; Kydyrbayeva, A.; Nigmatulla, G.; Keyer, V.; Kulatay, T.; Zauatbayeva, G.; Ingirbay, B.; Zhumabekova, M.; Zharlyganova, D.; Shustov, A.V. Suspension-Adapted HEK293FT Cells Enable High-Density Transfection for Efficient Lentiviral Vector Production in CAR-T Therapy. Bioengineering 2026, 13, 856. https://doi.org/10.3390/bioengineering13080856

AMA Style

Shevtsov A, Kydyrbayeva A, Nigmatulla G, Keyer V, Kulatay T, Zauatbayeva G, Ingirbay B, Zhumabekova M, Zharlyganova D, Shustov AV. Suspension-Adapted HEK293FT Cells Enable High-Density Transfection for Efficient Lentiviral Vector Production in CAR-T Therapy. Bioengineering. 2026; 13(8):856. https://doi.org/10.3390/bioengineering13080856

Chicago/Turabian Style

Shevtsov, Alexandr, Aitolkyn Kydyrbayeva, Gaziza Nigmatulla, Viktoriya Keyer, Tolganay Kulatay, Gulzat Zauatbayeva, Bakytkali Ingirbay, Maral Zhumabekova, Dinara Zharlyganova, and Alexandr V. Shustov. 2026. "Suspension-Adapted HEK293FT Cells Enable High-Density Transfection for Efficient Lentiviral Vector Production in CAR-T Therapy" Bioengineering 13, no. 8: 856. https://doi.org/10.3390/bioengineering13080856

APA Style

Shevtsov, A., Kydyrbayeva, A., Nigmatulla, G., Keyer, V., Kulatay, T., Zauatbayeva, G., Ingirbay, B., Zhumabekova, M., Zharlyganova, D., & Shustov, A. V. (2026). Suspension-Adapted HEK293FT Cells Enable High-Density Transfection for Efficient Lentiviral Vector Production in CAR-T Therapy. Bioengineering, 13(8), 856. https://doi.org/10.3390/bioengineering13080856

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Suspension-Adapted HEK293FT Cells Enable High-Density Transfection for Efficient Lentiviral Vector Production in CAR-T Therapy

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