Tri-droplet digital microfluidics system for editing mammalian cells

Making meaningful changes to genomes is revolutionizing patient care by making personalized cell therapies a reality. However, the delivery of gene-editing machinery in a manner that maximizes efficiency often comes at the cost of reduced cellular viability, particularly when working with low cell numbers ( < 10⁶ cells) or delicate cell types. Additionally, it is difficult to integrate current commercially available gene delivery devices into an automated gene editing workflow in a way that would allow for high throughput testing of different conditions (e.g., testing different guides or donor templates). Digital Microfluidics (DMF) exceeds at precisely controlling the movement of low quantities of cells in nanoliter droplets and has recently emerged as a promising technology for automating genetic engineering workflows. To meet the demand for an automated system capable of working with small cell populations, we have designed a novel electroporator that can be integrated into a dual plate DMF device allowing for automated, high viability transfection of mammalian cells in quantities as low as 10³ cells per reaction. To counteract cell death observed during conventional electroporation, we have created an innovative device geometry which we called the ‘tri-drop system’, that minimizes electrical currents, protects the cells from the production of harmful chemical reactions at the anode and cathode and does not require complicated fabrication techniques. In this system DMF actuation is used to merge three droplets into a sequential chain, the outer two droplets are comprised of high conductivity media and are in physical contact with gold electrodes and an inner droplet that contains cells and biomolecules suspended in low conductivity media. The design allows for high resistance, limiting current during EP to less than 25mA, while also generating a homogenous electric field throughout the middle droplet as confirmed through COMSOL simulations. Additionally, by isolating the metal electrodes from the cell-containing droplet, by-products of the harmful chemical reaction are kept separate from the biological payload allowing the pH experienced by the cells to remain neutral. Our platform has been able to electroporate as few as ~2,500 mammalian cells achieving ~90% viability while effectively inserting a GFP-tagged cas9 nuclease. Our current studies are focused on using primary human T cells to successfully generate CRISPR Cas9 edited cells from as few as 5,000-10,000 cells, a capability that no commercially available electroporation device can execute. This device will provide genetic engineers with a platform for automated gene editing where cells from a single donor can be used to test numerous conditions in parallel and at high throughput while limiting the requirements for other expensive reagents such as proteins, guide RNA, and buffer.