Biomodulatory Effects of Molecular Delivery in Human T Cells Using 3D-Printed Acoustofluidic Devices

Abstract

Objective: Cell-based therapies have shown significant promise for treating many diseases, including cancer. Current cell therapy manufacturing processes primarily utilize viral transduction to insert genomic material into cells, which has limitations, including variable transduction efficiency and extended processing times. Non-viral transfection techniques are also limited by high variability or reduced molecular delivery efficiency. Novel 3D-printed acoustofluidic devices are in development to address these challenges by delivering biomolecules into cells within seconds via sonoporation.

Methods: In this study, we assessed biological parameters that influence the ultrasound-mediated delivery of fluorescent molecules (i.e., calcein and 150 kDa FITC-Dextran) to human T cells using flow cytometry and confocal imaging.

Results: Low cell plating densities (100,000 cells/mL) enhanced molecular delivery compared to higher cell plating densities (p < 0.001), even though cells were resuspended at equal concentrations for acoustofluidic processing. Additionally, cells in the S phase of the cell cycle had enhanced intracellular delivery compared to cells in the G2/M phase (p < 0.001) and G0/G1 phase (p < 0.01), while also maintaining higher viability compared to G0/G1 phase (p < 0.001). Furthermore, the calcium chelator (EGTA) decreased overall molecular delivery levels. Confocal imaging indicated that the actin cytoskeleton had important implications on plasma membrane recovery dynamics after sonoporation. In addition, confocal imaging indicates that acoustofluidic treatment can permeabilize the nuclear membrane, which could enable rapid intranuclear delivery of nucleic acids.

Conclusions: The results of this study demonstrate that a 3D-printed acoustofluidic device can enhance molecular delivery to human T cells, which may enable improved techniques for non-viral processing of cell therapies.

Keywords: Acoustofluidics; Cell therapies; Lymphocytes; Microbubbles; Molecular delivery; Sonoporation; T cells; Ultrasound.