Deterministic cell pairing with simultaneous microfluidic merging and sorting of droplets.

Abstract

Cell-cell interactions drive immune activation, tissue repair, and stem cell fate, yet there are few methods that can create large numbers of pre-defined cell pairs to study cell crosstalk. Droplet microfluidics allows high-throughput compartmentalization of multiple cells, but random loading results in <1% of droplets containing the desired combinations. Here, we present P̲air I̲solation by C̲oalescence and S̲orting (PICS), a microfluidic platform that can generate specific cell pairs through droplet merging and sorting ('merge-sorting'). PICS detects target combinations using fluorescence and triggers simultaneous electrocoalescence and dielectrophoretic sorting. Using fluorescent dye-loaded droplets, we achieved 98.6% purity of merged and sorted droplets. In experiments using cells stained with three distinct dyes, >90% of desired cell pairs were recovered - compared to fewer than 1% when using random Poisson loading. To demonstrate the utility of PICS for extended co-culture studies, we merged cells in an alginate solution with calcium chloride droplets, producing monodisperse alginate hydrogels in which 93.3% of the beads contained target cell pairs that maintained viability over 18 hours. Compared to selective merger, this approach physically isolates desired droplets, eliminating unmerged contaminants and enabling cleaner downstream workflows. PICS allows off-chip pre-incubation of droplets before pairing, the merger of reagents for multi-step assays, and the rapid isolation of desired droplet pairs - capabilities not jointly accessible with existing approaches. In summary, PICS is a flexible platform to enrich specific combinations of droplets, cells, or particles for high-throughput studies of cell crosstalk.