In situ photopatterning of cell laden biomaterials for spatially organized 3D cell cultures in a microfluidic chip

• Published in: bioRxiv
• Main Authors: Ortiz-Cárdenas, Jennifer E, Zatorski, Jonathan M, Arneja, Abhinav, Montalbine, Alyssa N, Munson, Jennifer M, Chance John Luckey, Pompano, Rebecca R
• Format: Paper
• Language: English
• Published: Cold Spring Harbor Cold Spring Harbor Laboratory Press 10.03.2021
• Subjects: Biomaterials; Biomedical materials; Cell culture; Gelatin; Hydrogels; Lymphocytes T; Microfluidics; Micropatterning; Perfusion; Photolithography; Soft tissues
• DOI: 10.1101/2020.09.09.287870

Abstract Micropatterning techniques for 3D cell cultures enable the recreation of tissue-level structures, but their combination with well-defined, microscale fluidic systems for perfusion remains challenging. To address this technological gap, we developed a user-friendly in-situ micropatterning strategy that integrates photolithography of crosslinkable, cell-laden hydrogels with a simple microfluidic housing. Working with gelatin functionalized with photo-crosslinkable moieties, we found that inclusion of cells at high densities (≥ 107/mL) during crosslinking 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. The cell-laden precursor solution was flowed into a microfluidic chamber and exposed to 405 nm light through a photomask to generate the desired pattern. 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, a fragile cell type, primary human T cells, were patterned on-chip with high regional specificity. Viability remained high (> 85%) after overnight culture with constant perfusion. We envision that this technology will enable researchers to pattern 3D cultures under fluidic control in biomimetic geometries that were previously difficult to obtain. Competing Interest Statement The authors have declared no competing interest. Footnotes * Revised manuscript with new biomaterials characterization data, expanded characterization of micropatterning, and longer-term viability.