A Tri‐Droplet Liquid Structure for Highly Efficient Intracellular Delivery in Primary Mammalian Cells Using Digital Microfluidics

Abstract

Automated techniques for mammalian cell engineering are needed to examine a wide range of unique genetic perturbations especially when working with precious patient samples. An automated and miniaturized technique making use of digital microfluidics to electroporate a minimal number of mammalian cells (≈40 000) at a time on a scalable platform is introduced. This system functions by merging three droplets into a continuous droplet chain, which is called a triDrop. In the triDrop configuration, the outer droplets are comprised of high‐conductive liquid while an inner or middle droplet comprising of low‐conductivity liquid that contains the cells and biological payloads. In this work, it is shown that applying a voltage to the outer droplets generates an effective electric field throughout the tri‐droplet structure allowing for insertion of the biological payload into the cells without sacrificing long‐term cell health. This technique is shown for a range of biological payloads including plasmids, mRNA, and fully formed proteins being inserted into adherent and suspension cells which include primary T‐cells. The unique features of flexibility and versatility of triDrop show that the platform can be used for the automation of multiplexed gene edits with the benefits of low reagent consumption and minimal cell numbers.