Microparticle-enabled single cell multiparameter electronic immunophenotyping for selective electroporation

Abstract

Electroporation (EP) is one of the leading non-viral intracellular delivery methods used in various applications across research and cell therapy development and manufacturing. Currently widely used bulk EP methods, while they offer scalability, cost efficiency and simplicity, cannot be used for targeted or selective delivery to a defined subset of an input cell population. Here, we present a microparticle-enabled selectively permeabilizing impedance cytometer (ME-SPICy), a microfluidic single-cell EP platform that enables targeted EP of selected cell subpopulations based on their surface markers. Antibody conjugated microparticles (MPs) are used to label selected cell subpopulations within a larger heterogeneous sample. In this work, we demonstrate multiple multi-parameter impedance-based sensing schemes that enable real-time discrimination of non-labeled and labeled single cells within a mixed population as they flow through a 3D printed biconical micro-aperture. This allows for the system to analyze in real time if each single cell is a target cell, based on user set parameters, and selectively apply a low voltage (<16 V) for targeted single-cell EP. Simulations and experimental validation demonstrate that MP binding substantially alters cell impedance and phase signature, enabling accurate label-based discrimination. We demonstrated selective EP first using Jurkat cells by targeting either the labeled or non-labeled populations. Then we demonstrated targeted delivery with both dyes and EGFP mRNA in subpopulations of primary cells (peripheral blood mononuclear cells, activated T cells, and naïve T cells). ME-SPICy achieved high precision, with up to 98% purity and >5 fold enrichment in the electroporated cell population. This approach expands the capabilities of EP, offering a promising solution to decrease manufacturing complexity in both research and clinical cell engineering workflows.