Four-dimensional hydrogel-in-hydrogel bioprinting for the spatiotemporal control of organoid and organotypic cultures

Tissue architecture is a driving force for morphogenetic processes during development as well as for several physiological and regenerative responses. Far from being a passive static environment, tissue architecture is highly dynamic. Hydrogel technology reproduces in vitro geometrical and mechanical constrains that control the three-dimensional self-organization of (3D) organoids and organ-like cultures. This control is restricted to the initial culture conditions and cannot be adapted to the dynamic morphological changes of complex 3D cultures during their developmental trajectory. Here, we developed a method that overcomes this spatiotemporal limit. Using 2P crosslinking approach, high resolution 3D hydrogel structures can be fabricated within pre-existing hydrogel with spatiotemporal (fourdimensional, 4D) control relative to ex-vivo organotypic or organoid culture. This hydrogel-in- hydrogel bioprinting approach enables to continuously instruct the self-organization of the evolving 3D organ-like cultures.