Microfluidic-based mechanoporation for immune cell engineering: Principles, design, and applications in cell therapy

Cell therapy, particularly chimeric antigen receptor (CAR)-engineered immune cells, has emerged as a clinically validated strategy for tumor treatment, achieving remarkable outcomes in multiple malignancies. Despite this promise, a critical bottleneck in immune cell engineering lies in the efficient intracellular delivery of genetic materials into the cells. Viral vectors remain the current gold standard due to their high transfection efficiency and stable expression. However, they present significant drawbacks, most notably the risk of insertional mutagenesis caused by random genomic integration. Non-viral vectors, such as lipid- or polymer-based carriers, have been explored as alternatives, but their clinical application is limited by low transfection efficiency and poor reproducibility in primary immune cells. To address these limitations, vector-free mechanoporation strategies have emerged as promising alternatives, leveraging mechanical disruption of the plasma membrane to enable direct cytosolic delivery of exogenous cargo. This review highlights three major classes of microfluidic mechanoporation platforms: nanostructure-mediated penetration, constriction-based squeezing, and shear stress–driven hydroporation. For each strategy, the underlying mechanisms, device architectures, and delivery performance across diverse cargos and immune cell types are examined. Recent advances in device design to enhance delivery efficiency, as well as demonstrations of scalability and compatibility with clinically relevant workflows, are also discussed. The advantages, limitations, and translational potential of these platforms are evaluated, and future research directions are outlined, providing a practical guideline for future research and clinical translation. • Provides an extensive overview of microfluidic mechanoporation for cell engineering. • Compares nanostructure, constriction-based, and shear stress-driven platforms. • Discusses delivery efficiency, viability, and scalability across cell types. • Reviews integration with clinical workflows for cell and gene therapy. • Outlines challenges and future directions for next-generation mechanoporation