LigHTS: Biomimetic Hydrogel Photo-Fabrication for Imaging-Based Ultra-High-Throughput Screening.

Imaging-based ultra-high-throughput screening (UHTS) in pharma and biotech still relies on 384- and 1536-well plates whose rigid, flat substrates limit biological fidelity and screening efficiency. Highly biomimetic organoids and organs-on-chips improve relevance but at a cost that is incompatible with early screening campaigns. Photopatterned hydrogel scaffolds are potentially scalable, but current approaches lack plate-scale reproducibility and throughput. To address this gap, we present chemically-mediated Light-Intensity Gradients for structured hydrogel functionalization of ultra-High-Throughput Screening formats (LigHTS): an all-optical, in-well method for parallel photofabrication of biomimetic hydrogels in standard 384- and 1536-well plates. Adding ultraviolet-light absorber tartrazine to photo-curable solutions enables hydrogel thickness control by tuning illumination dose without focusing optics. Entire plates are functionalized in parallel with soft (∼1-10 kPa), thin (∼10-200 µm) hydrogels. Simultaneously, film photomasks that encode anisotropies enable control of well-level gel thickness and topography at UHTS scale. We demonstrate the utility of biomimetic gelatin-methacrylate-functionalized UHTS plates using mechanosensitive human cells (HT1080 fibrosarcoma cells, HaCaT epithelial cell colonies, and cardiomyocyte tissues derived from induced pluripotent stem cells), which display stiffness- and topography-dependent phenotypes. Finally, geometric uniformity across the plate meets industry requirements (coefficient of variation <20%), providing a readily available route to enhanced biomimicry in imaging-based UHTS pipelines.