What Can I Do To Get the Best Results from the Non-Physiologic Cell Handling Conditions of a Room Air BSC?

bp28 images01We posted previously about how the Biology of HIF Proteins Impacts the Outcome of Your Experiments. Of course, full-time control of cell handling conditions including oxygen, CO2, and temperature is the in vivocondition and this is best in vitro as well. Changes in HIF protein levels have been found within minutes of oxygen changes [1]. It can take up to 16 hours for cell culture to return to low oxygen levels in the incubator[2]. Cells Need Optimization and the longer the cells are out of optimal conditions, the more likely they are to be affected by those conditions. Adapting to Cytocentric cell culture techniques means that routine passaging of your cells is not business as usual.

I get it. I culture my cells in physiologic oxygen, but I have to handle my cells in a room air BSC (biological safety cabinet). What can I do to protect my cells from oxygen swings?

If you must handle cells in room air, we strongly recommend planning your experiments to minimize pericellular oxygen changes. In the incubator, pericellular oxygen levels are completely protocol-dependent. This is true in the BSC (biological safety cabinet) as well.

Pre-Equilibrate Cell Culture Media and Other Solutions

Unlike carbon dioxide[3], which acts more like a polar molecule in aqueous solutions, dioxygen (O2) is nonpolar with equal dipole moments. It does not dissolve well in polar aqueous solutions. This means that cell culture media are very slow to equilibrate with incubator oxygen in static culture (hours to days, depending upon medium depth, air-liquid interface area, and vessel type).

Pre-equilibrating cell culture media to the desired oxygen levels before using with your cells can give your incubator a better place to start for re-equilibration once the cells are safely back. If you are able to do so while maintaining sterility of your media, you can sparge medium with nitrogen gas to reduce the partial pressure of oxygen. In addition, pre-equilibrate plasticware you intend to use for your cells.

Factors that can influence the length of time needed for pre-equilibration or re-equilibration of medium include the type of vessel used, the starting concentrations of the chamber and the medium, the depth of the medium, and the surface area available for gas exchange. Lay flasks flat in the incubator, rather than stand them upright.

How Long? Overnight at least [24].

Is there a maximum time that I can have the cells out in the room air BSC (biological safety cabinet)?

We often get this question from people getting started in Cytocentric research. The oxygen exposure that your cells will suffer depends so much upon the specifics of the cell handling procedure that it is very difficult to set a maximum time that cells can be handled in room air without affecting their function. Reduce the amount of time that cells are out of optimum for the better results. Plan your protocol well and organize supplies ahead of time. Work smoothly without disruption so that cells, vessels and media are not in room air any longer than necessary.

Avoid mixing in room air

The more that cells, media and other liquid components are mixed with HEPA-filtered room air in the BSC (biological safety cabinet), the more the medium equilibrates with room air oxygen.

Pipette pre-equilibrated media carefully against the side or bottom of vessels to minimize mixing with HEPA-filtered room air in the BSC (biological safety cabinet).

Work fast and cold for HIF detection

HIF proteins are polyubiquinated and begin breaking down inside the cell within minutes of elevated oxygen exposure. Pre-chill wash buffer, conical tubes, and your centrifuge. Keep samples on ice as much as possible until they are used for analysis or placed in cryostorage.


Questions? Contact us This email address is being protected from spambots. You need JavaScript enabled to view it. at the Cytocentric blog. We will do our best to help you get started.

1.         1. Jewell, U.R., et al., Induction of HIF-1alpha in response to hypoxia is instantaneous. FASEB J, 2001. 15(7): p. 1312-4.

2.         2. Newby, D., L. Marks, and F. Lyall, Dissolved oxygen concentration in culture medium: assumptions and pitfalls.Placenta, 2005. 26(4): p. 353-357.

3.         3. Carroll, J.J., J.D. Slupsky, and A.E. Mather, The solubility of carbon dioxide in water at low pressure. Journal of Physical and Chemical Reference Data, 1991. 20(6): p. 1201-1209.

4.         4. Mantel, C.R., et al., Enhancing Hematopoietic Stem Cell Transplantation Efficacy by Mitigating Oxygen Shock. Cell, 2015. 161(7): p. 1553-65.


alicia author iconAbout the Author

Alicia D Henn, PhD, MBA

Alicia Henn has been the Chief Scientific Officer of BioSpherix, Ltd for two years. Previously, she was a researcher at the Center for Biodefense Immune Modeling in Rochester, NY. Alicia obtained her PhD in molecular pharmacology and cancer therapeutics from Roswell Park Cancer Institute in Buffalo, NY and her MBA from the Simon School at University of Rochester in Rochester, NY.