Hydrogel swelling drives cavity formation in multicellular spheroids

Mechanical forces play a fundamental role in shaping the complex architecture of organs that compose our body. While molecular motors are a primary source of biological forces, forces can also emerge without direct energy consumption. Entropic forces, such as those generated by macromolecular crowding, have emerged as a novel principle for cellular morphogenesis. However, the potential of entropic forces to drive tissue-scale morphogenesis remains unknown. Here we report that the swelling forces of a cell-synthesized extracellular hydrogel drives cavity formation in multicellular tissues. We induce the synthesis of hyaluronic acid by overexpressing the hyaluronic acid synthase HAS2 in HEK cell spheroids. Within 24 hours, spheroids harbor acellular cavities that are up to ~100 micron in diameter and enriched with hyaluronic acid. Upon enzymatic digestion of hyaluronic acid, the cavities shrink and disappear rapidly. This indicates that the swelling forces of hyaluronic acid support acellular cavities by counteracting cell contraction. Altogether our results demonstrate a new mechanism to generate acellular space within multicellular tissues based on the physics of hydrogel swelling. In the future, we will measure swelling forces and apply this physical principle to engineer tissue morphogenesis.