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Abstract Autologous cell therapy depends on T lymphocyte expansion efficiency and is hindered by suboptimal interactions between T cell receptors (TCR) and peptide‐MHC molecules. Various artificial antigen presenting cell systems that enhance these interactions are often labor‐intensive, fabrication costly, highly variable, and potentially unscalable toward clinical setting. Here, 3D centrifugation‐enabled priming of T cell immune‐synapse junctions is performed to generate tight T cell–Dynabead aggregates at a rate 200‐fold faster than that of conventional 24‐h bulk shaking. Furthermore, by forming T cell–Dynabead aggregates in the starting culture, two‐ to sixfold greater T cell expansion is achieved over conventional T cell expansion for cancer patient‐derived primary T cells while limiting over‐activation. Creating 3D T cell–Dynabead aggregates as the “booster” material enables highly efficient polyclonal T cell expansion without the need for complex surface modification of artificial antigen‐presenting cells (APCs). This method can be modularly adapted to existing T cell expansion processes for various applications, including adoptive cell therapies (ACTs). 

Abstract A high‐throughput non‐viral intracellular delivery platform is introduced for the transfection of large cargos with dosage‐control. This platform, termed Acoustic‐Electric Shear Orbiting Poration (AESOP), optimizes the delivery of intended cargo sizes with poration of the cell membranes via mechanical shear followed by the modulated expansion of these nanopores via electric field. Furthermore, AESOP utilizes acoustic microstreaming vortices wherein up to millions of cells are trapped and mixed uniformly with exogenous cargos, enabling the delivery of cargos into cells with targeted dosages. Intracellular delivery of a wide range of molecule sizes (<1 kDa to 2 MDa) with high efficiency (>90%), cell viability (>80%), and uniform dosages (<60% coefficient of variation (CV)) simultaneously into 1 million cells min⁻¹per single chip is demonstrated. AESOP is successfully applied to two gene editing applications that require the delivery of large plasmids: i) enhanced green fluorescent protein (eGFP) plasmid (6.1 kbp) transfection, and ii) clustered regularly interspaced short palindromic repeats (CRISPR)‐Cas9‐mediated gene knockout using a 9.3 kbp plasmid DNA encoding Cas9 protein and single guide RNA (sgRNA). Compared to alternative platforms, this platform offers dosage‐controlled intracellular delivery of large plasmids simultaneously to large populations of cells while maintaining cell viability at comparable delivery efficiencies.