Shin, S. W. | 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↗

bioRxiv2024

Disease-associated astrocyte epigenetic memory promotes CNS pathology

Astrocytes play important roles in the central nervous system (CNS) physiology and pathology. Indeed, astrocyte subsets defined by specific transcriptional activation states contribute to the pathology of neurologic diseases, including multiple sclerosis (MS) and its pre-clinical model experimental autoimmune encephalomyelitis (EAE)1-8. However, little is known about the stability of these disease-associated astrocyte subsets, their regulation, and whether they integrate past stimulation events to respond to subsequent challenges. Here, we describe the identification of an epigenetically controlled memory astrocyte subset which exhibits exacerbated pro-inflammatory responses upon re-challenge. Specifically, using a combination of single-cell RNA sequencing (scRNA-seq), assay for transposase-accessible chromatin with sequencing (ATAC-seq), chromatin immunoprecipitation with sequencing (ChIP-seq), focused interrogation of cells by nucleic acid detection and sequencing (FIND-seq), and cell-specific in vivo CRISPR/Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) used by the histone acetyltransferase p300 to control chromatin accessibility. ACLY+p300+ memory astrocytes are increased in acute and chronic EAE models; the genetic targeting of ACLY+ p300+ astrocytes using CRISPR/Cas9 ameliorated EAE. We also detected responses consistent with a pro-inflammatory memory phenotype in human astrocytes in vitro; scRNA-seq and immunohistochemistry studies detected increased ACLY+ p300+ astrocytes in chronic MS lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, MS. These findings may guide novel therapeutic approaches for MS and other neurologic diseases.

immunology↗