Toward a neurospheroid niche model: optimizing embedded 3D bioprinting for fabrication of neurospheroid brain-like co-culture constructs

Abstract A crucial step in creating reliable in vitro platforms for neural development and disorder studies is the reproduction of the multicellular three-dimensional (3D) brain microenvironment and the capturing of cell-cell interactions within the model. The power of self-organization of diverse cell types into brain spheroids could be harnessed to study mechanisms underlying brain development trajectory and diseases. A challenge of current 3D organoid and spheroid models grown in petri-dishes is the lack of control over cellular localization and diversity. To overcome this limitation, neural spheroids can be patterned into customizable 3D structures using microfabrication. We developed a 3D brain-like co-culture construct using embedded 3D bioprinting as a flexible solution for composing heterogenous neural populations with neurospheroids and glia. Specifically, neurospheroid-laden free-standing 3D structures were fabricated in an engineered astrocyte-laden support bath resembling a neural stem cell niche environment. A photo-crosslinkable bioink and a thermal-healing supporting bath were engineered to mimic the mechanical modulus of soft tissue while supporting the formation of self-organizing neurospheroids within elaborate 3D networks. Moreover, bioprinted neurospheroid-laden structures exhibited the capability to differentiate into neuronal cells. These brain-like co-cultures could provide a reproducible platform for modeling neurological diseases, neural regeneration, and drug development and repurposing.