Frequently asked questions in hypoxia research

Abstract

"What is the O₂ concentration in a normoxic cell culture incubator?" This and other frequently asked questions in hypoxia research will be answered in this review. Our intention is to give a simple introduction to the physics of gases that would be helpful for newcomers to the field of hypoxia research. We will provide background knowledge about questions often asked, but without straightforward answers. What is O₂ concentration, and what is O₂ partial pressure? What is normoxia, and what is hypoxia? How much O₂ is experienced by a cell residing in a culture dish in vitro vs in a tissue in vivo? By the way, the O₂ concentration in a normoxic incubator is 18.6%, rather than 20.9% or 20%, as commonly stated in research publications. And this is strictly only valid for incubators at sea level.

Keywords: Krogh tissue cylinder; gas laws; hypoxia-inducible factor; oxygen diffusion; partial pressure; tissue oxygen levels.

Figure 1

Composition of the gas phase in a tissue culture incubator. Notes: Input room air (left) is mixed with gaseous water and CO₂ to form the incubator’s gas mixture (right).

Figure 2

O₂ concentration gradients in cell culture medium. Notes: Normal 24-well tissue culture plates without (left) or with (right) confluent HeLa cell layers were removed from a normoxic 37°C incubator, and O₂ concentration profiles were determined under room air conditions at 25°C using a needle-type O₂ sensor (PreSens, Regensburg, Germany). Note that the change from incubator air to room air results in a higher pO₂ (no gaseous water, no pCO₂) and a better O₂ solubility (temperature change from 37°C to 25°C).

Figure 3

Pericellular O₂ concentration as a function of medium height and cell density. Notes: 24-Well SensorDish tissue culture plates were filled with different medium volumes and seeded with HeLa cells at various densities, resulting in 0%–100% confluency as indicated. The SensorDishes were removed from the 37°C incubator and the pericellular O₂ concentration determined under room air conditions at 25°C (resulting in a higher pO₂ and better O₂ solubility than within the incubator) using a SensorDish Reader (PreSens, Regensburg, Germany).

Figure 4

Krogh’s tissue cylinder. Notes: Overlapping longitudinal (convective) and radial (diffusive) pO₂ gradients form the physiological tissue O₂ distribution (calculated isobaric pO₂ profiles assuming constant tissue O₂ consumption). All cells located within this pO₂ profile are considered to be physiologically “normoxic”, despite the highly variable absolute pO₂ levels.

Figure 5

Organotypic characteristics in tissue oxygenation and O₂ metabolism. Notes: Combined spatial and temporal processes result in a broad spectrum of physiologically “normoxic” tissue pO₂ values. For details see section: What is the pO₂ in organs?