The extracellular matrix (ECM) plays a crucial role in organoid cultures by supporting cell proliferation and differentiation. A key feature of the ECM is its mechanical influence on the surrounding cells, directly affecting their behavior. Matrigel, the most commonly used ECM, is limited by its animal-derived origin, batch variability, and uncontrollable mechanical properties, restricting its use in 3D cell-model-based mechanobiological studies. Poly(2-alkyl-2-oxazoline) (PAOx) synthetic hydrogels represent an appealing alternative because of their reproducibility and versatile chemistry, enabling tuning of hydrogel stiffness and functionalization. Here, we studied PAOx hydrogels with differing compressive moduli for their potential to support 3D cell growth. PAOx hydrogels support spheroid and organoid growth over several days without the addition of ECM components. Furthermore, we discovered intestinal organoid epithelial polarity reversion in PAOx hydrogels and demonstrate how the tunable mechanical properties of PAOx can be used to study effects on the morphology and oxygenation of live multicellular spheroids.

Hydrogel Lab Department of Polymers Faculty of Chemical Technology University of Chemistry and Technology Technicka 1903 5 Prague 6 166 28 Czech Republic

Laboratory of Immunology Department of Translational Physiology Infectiology and Public Health Faculty of Veterinary Medicine Ghent University Merelbeke 9820 Belgium

Supramolecular Chemistry Group Centre of Macromolecular Chemistry Department of Organic and Macromolecular Chemistry Faculty of Sciences Ghent University Krijgslaan 281 S4 Ghent 9000 Belgium

Tissue Engineering and Biomaterials Group Department of Human Structure and Repair Faculty of Medicine and Health Sciences Ghent University C Heymanslaan 10 Ghent 9000 Belgium

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