Measuring Pericellular Oxygen Tension in Mammalian Cell Culture

Introduction
Mammalian cell culture is a technique in which scientists attempt to mimic conditions like those found in the body. Oxygen (O2), which plays a key role in tissue function and pathophysiology, is not controlled in conventional cell culture. To overcome this, incubators that control O2 in the gas phase that surrounds cells have been developed and are commercially available. For this method, it is generally presumed that the gas phase O2 concentration is equal to the pericellular O2 concentration, or the O2 that cells experience in cell culture media. Using O2 sensor spots, we examined the relationship between gas phase and pericellular O2 tensions in standard physioxic (5% O2) and hypoxic (1% O2) cultures.

Fig. 1: Measuring pericellular oxygen (O2) tension in mammalian cell culture using sensor spots. (A) Self-adhesive sensor spots (OXSP5-ADH) were placed on the surface on which cells were grown. 6-well plate wells are shown as an example. (B) Cable adapters (SPADBAS) were glued (SPGLUE) onto the backside of the cell culture vessel. Cables (SPFIB-BARE) were fastened within the adapters and connected to a meter (FSO2-C4).

Method
To measure pericellular O2 concentration in cell culture PyroScience FireSting technology was used. O2 sensor spots (OXSP5-ADH) were placed on the bottom of cell culture vessels – the surface on which cells are grown (Fig. 1A). Next, cable adapters (SPADBAS) were glued (SPGLUE) onto the backside of the vessel, aligned with the sensor spot (Fig. 1B). Cables (SPFIB-BARE) were placed within the adapters and connected to a meter (FSO2-C4).

Fig. 2: Cellular O2 consumption can induce hypoxia and anoxia in physioxic (5% O2) and hypoxic (1% O2) cell culture, respectively. (A-B) Pericellular O2 profiles (left) and average O2 tension values (right) of media, human mammary epithelial, MCF7, or MDA-MB-231 in a 24-well plate in a 5% O2 (A) or 1% O2 (B) incubator for 48h.

Results and Conclusion
Primary mammary epithelial cells, MCF7, or MDA-MB231 (breast cancer epithelial cell lines) were seeded sub-confluently onto wells containing sensor spots (21,000 cells/cm2) and let attach in normoxia overnight. Cultures were then placed in either physioxic (5% O2) or hypoxic (1% O2) incubators for 48h. Media (EMEM +10% FBS + 1% P/S) was included as a control. Media O2 tensions were in agreement with incubator setpoints, suggesting that the O2 sensor spots and the incubator O2 sensors were recording accurately (Fig. 2A-B). In 5% O2 culture, all three cell type cultures were below 1.5% O2 and MCF7 cultures were anoxic (anoxia defined as <0.5% O2) (Fig. 2A). In 1% O2 cultures, all cultures were anoxic (0.0% O2) (Fig. 2B).

In summary, we have devised a new method to measure pericellular O2 tension in O2-controlled cell culture. Using this method, we discovered that pericellular tensions are far below incubator setpoints in both standard physioxic and hypoxic cell culture protocols. To accurately control O2 in cell culture, we highly recommend measuring pericellular O2 tension. For more details please visit: https://www.biorxiv.org/content/10.1101/2023.10.02.560369v1.abstract

Northeastern University, Boston, MA, USA