Deterministic cell pairing with simultaneous microfluidic merging and sorting of droplets

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 Pair Isolation by Coalescence and Sorting (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.

Methods
Droplet microfluidic images and videos were collected using an AE31 inverted microscope and a Phantom Miro M110 high-speed camera. Droplet/gel collection images were collected on a Leica DMi8 inverted microscope using the brightfield and Leica GFP (11525314), Y3 (11525311), and Cy5-naEx (11536078) filter cubes to detect FITC/Calcein Green, Cell Trace Yellow, and Cell Trace Far-red, respectively. Length scale information was determined from the automatically generated scale bar from the Leica DMi8. Individual microscope image channels were then imported to ImageJ, where fluorescent intensity, droplet/gel size, cell pairing, and viability data were analyzed using the software plugins. The analyzed data were then input to GraphPad Prism to generate data visualizations. Device geometry was drafted in Autodesk AutoCAD. Additional experimental methods description can be found in the associated manuscript.