One Cell, One Drop, One Click: Hybrid Microfluidics for Mammalian Single Cell Isolation

Generating a stable knockout cell line is a complex process that can take several months to complete. In this work, a microfluidic method that is capable of isolating single cells in droplets, selecting successful edited clones, and expansion of these isoclones is introduced. Using a hybrid microfluidics method, droplets in channels can be individually addressed using a co‐planar electrode system. In the hybrid microfluidics device, it is shown that single cells can be trapped and subsequently encapsulate them on demand into pL‐sized droplets. Furthermore, droplets containing single cells are either released, kept in the traps, or merged with other droplets by the application of an electric potential to the electrodes that is actuated through an in‐house user interface. This high precision control is used to successfully sort and recover single isoclones to establish monoclonal cell lines, which is demonstrated with a heterozygous NCI‐H1299 lung squamous cell population resulting from loss‐of‐function eGFP and RAF1 gene knockout transfections. Combining droplet‐in‐channel with digital microfluidics enables efficient single cell trapping and on‐demand encapsulation. Droplets containing a single cell can be released, kept in traps, or merged with other droplets by on‐demand functionality. The advent of these on‐demand functions offers a rapid strategy to automate the gene‐editing pipeline for mammalian cells, specifically in the areas of isolating single clones and expansion.