Reproducibility and Variability in Critical Cell Culture Parameters - Temperature
Keep the cells in log-phase growth for batch after batch. Rinse and repeat. The cells should be the same every time you need them. Seems like a simple thing, right?
But it never is that easy to get consistent cell growth. That’s why we spend so much time and money checking the cells. Do they still express the right markers? Before researchers even get to the biologically question at hand, a lot of space in scientific reports is dedicated to the simple question; “Are the cells being used the type of cell that they are supposed to be?”
Here at the Cytocentric blog, we take the cell’s point of view. So what is really important to the cells in your care for consistent phenotype and function? Beyond cell density and viability, historically measurable parameters that have been used as indicators of cell culture health include: temperature, pH, osmolarity, lactate, carbon dioxide, and oxygen levels. Tests for contaminants such as mycoplasma, cross-contaminating cell types, and functional tests are also increasingly important for the monitoring of cell culture status. The more valuable the cells, the more important are all of these measures of culture integrity. Today we start with temperature. We will address other parameters in subsequent posts.
What Can I Do To Get the Best Results from the Non-Physiologic Cell Handling Conditions of a Room Air BSC?
We 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 vivo condition and this is best in vitro as well. Changes in HIF protein levels have been found within minutes of oxygen changes . It can take up to 16 hours for cell culture to return to low oxygen levels in the incubator. 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.
"Should We Call Physiologic Oxygen Hypoxia, Normoxia, Physioxia, or Something Else?"
How we describe oxygen in the microenvironment is important.
The use of physiologically relevant oxygen for in vitro cell culture is increasingly essential as cells grown in vitro become more clinically important. Oxygen levels are a critical cell parameter, just like carbon dioxide or temperature. Hyperoxia simply means too much oxygen, Normoxia means normal amounts, and Hypoxia means too little.
The term “Hypoxia” is used in two different frames of reference.
In the scientific literature, hypoxia is often used to describe physiologic oxygen levels that are lower than room air. Other researchers use same term for low oxygen conditions such as ischemia that are pathophysiologic. In these conditions, oxygen levels are too low for that particular tissue type in situ.
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.