The Effects of Temperature and Atmospheric Perturbation During Cell Culture: The Silent Variables

The Effects of Temperature and Atmospheric Perturbation During Cell Culture: The Silent Variables

The environmental confines of a laboratory incubator provide a controlled temperature and level of atmospheric carbon dioxide (CO2), in an attempt to replicate the cells’ native conditions; for mammalian cell culture, this mimics a cell’s environs in the host’s body – no small feat! This includes an aseptic environment with a specific atmospheric mix, maintained within a tightly controlled temperature and humidity range. When you stop to consider a typical cell-based assay workflow, it requires observation across several timepoints, and – more often than not – these timepoints require trips outside of the incubator for analysis. Have you ever stopped to think what these temperature and atmospheric excursions could be doing to your cells?


Did you know that even mild cold shock (25 ˚C) can permanently arrest cell proliferation?1 Based primarily on the body temperature of the host, mammalian cell growth is most efficient at 37 ˚C.2 In general, enzymatic activity and protein synthesis proceed optimally at 37 ˚C, however, a rise in temperature can cause sensitive proteins to denature, whereas a temperature drop can slow down catalysis and polypeptide initiation.3 Likewise, cellular growth too is retarded by low temperatures, whereas a more than doubling of the G1 stage, also referred to as the growth phase, has been observed at 31 ˚C.2 A lag in the G1 stage not only hastens initial cell growth, but delays eventual DNA synthesis and cell division. Although poorly insulated incubator doors and sample placement within the chamber also affect culture temperature, time spent out of the incubator represents a larger, more variable, factor with great potential to impact temperature cell health. 

A drop in temperature from 37 ˚C to typical laboratory temperatures (20 to 25 ˚C) can negatively impact your cells! While you may think the overall risk to your culture is minimal, the cumulative effects of time spent outside the incubator can add up. For example, the time spent outside will be greater for laboratories where the microscope or analytical equipment are not located in close proximity to the incubator. Taking into account the time spent imaging the cells and time in transit, it is not unreasonable to assume an out-of-incubator time of 15 to 20 minutes each time the cells are taken out. Should your cells require recurrent monitoring, this process may repeat every one to two hours until the desired result is achieved. As duration and frequency of non-incubator time increases, it becomes less likely that abbreviated incubation sessions will be able to return your samples to 37 ˚C.

Vessel volume and surface area can further influence rate of culture cooling. For instance, T-25 flasks hold up to 7.5 mL, while a T-75 flask holds as much as 22.5 mL. In terms of well culture, a single well on a 96-will plate can hold 200 μL, whereas 6-well plate contains up to 2.9 mL per well. As a general rule, more volume and less surface area will better retain heat. Consequently, one would expect plate-culture to cool quicker when compared to flask-culture, with high-numbered well plates being most prone to rapid cooling.

Atmospheric conditions

Neither flask- nor plate-based cultures are air tight, to allow gas exchange, so removal from the incubator exposes your culture to different atmospheric conditions. By volume, ambient atmosphere is composed of nearly 80% nitrogen, over 20% oxygen, and less than a half percent of CO2.4

Despite ambient oxygen being over 20%, very few cells in the human body are ever exposed to such a high concentration. In fact, the oxygenation of most human tissues lies between 1 and 8%.5 When tissues are overwhelmed with excessive levels of oxygen, cells may become damaged through the creation of reactive oxygen species. As such, setting incubator oxygen to 3% has been dramatically shown to increase cell proliferation, when compared to those cultured in 20% oxygen.6 High humidity, ideally at least 95%, is also important for efficient incubation. When humidity drops below 95% for an extended period, water will evaporate from the medium and will negatively affect the osmotic balance in the cells.   


Intracellular pH is determined by proton concentration and affects a myriad of cell processes, from ATP generation to protein folding. Accordingly, pH regulation is key to cell performance. In cell culture, most mammalian cell lines grow best at a pH of 7.4. Culture methodology generally employs a buffering system to keep pH within a target range, where an equilibrium between dissolved CO2 (bicarbonate ions) and a well-regulated (~5% (v/v)) CO2 atmosphere in the incubator is established. Supplying a constant level of CO2 ensures that equilibrium between water (H2O) and carbonic acid (H2CO3) in the culture media buffer is maintained. Although buffers slow the rate of pH movement, they cannot permanently arrest change.

Did you know that removing your cells from the incubator can have a profound effect on pH? Exposure to CO2-poor (<0.05%) room atmospheres causes bicarbonate (HCO3-) to leave the medium as gaseous CO2. The resultant decrease in hydrogen ions gives way to alkalinity and drives the pH up. Alkalinity is particularly toxic as it causes increased cell movement and cytoplasmic contraction with lethality established around 8.5.7,8 Generally, pH indicators such as phenol red are added to media so that changes in pH can be visualized in terms of color change. 

Other Culture Perturbations

While it seems obvious that removing your flask- or plate-cultures from the ideal incubation conditions of your incubator enhances the potential for error, the physical movement of cultures in itself constitutes risk. Although only applicable to adherent cultures, shear forces brought about from vessel jostling can negatively affect cell attachment.9

In addition to physical movement, laboratory lighting can have a negative impact on your culture. Stocks of culture media, culture additives, and prepared media are frequently kept in dark storage as they are sensitive to photodegradation, making frequent removal of cells from the incubator damaging under fluorescent lighting.10

Now that you’re tuned in to the realities of cell analysis assays, you’re probably keen to find a workaround for the silent variables. It’s time to consider the Essen IncuCyte!

Learn more about IncuCyte Live-Cell Analysis system


  1. Neutelings T et al: Effects of mild cold shock (25°C) followed by warming up at 37°c on the cellular stress response. PLOS ONE 8, e69687.
  2. Watanabe I et al: Effects of temperature on growth rate of cultured mammalian cells (L5178Y). The Journal of Cell Biology 32, 309–323.
  3. Craig N: Effect of reduced temperatures on protein synthesis in mouse L cells. Cell 4, 329–335.
  4. Composition of the atmosphere | Climate education modules for K-12. Available at:
  5. Lloyd D et al: Avoid excessive oxygen levels in experiments with organisms, tissues and cells In: Advances in Microbial Physiology 67, 293–314.
  6. Parrinello S et al: Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts. Nature Cell Biology 5, 741–747.
  7. Mackenzie CG et al: The effect of pH on growth, protein synthesis, and lipid-rich particles of cultured mammalian cells. The Journal of Biophysical and Biochemical Cytology 9, 141–156.
  8. Taylor AC: Responses of cells to pH changes in the medium. The Journal of Cell Biology 15, 201–209.
  9. Tchao R: Fluid shear force and turbulence-induced cell death in plastic tissue culture flasks. Toxicology in Vitro. 9, 93–100.
  10. Wang RJ: Effect of room fluorescent light on the deterioration of tissue culture medium. In Vitro 12, 19–22.
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