What is Hypox­ia in Cell Cultures?

In standard CO₂ incubators, oxygen levels are typically maintained at 138 - 160 mmHg (~18–21%)—far higher than the physiological conditions most cells experience in tissues. Hypoxia and physoxia in cell culture involves intentionally reducing oxygen to mimic in vivo environments, such as those in tumors, ischemic tissues, or stem cell niches. By replicating these oxygen-deprived states, researchers can investigate cellular adaptation mechanisms, survival strategies, and failure pathways critical to understanding diseases like cancer, stroke, and chronic wounds.

Hypoxia and physoxia are not merely about oxygen scarcity; they represent dynamic shifts in cellular physiology. For instance, under hypoxia, cells transition from oxygen-dependent oxidative phosphorylation to anaerobic glycolysis—a metabolic "gear shift" akin to a car switching to a backup engine. This reprogramming alters gene expression, protein synthesis, and even epigenetic regulation, with downstream effects on processes like angiogenesis, apoptosis, and immune evasion.

Studying hypoxia is vital for improving the translational relevance of in vitro models. For example, cancer cells cultured under normoxia exhibit vastly different behaviors compared to those in hypoxic tumor microenvironments. Similarly, stem cells require precise oxygen tensions to maintain pluripotency or differentiate into functional tissues. Accurately modeling these conditions bridges the gap between lab experiments and human physiology.

This article clarifies the distinctions between hypoxia (pathological oxygen deprivation) and physoxia (tissue-specific physiological oxygen), critiques the limitations of standard CO₂ incubators, and advocates for specialized hypoxia workstations to achieve experimental rigor.

Hypoxia occurs when oxygen levels fall below a cell’s physiological requirements, typically ≤30 mmHg (≈4% O₂ at sea level). This mimics pathological states like solid tumors or ischemic injury and triggers adaptive responses, such as HIF-mediated shifts to anaerobic glycolysis.

Physoxia (or physioxia), in contrast, describes the tissue-specific oxygen tension found in vivo, ranging from 30–70 mmHg (≈4–9% O₂). For example, stem cell niches and liver tissues operate at ~30–40 mmHg, while well-perfused organs like the lungs experience higher tensions. Researchers must consult tissue-specific data (e.g., Keeley & Mann, 2019) to select appropriate oxygen levels for in vitro models.

Note: As physoxia could describe a wide range of oxygen tensions, it’s important for the reader to understand these definitions and recognise the pressures we use to describe hypoxia and physoxia are estimates here. These could be shifted entirely as one looks at a specific tissue, organ or pathological state.

Under low oxygen conditions, cells face an energy crisis. Oxygen is essential for oxidative phosphorylation, which generates 36 ATP molecules per glucose molecule. When oxygen drops, cells switch to anaerobic glycolysis, producing only 2 ATP per glucose and accumulating lactate.

This metabolic shift is orchestrated by HIFs, which:

• Increase glucose uptake via GLUT1 transporters.
• Upregulate glycolytic enzymes like hexokinase and lactate dehydrogenase.
• Inhibit mitochondrial activity by inducing pyruvate dehydrogenase kinase (PDK1).

Hypoxia also triggers non-metabolic adaptations:

• Angiogenesis:
  HIFs upregulate VEGF to stimulate blood vessel growth.
• pH regulation:
  Lactate export acidifies the extracellular microenvironment, influencing cell-cell signaling.
• DNA repair:
  Hypoxia reduces ROS production, temporarily protecting cells from oxidative damage.

Cell-type-specific responses:

• Cancer cells:
  Exploit hypoxia to upregulate drug-resistance genes (e.g., MDR1) and metastasize.
• Neurons:
  Undergo apoptosis within hours of oxygen deprivation.
• Immune cells:
  Hypoxia polarizes macrophages toward pro-tumorigenic (M2) phenotypes.

Hypoxia arises in cell cultures due to:

• Diffusion limitations:
  Oxygen gradients form in 3D models (e.g., spheroids, organoids), where surface cells consume oxygen before it reaches the core. For instance, in tumor spheroids >500 µm in diameter, core oxygen levels can drop to <10 mmHg, mimicking in vivo necrotic regions.
• High metabolic demand:
  Rapidly proliferating cells (e.g., cancer lines) deplete oxygen faster than diffusion replenishes it.
• Experimental design:
  Researchers directly control oxygen levels to model diseases (e.g., myocardial infarction, tumor cores) or stem cell niches.

Hypoxia vs. normoxia in cell cultures

Standard CO₂ incubators are only designed to regulate temperature, humidity, and CO₂ levels and often end up with internal oxygen concentration around 138 mmHg (18%), which is lower than ambient air (normoxia) but still far higher than the actual oxygen tensions found in most tissues. These higher oxygen levels would be considered hyperoxic for nearly all cell types and as such do not mimic a normal physiological state.

To experimentally induce physoxia or hypoxia, researchers should employ specialized hypoxia chambers or workstations that can precisely lower oxygen tension. These systems are engineered to create and maintain oxygen levels as low as 1 mmHg (0.1%), enabling studies that more accurately model the low-oxygen conditions present in any pathophysiological states. Such specialized equipment overcomes the inherent limitations of standard incubators and ensures that oxygen tensions are controlled with high precision—a necessity for robust and reproducible experimental design.

While researchers can induce “chemical hypoxia” this often has a limited scope of studies it can support and we have written about this extensively in our article “Simulating In Vivo Oxygen Conditions: A Guide for Cell Culture Protocols”.

The Importance of Accurate Oxygen Tension

While percentage measurements remain a standard, researchers should consider using or converting to oxygen partial pressure, as these absolute measurements offer a more accurate, reproducible measure of what cells experience:

• Atmospheric Variability:
  Oxygen expressed as a percentage does not account for differences in atmospheric (barometric) pressure caused by weather or altitude. This means that a chamber set to, for example, 5% O₂ might deliver a different actual oxygen concentration across days as the weather changes, but 38 mmHg will remain the same in all conditions.
• Physiological Relevance:
  Cells in the body are exposed to oxygen levels defined by their partial pressure (typically measured in mmHg or kPa), not by percentage. Matching these conditions exactly in vitro leads to more accurate models of in vivo tissue environments.
• Enhanced Reproducibility:
  By measuring and controlling oxygen in terms of its partial pressure, researchers can eliminate a major source of variability—ensuring that a specified oxygen level is consistent daily regardless of external conditions.
• Translational Consistency:
  Reporting oxygen in unified units helps align cellular laboratory research with physiological data, improving the relevance and comparability of experimental outcomes.

The HypoxyLab™ hypoxia workstation overcomes the limitations of standard incubators with:

• Partial pressure calibration:
  Direct measurement in mmHg ensures accuracy, regardless of altitude or weather.
• Rapid deoxygenation:
  Achieves 5% O₂ (<10 mmHg) in <20 minutes and near-anoxia (1–2 mmHg) in <45 minutes.
• Real-time analytics:
  Touchscreen interface monitors O₂, CO₂, temperature, and humidity.
• Controlled gas flow:
  Reduces nitrogen consumption by 50% compared to traditional hypoxia chambers.
• Sterility:
  HEPA filtration and UV-compatible materials prevent contamination in long-term cultures.
• Media oxygen validation:
  Optional OxyLite™ sensor to measures dissolved oxygen in media, confirming physiological conditions.