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March 15, 2011 (Vol. 31, No. 6)

Oxygen Monitoring in Cell Cultures

Real-Time, Noninvasive Measurement in Multiwell Plates Is Now Possible During Incubation

  • Cell culture has historically been performed at near ambient atmospheric (18–21%) partial pressure of oxygen (pO2). Special instrumentation is needed to maintain physiological levels of oxygen tension. In addition, hypoxia, defined as a lower than normal pO2 at the cell or tissue level, has long been known to be detrimental to cell metabolism and viability.

    Culturing cells in ambient atmosphere (21% pO2) insures the cells have “enough” oxygen but most cells experience an in vivo pO2 between 0.7 and 7%. Recent studies have shown that cells grown at 21% pO2 have altered phenotypes and gene-expression levels as compared to cells grown at a more physiologically normoxic level.

    Incubators and glove boxes can be set for a relevant physiologic pO2 atmosphere, but because cellular respiration removes O2 from the liquid media (rather than the gaseous atmosphere), the gaseous pO2 level for the incubation is not a good estimation of the pO2 the cells are actually experiencing. Instead, there is a need to measure pO2 at the level of the cellular monolayer, the pericellular pO2, to know the precise oxygen conditions to which the cells are being exposed.

  • Noninvasive Oxygen Detection

    Click Image To Enlarge +
    Figure 1. (A) SensorDish Reader with a 24-well multidish (OxoDish) for real-time monitoring of dissolved oxygen in a (B) cabinet, (C) glove box, and (D) incubator

    The SensorDish® Reader is a small apparatus that can optically detect pericellular oxygen or pH levels in 6- or 24-well plates during incubation (Figure 1). The plates (OxoDish® for O2) contain an integrated sensor spot at the bottom of each transparent well. The spots are illuminated by the Reader, which interprets the resulting phase angle shift in relation to O2 levels enabling noninvasive continuous monitoring of pericellular pO2 levels and can be a representation of cellular oxygen consumption.

    Real-time measurements of oxygen and pH in mammalian cell cultures are important to understanding the metabolic dynamics in cell cultures. The SensorDish Reader PLUS (SDR), available from Coy Laboratory Products, includes the tools for measuring temperature and pressure that are needed for the SensorDish Reader to measure pericellular pO2.

    The SDR allows the time course of cellular oxygen consumption to be followed in response to environmental conditions or toxicological insults. This type of treatment-effect monitoring could also be valuable for other applications such as tissue engineering and stem cell research.

  • Click Image To Enlarge +
    Figure 2. Average pericellular pO2 levels of MEF cells plated at differing densities (100,000, 33,000, 10,000, and 0 cells/cm2) and exposed to (A) ~19%, and (B) 7% ambient pO2 as controlled by a Coy O2 Controlled Glove Box. n=3 for each treatment. 1 mL media/well. At both 19% and 7% O2, diffusion is unable to keep up with the O2 loss in media due to respiration. Note the need to pre-equilibrate media to obtain the desired O2 level in the liquid.

    In the present work, the SensorDish Reader was utilized to monitor pericellular pO2 for cells plated at three different cell densities and varying gaseous oxygen tensions [19% pO2 (Figure 2A) and 7% pO2 (Figure 2B)].

  • Click Image To Enlarge +
    Figure 3. Average pO2 levels in the culture media of MEF cells exposed to antimycin, DMSO, and CCCP at four hours: n=4 for each treatment: 1 mL media/well with 1 mL/well of mineral oil to limit gaseous diffusion. Together the SDR and O2 Controlled Glove Box enable in-culture comparative studies of O2 utilization.

    In addition, the oxygen consumption rates in response to mitochondrial modulators (Figure 3) were also assessed. Mouse embryonic fibroblasts (MEFs) were used as a representative mammalian cell type, carbonyl cyanide m-chlorophenylhydrazone (CCCP) was used as a mitochondrial uncoupler, antimycin as an inhibitor of Complex III of the electron transport chain, and dimethyl sulfoxide (DMSO) as the solvent control.

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