Three-dimensional organoid cultures based on hydrogels can be applied to study development, regeneration, and disease in vitro. However, control over the engineered hydrogel’s composition, mechanical properties, and geometric constraints is typically limited to the initial stage of fabrication. Modulating the hydrogel’s characteristics over time in response to the evolving culture is often not possible.
Here, we overcome these limitations by developing a live hydrogel-in-hydrogel bioprinting approach that enables the dynamic fabrication of instructive hydrogel elements within pre-existing hydrogel-based organoid cultures. This can be achieved through the crosslinking of photosensitive hydrogels via two-photon absorption at any point during culture.
We show that such instructive hydrogels guide axonal directionality in growing organotypic spinal cord cultures and that the geometry and mechanical properties of the hydrogel control differential cell migration in developing tumor organoids. Finally, we demonstrate that hydrogel constraints promote cell polarity in hepatic organoids, guide morphogenesis in small intestinal organoids, and control lung bud bifurcation according to the hydrogel’s composition and shape.
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