Sequential Patterning of Photoresponsive Hydrogels Directs Crypt Fission and Reveals the Role of Epithelial Curvature on Fission Symmetry.

Fission increases the number of crypts in the intestine during neonatal growth and also restores crypt density after injury by bifurcation of a pre-existing parent crypt into daughter crypts. While fission is typically symmetric in healthy crypts, it is more asymmetric in diseases, and the relationship between parent crypt shape and daughter crypt (a)symmetry is difficult to study as crypt budding and fission are stochastic in organoid models and difficult to control in vivo. Here, a photoresponsive hydrogel is introduced to spatiotemporally control daughter crypt emergence from mature parent crypts in intestinal organoids, enabling longitudinal tracking of crypt bifurcation in vitro. Variation of the photopatterned dimensions tunes parent crypt shape and reveals that both fission efficiency and crypt symmetry depend on parent crypt geometry. Epithelial boundary analysis identified parent crypt curvature as a key factor influencing daughter crypt symmetry. High-curvature or narrow crypts yielded symmetric daughter crypts, whereas wider parent crypts with lower epithelial curvature generated progressively more asymmetric crypts. Mechanistically, non-muscle myosin IIA acts as one key regulator of crypt symmetry. Overall, this work introduces a reproducible and spatiotemporally controllable in vitro model of crypt fission, allowing identification of mechanical determinants of fission that influence intestinal regeneration and development.