Hollow spheroid formation by cells at the mechanical interface of hyaluronic acid hydrogels in three dimensions

Extracellular matrix (ECM) is a non-cellular material that offers not only structural/mechanical support but also chemical and physical guidance cues for cells. Cellular self-organization is highly related to the property of ECM. Here, we investigate the 3D hollow spheroid formation process of vascular mesenchymal cells (VMCs) at the interfaces between hydrogel ECMs with different mechanical properties. The combined hydrogel geometrically consisted of two subparts differing in their stiffness and had geometric interfaces between those two subparts. The experimental results show that 3D hollow spheroids with large cavity generated around interfacial regions of hydrogels, which were not observed in a common 3D culture with sole-stiffness hydrogel. The hollow spheroids emerged because of the tailored groove dimensions of hydrogel interface and the concentration gradients of proteins and cells across the hydrogel interfaces. This experimental demonstration provides a potential method to improve the biological properties of multicellular spheroids for fundamental and therapeutic applications.