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

With the increased realization of the effect of oxygen (O₂) deprivation (hypoxia) on cellular processes, recent efforts have focused on the development of engineered systems to control O₂ concentrations and establish biomimetic O₂ gradients to study and manipulate cellular behavior. Nonetheless, O₂ gradients present in 3D engineered platforms result in diverse cell behavior across the O₂ gradient, making it difficult to identify and study O₂ sensitive signaling pathways. Using a layer-by-layer assembled O₂-controllable hydrogel, the authors precisely control O₂ concentrations and study uniform cell behavior in discretized O₂ gradients, then recapitulate the dynamics of cluster-based vasculogenesis, one mechanism for neovessel formation, and show distinctive gene expression patterns remarkably correlate to O₂ 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.