High-definition electroporation: Precise and efficient transfection on a microelectrode array

Intracellular delivery is critical for a plethora of biomedical applications, including mRNA transfection and gene editing. High transfection efficiency and low cytotoxicity, however, are often beyond the capabilities of bulk techniques and synonymous with extensive empirical optimization. Moreover, bulk techniques are not amenable to large screening applications. Here, we propose an expeditious workflow for achieving optimal electroporation-based intracellular delivery. Using the multiplexing ability of a high-definition microelectrode array (MEA) chip, we performed a sequence of carefully designed experiments, multiple linear regression modelling and validation to obtain optimal conditions for on-chip electroporation of primary fibroblasts. Five electric pulse parameters were varied to generate 32 different electroporation conditions. The effect of the parameters on cytotoxicity and intracellular delivery could be evaluated with just two experiments. Most successful electroporation conditions resulted in no cell death, highlighting the low cytotoxicity of on-chip electroporation. The resulting delivery models were then used to achieve dosage-controlled delivery of small molecules, delivery of Cas9-GFP single-guide RNA complexes and transfection with an mCherry-encoding mRNA, resulting in previously unreported high-efficiency, single-cell transfection on MEAs: cells expressed mCherry on 81% of the actuated electrodes, underscoring the vast potential of CMOS MEA technology for the transfection of primary cells. [Display omitted] •We use a CMOS microelectrode array chip for single-cell electroporation (EP) of primary fibroblasts.•32 EP conditions were designed by varying 5 pulse parameters, following Design of Experiment principles.•The EP conditions were tested simultaneously in highly parallelized screening experiments.•The EP parameters' effect on intracellular delivery and cell death was modelled through linear regression.•The model enabled dosage-controlled delivery of dextran, delivery of Cas9, and mRNA transfection with above 80% efficiency.