Invasin-functionalized PIC hydrogels enable long-term 3D culture of epithelial organoids

Tissue stem cell (TSC)-derived epithelial organoids are typically cultured in Matrigel [T. Sato <i>et al.</i>, <i>Nature</i> <b>459</b>, 262-265 (2009)], an extracellular matrix-like hydrogel produced from Engelbreth-Holm-Swarm sarcoma cells. This tumor is grown in the mouse abdomen [R. W. Orkin <i>et al.</i>, <i>J. Exp. Med.</i> <b>145</b>, 204-220 (1977)]. Previously, we demonstrated that the <i><i>Yersinia</i></i> membrane protein Invasin, coated on transwells, replaces Matrigel by activating β1-integrins, allowing long-term expansion of primary epithelial cells as 2D organoid sheets [J. J. A. P. M. Wijnakker <i>et al.</i>, <i>Proc. Natl. Acad. Sci. U.S.A.</i> <b>122</b>, e2420595121 (2025)]. Here, we functionalize a synthetic polyisocyanide (PIC) hydrogel with the integrin-activating domain of Invasin (INV). PIC hydrogels are soluble at 4 °C and form a gel at 37 °C [P. H. J. Kouwer <i>et al.</i>, <i>Nature</i> <b>493</b>, 651-655 (2013)]. When INV is covalently linked to PIC, the resulting hydrogel supports multipassage 3D growth of human intestinal and airway organoids. Self-renewal, polarization, and differentiation are maintained. The 3D swelling assay for cystic fibrosis drug testing (S. F. Boj <i>et al.</i>, <i>J. Vis. Exp.</i> (2017), 10.3791/55159] was validated using PIC-INV. With PIC-INV hydrogels, we establish a fully defined and animal-free system for 3D TSC-derived organoid culture.