Genetically Stable and Scalable Nanoengineering of Human Primary T Cells via Cell Mechanoporation

Creators: Hur, Jeongsoo; Kim, Hyelee; Kim, Uijin; Kim, Gi-Beom; Kim, Jinho; Joo, Byeongju; Cho, Duck; Lee, Dong-Sung; Chung, Aram J.

Abstract

Effective tumor regression has been observed with chimeric antigen receptor (CAR) T cells; however, the development of an affordable, safe, and effective CAR-T cell treatment remains a challenge. One of the major obstacles is that the suboptimal genetic modification of T cells reduces their yield and antitumor activity, necessitating the development of a next-generation T cell engineering approach. In this study, we developed a nonviral T cell nanoengineering system that allows highly efficient delivery of diverse functional nanomaterials into primary human T cells in a genetically stable and scalable manner. Our platform leverages the unique cell deformation and restoration process induced by the intrinsic inertial flow in a microchannel to create nanopores in the cellular membrane for macromolecule internalization, leading to effective transfection with high scalability and viability. The proposed approach demonstrates considerable potential as a practical alternative technique for improving the current CAR-T cell manufacturing process.

Copyright and License

Acknowledgement

This study was supported by the Samsung Research Funding and Incubation Center for Future Technology (Grant SRFC-IT1802-03), the National Research Foundation of Korea (NRF) funded by the Korean government (MSIT; Ministry of Science and ICT; 2021R1A2C2006224, RS-2023-00242443, and RS-2023-00218543), the Technological Innovation R&D Program (RS-2023-00262758) funded by the Ministry of SMEs and Startups (MSS, Korea), and the Materials/Parts Technology Development Program (20020278) funded by the Ministry of Trade, Industry & Energy (MOTIE, Korea) (all to A.J.C.) and Institute of Information & Communications Technology Planning & Evaluation (IITP) of Korea Grant IITP2022-0-006240101005 and National Research Foundation (NRF) of Korea Grants NRF2021R1C1C1006798 and NRF2022M3H9A108101112 to D.-S.L.

Contributions

H.K. and U.K. contributed equally to this work. J.H. and A.J.C. conceived the research, and all authors contributed to the data analysis and experimental design. J.H., H.K., G.-B.K., and B.J. conducted the experiments and performed fluorescence imaging. U.K. carried out the RNA-seq analysis. J.K. isolated and prepared human primary T cells. J.H. and A.J.C. wrote the manuscript, and all authors participated in data discussion and provided feedback. A.J.C. and D.-S.L. supervised the work.

Data Availability

Materials and methods; optimization of the microfluidic channel design; additional fluorescence histogram, MFI fold change, and viability data; and a comparison of the transfection yields of the hydroporator and conventional methods (PDF)
T cell deformation via the hydroporator (Movie S1) (MP4)

Conflict of Interest

The authors declare the following competing financial interest(s): G.-B.K., B.J., and A.J.C. have a financial interest in MxT Biotech, which is commercializing the hydroporator.

Files

nl3c01720_si_001.pdf

Files (1.3 MB)

Name	Size	Download all
nl3c01720_si_002.mp4 md5:3b65e3c198bca91ce49c504ddc0d6fcb	831.8 kB	Download
nl3c01720_si_001.pdf md5:8a61eedf71a04293402ce5cdcaf48d38	450.5 kB	Preview Download

	All versions	This version
Views	1	1
Downloads	0	0
Data volume	0 Bytes	0 Bytes