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bioRxiv · 10.1101/2025.10.23.683892

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

Enrico, A.·Rigolli, S.·Zimmermann, J.·Pezzotti, M.·Torchia, E.·Di Sante, M.·Auricchio, F.·Pasqualini, F. S.
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Abstract

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 {micro}m. 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.

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Enrico, A., Rigolli, S., Zimmermann, J., Pezzotti, M., Torchia, E., Di Sante, M., Auricchio, F., Pasqualini, F. S.. 2025-10-24. LigHTS: Massively Parallel Biomimetic Photo-Functionalization for Imaging-Based Ultra-High-Throughput Screening. https://doi.org/10.1101/2025.10.23.683892

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