Imaging oxygen microenvironment in hydrogel microwell array

Hydrogel microwell arrays (HMAs) have been wildly used for engineering cell microenvironments by providing well-controlled biophysical and biochemical cues (e.g., three-dimensional (3-D) physical boundary, biomolecule coating) for cells. Among these cues, the oxygen microenvironment has shown great effect on the cellular physiological processes. However, it is currently technically challenging to characterize the local oxygen microenvironment within HMAs. Here, we prepared HMAs with different cross-linking concentrations to adjust the structural and physical properties of HMAs. Then we introduced a scanning electrochemical microscopy (SECM)-based electrochemical method to map the surface topography and oxygen microenvironment around HMAs. The SECM results show both the 3-D topography and the oxygen permeability of HMAs in aqueous solution. The obtained oxygen permeability of HMAs increases with increasing the cross-linking concentration, and the microwell boundaries show the highest oxygen permeability throughout HMAs. This work demonstrates that SECM offers a high spatial resolution and in situ method for characterization of the topography and the local oxygen permeability of HMAs, which can provide useful information for better engineering cell microenvironments through optimizing HMAs design.