CellTrap: an instrument-free microfluidic platform for cell-cell interactions at stochastically generated effector-to-target ratios.

Immune-cancer cell interactions play a central role in understanding antitumor responses and evaluating immunotherapies. However, long-term, single-cell-level analysis of these interactions remains challenging. To address this, we developed CellTrap, an instrument-free, perfusion-capable microfluidic device featuring 1024 parallel traps. Each trap is equipped with a filter constriction to stably retain cells under hydrostatic flow, sustain continuous medium perfusion, and minimize trap-to-trap crosstalk. By intentionally leveraging stochastic Poisson loading, a single seeding step simultaneously generates perfectly matched internal controls alongside variable effector-to-target (E : T) ratios across the array. Device characterization using 10 µm fluorescent beads and cells validated the predictable trap occupancy governed by Poisson statistics. Initial proof-of-concept experiments using primary human PBMCs against GFP-expressing U87 (U87GFP) glioblastoma cells successfully demonstrated targeted, immune-mediated cytotoxicity over 14 hours. To decouple donor-derived biological heterogeneity from technical validation, we subsequently employed IL-2-stimulated Natural Killer cells (NK92IL2) as a standardized effector population against U87GFP, K562 leukemia, and LS174T adenocarcinoma targets. Continuous time-lapse imaging seamlessly linked early transient intracellular calcium fluxes (indicating target engagement) to distinct, contact-dependent cytotoxic outcomes. Our data demonstrate that increasing E : T ratios consistently enhances immune-mediated target lysis, highlighting the platform's robust utility for dissecting complex immune-cancer dynamics and guiding the development of personalized immunotherapies.