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

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.

Highly Efficient Transfection of Human Primary T Lymphocytes Using Droplet-Enabled Mechanoporation.

Whole-cell-based therapy has been extensively used as an effective disease treatment approach, and it has rapidly changed the therapeutic paradigm. To fully accommodate this shift, advances in genome modification and cell reprogramming methodologies are critical. Traditionally, molecular tools such as viral and polymer nanocarriers and electroporation have been the norm for internalizing external biomolecules into cells for cellular engineering. However, these approaches are not fully satisfactory considering their cytotoxicity, high cost, low scalability, and/or inconsistent and ineffective delivery and transfection. To address these challenges, we present an approach that leverages droplet microfluidics with cell mechanoporation, bringing intracellular delivery to the next level. In our approach, cells and external cargos such as mRNAs and plasmid DNAs are coencapsulated into droplets, and as they pass through a series of narrow constrictions, the cell membrane is mechanically permeabilized where the cargos in the vicinity are internalized via convective solution exchange enhanced by recirculation flows developed in the droplets. Using this principle, we demonstrated a high level of functional macromolecule delivery into various immune cells, including human primary T cells. By utilizing droplets, the cargo consumption was drastically reduced, and near-zero clogging was realized. Furthermore, high scalability without sacrificing cell viability and superior delivery over state-of-the-art methods and benchtop techniques were demonstrated. Notably, the droplet-based intracellular delivery strategy presented here can be further applied to other mechanoporation microfluidic techniques, highlighting its potential for cellular engineering and cell-based therapies.