Joslin, K. M. | bioRxiv Science

bioRxiv2025

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 cell combinations. Here, we present a microfluidic device capable of deterministically isolating specific cell pairs using droplet merging and sorting ( merge-sorting). The system 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 this platform for extended co-culture studies, we merged cells in an alginate solution with calcium chloride droplets, producing monodisperse alginate hydrogels in which 92% 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. The device allows off-chip pre-incubation of droplets before pairing, the merger of reagents for multi-step assays, and the isolation of desired droplet pairs -- capabilities not jointly accessible with existing approaches. In summary, merge-sort is a flexible platform to enrich specific combinations of droplets, cells, or particles for high-throughput studies of cell crosstalk.

bioengineering↗

bioRxiv2025

The Immunophenotype and Proviral Landscape of HIV-infected CD4 T Cells During Antiretroviral Therapy

In individuals on effective antiretroviral therapy, integrated HIV proviruses persist within CD4 T cells, forming a viral reservoir that rebounds if treatment is stopped. Identifying and targeting these rare, infected cells is critical for advancing therapies, but methods to study reservoir cells are limited and their unique properties remain largely unknown. We applied DAb-seq, a high-throughput method that combines single-cell DNA and surface protein sequencing, to profile over five hundred and twenty thousand CD4 T cells from the blood of six individuals on ART. Infected cells were unequally distributed in T cell subsets, and differential protein expression between infected and uninfected cells revealed significant heterogeneity across cell subsets. Attempts to identify surface markers that differentiate infected from uninfected cells found antigens that mirrored the enrichment of HIV in central memory subsets. However, while central memory T cells harbored the majority of HIV, cells with intact provirus were enriched relative to their defective counterparts in Naive and Regulatory T cell subsets, suggesting that they differentially maintain intact proviruses. In summary, we developed DAb-seq as an open-source platform for linking the proviral landscape to diverse cellular phenotypes, revealing heterogeneity in surface protein expression and provirus maintenance across infected subsets.

genomics↗