Cytocentric Blog

What Oxygen Level Does My Cell Culture Actually Experience?



Well, it depends upon your protocol.

Just like in the perfect cookie recipe1, there are many factors in cell culture that affect your results. There is one set of ingredients or reagents that we don’t often think about when placing cultures into the incubator and shutting the door: the gasses. Even the 5% CO2 reading on the incubator isn’t what your cells are actually experiencing; not until the cell cultures have equilibrated with the incubator.

That takes time: time that your cells are out of optimum conditions.

Likewise, with an oxygen-controlled incubation chamber, it takes time for cell culture to equilibrate with the incubator oxygen levels. There are multiple levels that have to come to equilibrium first. There is the gas phase outside of the vessel, the headspace within the vessel, the gas/medium interface, and the medium at the pericellular level.

At each of these levels, different factors, determined by your protocol, change the exchange rate and the time that your cells are out of optimum. Let’s break it down.


The Biology of HIF Proteins Impacts the Outcome of Your Experiments in Physiologic Oxygen: Considerations for protocol design


We recently posted an article “What Oxygen Level Should I Use for My Cells In Vitro?” in response to questions that we have fielded from customers getting started in cytocentric research. To expand on that topic, we are posting here a few more resources for information on the mechanism of how changes in oxygen levels impact cells in culture.

Cellular responses to oxygen are mediated in part by hypoxia-induced factors (HIF-1 and -2 protein families). These transcription factors control a wide variety of cell functions, and are among the most rapidly regulated proteins known to date. If you culture your cells at physiologic oxygen levels and expose them to supraphysiologic oxygen during room air handling, it can affect your results.


Mesenchymal Stem Cells for Cellular Therapies and Regenerative Medicine: Cytocentric oxygen control makes sense from the molecular level through to the bottom line.


Mesenchymal stem cells (MSC) have shown tremendous promise as a cellular therapy for indications as varied as arthritis [1], diabetes [2], cardiac disease [3], wound repair [4], graft-versus-host disease [5], ALS [6], spinal cord injury [7], even infectious disease [8, 9]. MSC also have the potential to transdifferentiate into multiple cell lineages for regenerative medicine applications [10]. But for MSC to live up to that promise, the best conditions for expanding and studying these critical cells in the laboratory must be established.



What Oxygen Level Should I Use for My Cells In Vitro?


Well, it depends.

Are you looking for physiological relevance or comparison with cells cultured in a standard room-air incubator?

First, you should know that there is nothing normal or “normoxic” about room air oxygen for cells that normally reside inside the body.In vivo, as soon as air enters the body, it mixes with CO2 being expired and the percentage of oxygen decreases.


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Biocontainment: Cells Need Technician Protection

“I’ve been handling cells this way for years and I’VE never gotten sick.”

How many times have you heard that, and doubted it?

Confidence in handling potentially dangerous cells, tissues, and vectors, can easily become complacency. Now in an uncertain era of CRISPR/Cas GMOs and gene drives, biocontainment and biosafety have taken on more urgency.