LigHTS: Massively Parallel Biomimetic Photo-Functionalization for Imaging-Based Ultra-High-Throughput Screening

Imaging-based ultra-high-throughput screening (UHTS) in pharma and biotech still runs on 384/1536-well plates whose stiff, flat substrates limit biological fidelity and screening efficiency. Highly biomimetic organs-on-chips and organoids improve relevance but lack reproducibility and plate-scale throughput. Biomimetic hydrogel scaffolds can be produced at scale through photopolymerization, which yet uses focused optics to define micrometer-resolved geometries, constraining scalability. To address the technical challenge of truly scalable biomimetic substrates featuring anisotropies, this study presents LigHTS, an all-optical, in-well method that replaces focused with collimated illumination to photofabricate structured hydrogels in standard 384/1536-well plates. Adding food dye tartrazine to gelatin-methacrylate (GelMA) solutions increases hydrogel thickness sensitivity to UV dose by ∼10x, allowing uniform control of film thickness without lenses. Entire plates are functionalized in parallel with soft hydrogels (∼1-10 kPa) whose thickness is tunable from 10 to 100 µm. Simultaneously, simply interposing film photomasks encoding anisotropies enables orthogonal control of thickness and topography at UHTS throughput. Biological effect is demonstrated with mechanosensitive HT1080 cells, which display stiffness- and topography-dependent spreading and contact-guided migration on LigHTS-produced grooved substrates. Geometric uniformity across the plate (coefficient of variation <20%) meets HTS reproducibility standards, providing a readily available solution with enhanced biomimicry for imaging-based UTHS pipelines.