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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. Continue reading to learn about maintaining cell culture integrity through temperature.

Temperature Swings Affect Cellular Function

We have known since the 1960s that changing the temperature higher or lower that 37ºC has a dramatic effect on mammalian cell growth kinetics [1]. The cells do not have to come all the way to room temperature during cell culture for there to be an effect on cellular function. Changes in mammalian gene expression can be detected after one hour at 32ºC [2].

Cell Culture Temperature and Time Out of Optimum

Human internal body temperature has been the starting point for incubation of human cell cultures for about 50 years, although there are some applications for lower or higher incubation temperatures. When we put cells in the incubator and close the door, it is easy to see the effect on the incubator. The temperature drops and the incubator compensates by adding heat. Incubators are tuned by the manufacturer to minimize any over-reaction to temperature drops that might drive temperatures above optimum. This means that the temperature has to come up slowly to avoid overshooting. So obviously, the more time the incubator doors are open, the more time it takes for the incubator to return to optimum temperature.

Variable Time Out of Optimum Can Add Variability to Your Experiments

Once the door is shut and the cells are back in the incubator, safe and sound, after handling, it is easy to think that they are at optimum. It seems like as soon as the indicator on the exterior of the room-air incubator reads “37.0” again, that the cells inside are experiencing physiologic temperature. However, it takes time for static cultures to equilibrate with the incubator.

The time that it takes for cell cultures to return to temperature after being manipulated outside of the incubator can vary depending upon many, many factors. These include how long the cells were out of the incubator and the heat transfer geometries of the culture, the vessels, and the incubator. The starting temperature of any fresh vessels and medium used, and how many vessels are new to the incubator also can affect the mass that has to be warmed, and therefore how long the cells are out of optimum.

Even after the indicator on the incubator reads “37.0” again.

All of these factors can also add variability from experiment to experiment if they are not the same over time. Is there the same number of flasks in your incubator this week as last week? How often has the door been opened today as compared with over a holiday? Some of these factors are very difficult to control, especially for a shared room-air incubator.

Reducing Time for Temperature Equilibration

Pre-warming all media to 37ºC is one way to help reduce time out of optimum in culture temperature. Many cell culturists put their medium in a waterbath to bring it to temperature before use. This is great temperature-wise, but introduces problems such as contamination from immersion of media flasks up to the neck in the swamp-like waterbath that many research labs maintain (or don’t maintain).

Pre-incubation of smaller amounts of medium (just the volume you need) in a filtered flask in the incubator can be a less risky option. In the incubator, not only do you avoid the swamp, but pH and gas content can stabilize along with the temperature before the cells are exposed. In addition, medium isn’t subjected to repeated episodes of warming before the whole bottle of medium is consumed. However, there is the risk of chilling cell cultures that share that incubator with your pre-equilibrating medium.

The best option is to pre-incubate your medium to optimum conditions in an extra incubator, sans cell cultures. There the medium can come to optimum temperature without disturbing neighboring cell cultures.

Working with Cells in a HEPA-filtered Room-Air BSC Can Chill Cultures

Room air is hypothermic, hypocapnic, and hyperoxic for cells. The more time it takes you to work with them out in room air, the more time they are out of optimum for all of these critical cell parameters. Full-time optimization of the cell culture workspace is what cells need. But if you have to work with your cells in a room-air BSC, organize all needed materials before work starts, and then work quickly and smoothly, minimizing the mixing of liquids with room air.

Any questions? Contact us here at the Cytocentric Blog.

1.         1. Watanabe, I. and S. Okada, Effects of temperature on growth rate of cultured mammalian cells (L5178Y). J Cell Biol, 1967. 32(2): p. 309-23.

2.         2. Fujita, J., Cold shock response in mammalian cells. J Mol Microbiol Biotechnol, 1999. 1(2): p. 243-55.


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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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