In vitro Cell Culture pO 2 is Significantly Different from Incubator pO 2 L. L. Bambrick Dept. of Anesthesiology, University of Maryland School of Medicine, Baltimore. MD 21201 Y. Kostov and G. Rao Center for Advanced Sensor Technology, Dept. of Chemical and Biochemical Engineering, University of Maryland Baltimore County, Baltimore, MD 21250 DOI 10.1002/btpr.622 Published online May 26, 2011 in Wiley Online Library (wileyonlinelibrary.com). Continuous noninvasive monitoring of peri-cellular liquid phase pO 2 in adherent cultures is described. For neurons and astrocytes, this approach demonstrates that there is a signifi- cant difference between predicted and observed liquid phase pO 2 . Particularly at low gas phase pO 2 s, cell metabolism shifts liquid phase pO 2 significantly lower than would be pre- dicted from the O 2 gas/air equilibrium coefficient, indicating that the cellular oxygen uptake rate exceeds the oxygen diffusion rate. The results demonstrate the need for direct pO 2 measurements at the peri-cellular level, and question the widely adopted current practice of relying on setting the incubator gas phase level as means of controlling pericellular oxygen tension, particularly in static culture systems that are oxygen mass transfer limited. V V C 2011 American Institute of Chemical Engineers Biotechnol. Prog., 27: 1185–1189, 2011 Keywords: oxygen, neurons, astrocytes, sensor, adherent cells, cell culture Introduction In vitro cell culture technology is little changed from its inception. An incubator with temperature/humidity/CO 2 con- trol and simple plastic disposable vessels filled with sterile media are typically employed. The vast majority of cultures are done under static conditions, where the vessels are sim- ply placed in the incubator. Recently, techniques to dynami- cally regulate the incubator atmosphere and thereby the conditions experienced by cells have become available. However, it is unclear to what extent the incubator atmos- phere affects the pericellular environment of static culture vessels. This question is the subject of the present study. Cell survival, proliferation, and differentiation are all regu- lated by the extracellular pO 2 . In vivo, arterial blood pO 2 is 90–110 mm Hg while in venous blood it is 35–40 mm Hg. The extracellular pO 2 seen by cells outside the vasculature varies from tissue to tissue and depends on blood flow, dis- tance from the vasculature and tissue oxygen consumption. Mean tissue pO 2 is about 22 mm Hg (3%) in adult, less in embryo. 1 Measurements with oxygen microelectrodes have given values of 10–40 mm Hg in brain, depending on brain region. 2 Because pO 2 regulates differentiation, gene expression, and metabolism, there is a need to measure and control this critical variable. This may be particularly true when consid- ering the potential differences between the pO 2 experienced by a cell in vivo and the pO 2 seen by the cell in tissue cul- ture medium exposed to room air (20.8% oxygen). Most in vitro cell studies are done in a 95% air, 5% CO 2 incubator environment. In the absence of cells, the resulting 20% O 2 gives a liquid phase pO 2 of 156 mm Hg—which would mean that cultured cells are exposed to extremely hyperoxic conditions. In fact, the actual pO 2 in close proximity to the cells, the pericellular pO 2 , is dependent on two variables: ox- ygen supply to the cells by diffusion through the media/ves- sel walls and oxygen consumption by the cells. For example, one study found that although primary cultures of endothelial and mesangial cells were hyperoxic (78–110 mm Hg), sev- eral passaged cell lines were hypoxic in a 20% O 2 , atmos- phere due to their higher density and higher metabolic rates. 3 In another study, microelectrodes were used to record depth profiles for oxygen in cell cultures under static condi- tions, and the authors found significant differences between measured pericellular oxygen tension and that in ambient air. 4 Typically, the actual pO 2 is an unmonitored and uncon- trolled variable in most in vitro experiments. Standard methods for measuring pO 2 in vitro have included the Clarke-type electrode and, more recently, the use of optical probes with immobilized fluorophores on the tip of an optical fiber. Another option is the use of oxygen sensitive fluorophores dissolved in the medium (see, e.g., Ref. 5 ). Both optical probes and Clarke-type electrodes have the disadvantage of needing to be positioned in the tissue culture dish (or flask) with an open connection back to their recorder, exposing the cells to contamination. Furthermore, their reading reflects the concentration in their immediate vi- cinity (i.e., at the probe immersion depth) and may not be representative of the actual oxygen concentration around the Conflicts of interest: G.R. and Y.K. have an equity position in Fluoro- metrix, Corporation. Correspondence concerning this article should be addressed to G. Rao at grao@umbc.edu. V V C 2011 American Institute of Chemical Engineers 1185