# Transient Single Cell Hypoxia Induced by Localized Galvanostatic Oxygen Challenge

## TL;DR

A new method uses a microelectrode to create localized hypoxia in single cells, allowing real-time study of cellular responses to low oxygen.

## Contribution

A novel galvanostatic oxygen scavenging technique enables rapid and localized hypoxia in individual adhered cells.

## Key findings

- The microelectrode creates a steady-state oxygen depletion zone within seconds.
- Cells under the microelectrode show increased hypoxia-related fluorescence.
- The method reveals heterogeneous responses among cells in a population.

## Abstract

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.