Fabrication of mechanically-tunable multilayered 3D cell co-culture hydrogel system for high-throughput invetigation of complex cellular behavior

Abstract

Hydrogels are widely used as a 3D cell coculture platform, as they can be tailored to provide suitable microenvironments to induce cellular phenotypes with physiological significance. Herein, programmable multilayer photolithography is employed to develop a 3D hydrogel-based co-culture system in an efficient and scalable manner, which consists of an inner microgel array containing one cell type covered by an outer hydrogel overlay containing another cell type. In particular, the mechanical properties of microgel array and hydrogel overlay are independently controlled in a wide range, with elastic moduli ranging from 1.7 to 31.6 kPa, allowing the high-throughput investigation of both individual hydrogel mechanics and mechanical gradients generated at their interface. Utilizing this system, it was demonstrated that macrophage phenotypical changes (i.e. proliferation, spheroid formation and M ϕ polarization) were substantially influenced by the direction and degree of mechanical gradient, as well as the presence of co-cultured fibroblasts in the vicinity. Furthermore, the paracrine effect between the macrophages in different microgels was clearly mediated by their inter-distance.