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bp10 headerThe Reproducibility Crisis and the CytoCentric Environment

There has been a lot of public attention directed at scientific reproducibility, most recently in articles in PLOS Biology, and in the Washington Post. A National Academy of Sciences workshop, and a University College London event, have also focused public attention on the etiology of irreproducible biomedical studies. The inability to get the same results in study after study has tremendous economic and social impact on our country. Continue reading our post below to learn why a cell focus in research is so critical and how cytocentricity helps scientific reproducibility.

There are different levels at which processes, materials and data can be compared and there is an associated level of variability between each of them:

• Intra-Laboratory Experiment to Experiment or Batch to Batch

• Intra-Laboratory Researcher to Researcher

• Inter-Laboratory

• Translation Pre-clinical to Clinical Phase

• Translation Phase I/II to Phase III

While there is a lot agreement that variable starting materials and poorly written methods contribute a great deal to the generation of irreproducible studies, there has not been a lot of discussion about the contribution of the cell culture environment to variability.

In most cell culture laboratories, incubators are shared. See our previous post about how “Cells Need Protocol” for more discussion of simple ways to reduce risks to individual cultures in shared incubators.

Cells in vitro are completely dependent upon the local environment for oxygen and carbon dioxide. There are multiple levels of environmental gas exchange in vitro:

• Between cells and cell culture media

• Between media and any headspace in the cell culture vessel

• Between the vessel and the incubator

• Between different regions of the incubator

• Between the incubator and the outside environment

When the incubator environment is disturbed, the cells can be affected. The frequency of disturbance can vary tremendously from experiment to experiment, batch to batch, incubator to incubator, lab to lab.

Let’s start with just one level of gas exchange – between the incubator and the outside environment.

What happens to oxygen and CO2 levels when the incubator door is opened?

We did some experiments in which we answered this question with different sized oxygen-controlled incubators.

This is what happens when we turned off the oxygen control in a large incubator and opened the door twice – just long enough to remove a T-75 flask from a shelf and 20 minutes later, to replace it.

The average oxygen level we have found in standard CO2 -controlled incubators is 17.2 +/-0.3% (n=4 incubators in 3 different institutions) after a single, brief door opening event. This level slowly drifts up over several days to about 19%, if the door is kept closed.

This was a large incubator that was less sensitive to large swings in gas levels when the door was opened briefly. Here are the swings in O2 and CO2 experienced in a small incubator that is opened frequently:

Here the oxygen level is initially diminished by the CO2 charge. Subsequent incubator breaches drive oxygen up as room air (~20.8% O2) enters, and then back down again as CO2 levels are restored.

These findings have two important implications:

1. Incubator oxygen levels are not equivalent to room air oxygen levels.

2. Incubator oxygen levels are quite variable due to incubator breaches.

Was your incubator opened the same number of times this week as last week? That is hard to determine or control, especially in a standard shared-lab setting.

Could the variation in incubator gas levels be a source of variability in experiments?


So is there a way to reduce the variability in the in vitro cellular environment and so, reduce irreproducible results from cells?

One way is to enforce strictly reproduced incubation conditions on every experiment, even down to the frequency and timing of incubator breaches. This requires extremely dedicated staff and the maintenance of identical conditions if the process is ever transferred, scaled-up, or scaled-out. Not very practical.

An easier way is to open the incubator only into an atmospherically-controlled workspace, so that these swings in atmospheric content don’t occur in the first place.

Using these engineering controls to maintain constant incubator conditions is the Cytocentric approach to controlling the variability in the cellular environment that can add to irreproducibility of in vitro studies.


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About The Author

Alicia D Henn, PhD, MBA

Alicia D Henn, PhD, MBA

Chief Scientific Officer of BioSpherix, Ltd



Alicia Henn has been the Chief Scientific Officer of BioSpherix, Ltd since 2013. 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.



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