Hyperoxia Cell Oxidative Model System

Constructing various cell models of oxidative stress is the basis of cell aging, antioxidants and so on. The in vitro test system using cultured cells will be a useful intermediate screening test, which can avoid unnecessary animal experiments. Generally speaking, it is necessary to evaluate the substance with a simple in vitro test system, and then confirm the results with in vitro and in vivo test systems.

Hyperoxia can be defined as oxygen concentration "higher than normal", so it is a relative measurement. Generally speaking, 20% O₂ is "normal oxygen", and exceeding this value (40% O₂ or 60% O₂) can be considered as high oxygen environment. High levels of oxygen metabolism will produce a large number of reactive oxygen species intermediates (ROI), which will lead to cellular oxidative stress.

Unlike most methods of acute and high-level administration using chemical oxidants, hyperoxia environment is more suitable for chronic and low-level oxidative stress induction. In order to help our customers study and culture various cell types under specific oxidative stress conditions, Creative Bioarray has established a set of workflow of inducing cell oxidative stress with hyperoxia, which can establish various types of oxidative stress models for customers.

Induction of Cellular Oxidative Stress

Hyperoxia Cell Oxidative Model System

Exposure to hyperoxia is generally performed by incubating cell cultures to 98% O₂ + 2% CO₂ at 1 atm. (normobaric hyperoxia) for several days.

We will monitor the actual oxygen tension in the incubator and avoid atmospheric disturbance caused by the opening of the door. In many cases, we also culture cells in glass culture bottles, rinse them with the required gas mixture, and then seal them with silica gel plugs. In such systems, the oxygen tension is stable because the oxygen consumption of cells is usually negligible.

SOP for Cell Stress Model Evaluation

We will evaluate the oxidative stress of cells through the following parameters

Loss of clonal cell survival, inhibition of DNA synthesis, decrease in NAD levels, decrease in ATP levels, DNA strand breaks, chromosomal aberrations, sister-chromatid exchanges, mutations.
The growth changes of cells treated with hyperoxia were indirectly measured by measuring the absorbance of MTT dye.
The intracellular ROS level was detected by oxidation sensitive fluorescent probe dye dihydroethidium.
Intracellular glutathione levels were analyzed using 5-chloromethylfluorescein diacetate.
The activities of SOD and catalase were measured.
Morphological observation. During the exposure of Chinese hamster cells to hyperoxia, some morphological changes were observed: the number of lysosomes increased, pyknosis and vacuolation increased, and mitochondria were damaged, which were characterized by ridge disorder, swelling, electron density and membrane gap reduction.

Comparison of Cell Stress Induced by H₂O₂ and Hyperoxia

Effect	H₂O₂	Hyperoxia
Loss of clonal cell survival	+	+
Inhibition of O₂ consumption	nd	+
Inhibition of glycolysis	nd	-
Inactivation of mitoehondrial enzymes	nd	+
Inhibition of DNA synthesis	+	nd
Decrease in NAD levels	+	-
Decrease in ATP levels	+	+
DNA strand breaks	+	-
Chromosomal aberrations	+	+
Sister-chromatid exchanges	+	+
Mutations	+	-

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