Reversible Microfluidic Platform for Spheroid Culturing, Downstream Characterization, and Dynamic Anticancer Susceptibility Testing.

Despite the potentialities of three-dimensional (3D) spheroids to improve the predictive capability of preclinical in vitro assays, their translation into microfluidic drug screening pipelines remains limited by insufficient operational standardization, restricted access to downstream analyses, and static exposure regimes that inadequately capture in vivo mass transport. To address these downsides, we here introduce a reversibly bonded polydimethylsiloxane microfluidic platform that provides highly reproducible microwell-enabled spheroid generation, nondestructive spheroid retrieval for advanced off-chip characterization, and dynamic perfusion-based drug-susceptibility testing. A standardized operational workflow addressing practical aspects of microfluidic 3D culture (i.e., bubble suppression, surface passivation, and controlled media handling) is delivered, ultimately enabling reliable, user-friendly spheroid formation into daily practice across distinct biological systems. To date, the system has been successfully interrogated for the generation of spheroids derived from breast tumor and zebrafish liver cells. Importantly, as-produced spheroids could be retrieved from the reversible microfluidic device for downstream analyses, as demonstrated by transmission electron microscopy (TEM). TEM analysis of retrieved spheroid sections revealed preserved cellular ultrastructure, including well-defined nuclei and organelles. This data highlights the suitability of the microfluidic platform for downstream high-resolution characterization of as-cultured spheroids. Finally, doxorubicin susceptibility experiments under physiologically relevant perfusion conditions revealed enhanced cytotoxicity compared with static conditions, highlighting the critical role of flow-driven mass transport in generating more predictive drug-response profiles. Collectively, this work yields a scalable, reproducible, and accessible microfluidic framework that bridges spheroid culturing, downstream characterization, and dynamic pharmacology, expanding the analytical potential of in vitro microphysiological models for susceptibility testing. These assays can be broadly leveraged beyond drug discovery, including applications in toxicity testing of (bio)-chemicals and (nano)-materials toward new approach methodologies, safety research, and environmental assessments.