Effects of Cell Density
The first experiment used the Sensor-Dish Reader to investigate the effects of cell density on the cellular oxygen consumption rate and subsequent pericellular pO2 levels. The oxygen profile (Figure 2A) clearly shows an effect of cell density on the oxygen consumption level and suggests that diffusion of O2 from the atmosphere into the media is a limiting step. There is a proportional deviation of pericellular pO2 from the media-only control based on the number of cells initially plated during the course of the incubation, implying that individual cellular respiration is equivalent at all the cell densities tested.
The second experiment tested the Reader at lower physiological ambient oxygen levels on the cell density induced observations of the first experiment. The oxygen profiles confirm the effect of cell density on oxygen consumption and the resulting pericellular O2 level (Figure 2B).
In the 7% pO2 media only treatment, it takes seven hours or more to reach equilibrium with the glove-box oxygen concentration. Pre-equilibrating media for experimental use may be critical to obtain and maintain the desired liquid pO2. The pericellular pO2 in the highest cell densities dropped below the gaseous incubation pO2 (7%) after only 1½ hours and began to level off at ~2% pO2. Both of these experiments imply that pericellular pO2 levels may be much lower than anticipated after a few hours of incubation, which could compromise experimental results.
Effects of Mitochondrial Modulators
The third experiment was designed to investigate the effects of specific mitochondrial modulators on cellular oxygen consumption and subsequent pO2 levels in the media. The oxygen profile shows a pronounced effect on cellular oxygen consumption (Figure 3).
Measurements at the three- to four- hour time points indicate that there was little variation in oxygen consumption prior to receiving the dosed media. Within 30 minutes of dosing, observable differences emerge in the oxygen consumption curves between the treatments. DMSO, the vehicle control, caused little variation in the average pericellular pO2 level in the MEF cells. Adding antimycin produced an observable decrease in oxygen consumption, while treatment with CCCP resulted in an observable increase in cellular oxygen consumption.
Using the SensorDish Reader, we monitored pericellular pO2 levels in mammalian cell cultures and showed cell density dependent oxygen consumption and variation in cellular oxygen demand in response to toxic compounds. The density dependent differences observed at 19% pO2 were also observed at 7% pO2, a more physiologically relevant oxygen level. At higher cell densities, in both pO2 levels, diffusion of O2 from the atmosphere into the media was insufficient to compensate for O2 utilization by the cells. This is important for cellular studies as numerous examples show cellular response varies with localized O2 levels.
In addition, cell culture media without any cells took at least seven hours to reach equilibrium when exposed to a 7% pO2 atmosphere. Therefore, pre-incubation of the media at the desired oxygen level is recommended and utilization of the Coy O2 Control Glove Box enables control and maintenance of the desired oxygen level during pre-incubation, set-up, incubation, and treatment.
Also, gaseous pO2 during incubation is a poor measure of the pericellular pO2 to which the cells are actually exposed. The SDR allows for constant and rapid quantification of pericellular pO2 levels that are a more realistic measure of the oxygen concentration that cells are actually experiencing. This, in turn, can be used as a measure of cellular oxygen demand and metabolic function.