A parametric study of mechanoporation through microfluidic design to modulate shear, compressive, and adhesion forces...

Efficient and reproducible intracellular delivery is critical for manufacturing next generation cell therapies. Mechanoporation employs mechanical forces, including shear loading, adhesion, and compressive strain, to transiently permeabilize cell membranes and enable cargo transport. However, the influence of microsecond-scale unsteady forces and the origins of variability in delivery and viability remain insufficiently characterized. Here, we performed a parametric investigation of microfluidic mechanoporation using parallelized channel designs of varied widths to systematically modulate pre-compression shear loading and strain rates under constant volumetric flow. Narrow channels were found to promote a more uniform pre-constriction loading and compressive dynamics, leading to improved reproducibility of delivery outcomes. High-speed video analysis revealed greater cell focusing and computational fluid dynamics (CFD) confirmed higher pre-constriction shear loading rates and higher asymmetric biaxial forces prior to ridges, yielding a substantial improvement in delivery efficiency in both adherent B16F10 melanoma cells and suspended T-cells. Modulating cell-surface adhesion by adjusting surface chemistry showed that adhesive coatings slightly increase delivery efficiency at the expense of viability. Changing cell stiffness with pharmacological softening caused a decline in delivery efficiency. These trends indicate that mechanoporation outcomes are governed more strongly by the kinetics of loading dictated by fluid-driven acceleration and strain rate rather than by absolute strain or adhesion magnitude. Principal component and multivariate analyses identified two significant predictors of delivery and viability: strain rate and Basset-Boussinesq history (BBH) forces. Both predictors were consistently elevated in narrow multichannel architectures that showed higher delivery and lower viability. Together, these findings demonstrate that narrow channel designs establish a geometry-driven acceleration regime characterized by elevated strain rates and BBH forces that enhances delivery efficiency while imposing a viability tradeoff.