A high-throughput, 3D microtissue platform for multiparametric analysis of tissue remodeling.

Abstract

Extracellular matrix (ECM) remodeling and force generation are fundamental drivers of tissue morphogenesis and repair, yet scalable methods to quantitatively interrogate these dynamic mechanical processes remain limited. Here, we present a high-throughput screening platform that integrates engineered three-dimensional (3D) microtissues within a standardized 96-well format. We introduce a robust mold-casting fabrication process and a layer-by-layer surface modification strategy, that ensures long-term tissue stability and prevents detachment (95% tissue formation success; stable in culture for more than 10 days). This system enables simultaneous, longitudinal quantification of tissue closure, tissue contractility, and tissue compaction from a phase-contrast imaging modality. The computational data analysis tools that accompany this framework ensure reproducibility through deterministic computation and accelerate data extraction 80-fold relative to manual annotation. Using pharmacological compounds, we show that tissue closure dynamics, force generation, and compaction represent independent variables of ECM-driven tissue remodeling, challenging assumptions embedded in commonly used contraction-based assays. Furthermore, benchmarking against reported clinical drug responses demonstrates that the 3D platform better aligns with clinical outcomes (Kendall{tau} -b = 0.72, p=0.045, n =8/10) than a conventional two-dimensional scratch wound assay (Kendall{tau} -b = 0.52, p=0.25, n =4/10). Together, this work establishes a scalable assay for functional screening and quantitative assessment of tissue remodeling dynamics in three-dimensional systems.