Fiber Diameter and Architecture Direct Three-Dimensional Assembly of Pericytes into Spheroids

Due to their physiological relevance, multicellular 3D spheroids are actively replacing standard 2D monolayer cultures. How spheroids are formed through the assembly of individual cells in natural fibrous environments that include a mix of diameters and architectures in vivo remains unknown. Here, we demonstrate that the spontaneous assembly of human vasa vasorum-derived pericytes in 3D spheroids depends on the fiber diameter and network architecture. A parallel arrangement of suspended fibers of all tested diameters (200, 500, and 800 nm) leads to the formation of spheroids, while on crosshatch networks, spheroid assembly on larger diameters is absent. The design of fibrous networks of a mix of diameters and architectures leads to the patterning of spheroids in desired locations. Fiber remodeling in parallel arrangements serves as force sensors providing mechanical insights into the assembly dynamics of spheroids and subsequent cell sprouting from spheroids. Translocation and merger of spheroids occur predominantly on parallel fiber networks, while on crosshatch networks, a cellular exchange is observed between spheroids connected with remodeled fibers. Rho kinase inhibition by Y27632 and subsequent wash-off leads to spheroid disintegration and reassembly, thus, highlighting the role of cell contractility in the assembly and integrity of 3D spheroids. Overall, using extracellular mimicking fiber networks of varying diameters and architectures, we report new insights into the 3D dynamics of spheroids which may inform pericytes role in vasculogenesis, and (patho)physiological angiogenesis