Discretizing Three‐Dimensional Oxygen Gradients to Modulate and Investigate Cellular Processes

With the increased realization of the effect of oxygen (O(2)) deprivation (hypoxia) on cellular processes, recent efforts have focused on the development of engineered systems to control O(2) concentrations and establish biomimetic O(2) gradients to study and manipulate cellular behavior. Nonetheless, O(2) gradients present in 3D engineered platforms result in diverse cell behavior across the O(2) gradient, making it difficult to identify and study O(2) sensitive signaling pathways. Using a layer‐by‐layer assembled O(2)‐controllable hydrogel, the authors precisely control O(2) concentrations and study uniform cell behavior in discretized O(2) gradients, then recapitulate the dynamics of cluster‐based vasculogenesis, one mechanism for neovessel formation, and show distinctive gene expression patterns remarkably correlate to O(2) concentrations. Using RNA sequencing, it is found that time‐dependent regulation of cyclic adenosine monophosphate signaling enables cell survival and clustering in the high stress microenvironments. Various extracellular matrix modulators orchestrate hypoxia‐driven endothelial cell clustering. Finally, clustering is facilitated by regulators of cell–cell interactions, mainly vascular cell adhesion molecule 1. Taken together, novel regulators of hypoxic cluster‐based vasculogenesis are identified, and evidence for the utility of a unique platform is provided to study dynamic cellular responses to 3D hypoxic environments, with broad applicability in development, regeneration, and disease.