Transient Single Cell Hypoxia Induced by Localized Galvanostatic Oxygen Challenge

Studying cells exposed to low and controllable oxygen levels is key to investigating various fundamental aspects of pathological states, such as stroke and cancer. At present, available methodologies applied in vitro focus on large groups of cells exposed to low oxygen conditions through slow-time approaches, such as environmental incubators or microfluidic devices. Here, we demonstrate a novel approach for titrating the local oxygen concentration around individual adhered PC12 cells, enabling single cells within a population to be exposed to hypoxic-like conditions. A 25 μm diameter platinum disk microelectrode performing the oxygen reduction reaction (ORR) at constant current (galvanostatic control) is used as a microscale oxygen scavenger that can be positioned precisely over individual cells. By coupling the galvanostatic oxygen challenge with confocal laser scanning microscopy (CLSM) and a commercially available hypoxia dye (Image-iT Green hypoxia reagent), we monitor the response of single cells when exposed to depleted oxygen concentrations over time. Numerical simulations are used to characterize the oxygen and pH gradient imposed by the microelectrode at different cathodic currents, revealing that within seconds, the oxygen depletion zone reaches a steady-state condition, extending a few microelectrode radii into solution, while the corresponding pH gradient is strongly compressed by the buffer solution. Cells under the microelectrode show a marked increase in average fluorescence rate relative to control, reporting their hypoxic conditions and demonstrating the effectiveness of the proposed method. Heterogenous cell response in a challenged group is also observed, highlighting the ability of this approach to investigate the natural heterogeneity in cell populations. This work provides a platform and roadmap for future studies of cellular systems where the ability to control and vary oxygen concentration on a rapid time scale would be beneficial.