Towards spatially-organized organs-on-chip: Photopatterning cell-laden thiol-ene and methacryloyl hydrogels in a...

Micropatterning techniques for 3D cell cultures enable the recreation of tissue-level structures, but the combination of patterned hydrogels with organs-on-chip to generate organized 3D cultures under microfluidic perfusion remains challenging. To address this technological gap, we developed a user-friendly in-situ micropatterning protocol that integrates photolithography of crosslinkable, cell-laden hydrogels with a simple microfluidic housing, and tested the impact of crosslinking chemistry on stability and spatial resolution. Working with gelatin functionalized with photo-crosslinkable moieties, we found that inclusion of cells at high densities (≥ 10⁷/mL) did not impede thiol-norbornene gelation, but decreased the storage moduli of methacryloyl hydrogels. Hydrogel composition and light dose were selected to match the storage moduli of soft tissues. To generate the desired pattern on-chip, the cell-laden precursor solution was flowed into a microfluidic chamber and exposed to 405 nm light through a photomask. The on-chip 3D cultures were self-standing and the designs were interchangeable by simply swapping out the photomask. Thiol-ene hydrogels yielded highly accurate feature sizes from 100 - 900 μm in diameter, whereas methacryloyl hydrogels yielded slightly enlarged features. Furthermore, only thiol-ene hydrogels were mechanically stable under perfusion overnight. Repeated patterning readily generated multi-region cultures, either separately or adjacent, including non-linear boundaries that are challenging to obtain on-chip. As a proof-of-principle, primary human T cells were patterned on-chip with high regional specificity. Viability remained high (> 85%) after 12-hr culture with constant perfusion. We envision that this technology will enable researchers to pattern 3D co-cultures to mimic organ-like structures that were previously difficult to obtain.