In mammalian cells, changes in gene expression can already be detected after one hour at 32°C. In addition, modifications in cellular architecture, cell cycle progression, mRNA stability, protein synthesis, and protein degradation have been observed in different cell lines.
Temperature is an important parameter in cell culture because it can strongly affect the reproducibility of experimental results.
As early as 1960, Watanabe and Okada showed that temperature changes above or below 37°C have a marked effect on the growth kinetics of mammalian cells, similarly to what is observed in bacterial systems. (see here).
Effects of temperature on cell physiology
In mammalian cells, changes in gene expression can already be detected after one hour at 32 °C (see here or here) . In addition, modifications in cellular architecture, cell cycle progression, mRNA stability, protein synthesis, and protein degradation have been observed in different cell lines. (see here).
In addition to directly influencing cell growth, temperature changes also affect the pH of the culture medium.
This is because the solubility of CO₂ increases at lower temperatures, which in turn impacts the bicarbonate buffering system (it increases at lower temperatures, see here).
Optimal temperature and tolerance
Temperature requirements depend on the organism and the tissue of origin.
For example birds have a physiological temperature around 38.5°C and skin cells are often adapted to slightly lower temperatures than internal tissues.
However, overheating is generally more harmful than underheating.
Cultured mammalian cells can tolerate significant temperature reductions:
- they can survive at 4°C for several days
- they can be stored at −196°C for preservation (with appropriate protocols)
⚠️ However:
- cells generally cannot tolerate more than ~2°C above optimal temperature for extended periods
- they may die rapidly at temperatures around 40°C or higher.
Importance of temperature stability
For reproducibility, it is essential to maintain temperature as constant as possible, both during culture and during handling outside the incubator.
Cells should not be allowed to reach room temperature during manipulation, as this can alter multiple cellular functions.
Temperature variability in the incubator
You may have noticed that when the incubator door is opened, the temperature drops below the set value (usually 37°C).
After closing the door, the incubator temperature typically recovers within a few minutes.
However, the temperature of the culture medium does not recover as quickly.
Static cultures require time to equilibrate with the incubator temperature.
Factors affecting temperature recovery
Several factors influence how quickly cultures return to optimal temperature:
- duration of exposure outside the incubator
- type and geometry of culture vessels
- number of vessels inside the incubator
- initial temperature of media and containers
Direct contact of culture vessels with metal shelves can influence heat transfer. 👉 It is therefore recommended to avoid stacking vessels and to place each one directly on the incubator shelf.
How to minimize temperature fluctuations
Even if it is not always possible to control all variables (especially in shared incubators), fluctuations should be minimized as much as possible.
🧴 Medium preheating
Some researchers preheat the entire bottle of medium in a water bath at 37°C before use.
While this ensures correct temperature, it can introduce contamination risks due to contact with water.
👉 The use of antimicrobial additives in the water bath is recommended.
A better approach is to:
- divide the medium into smaller aliquots (e.g. sterile 50 mL tubes)
- place them in the incubator before use
👉 This reduces contamination risk and avoids repeated heating of the entire bottle.
An even better option is to place the medium in a sterile flask with a filter cap.
👉 This allows both temperature equilibration and pH stabilization.
Ideally, a dedicated incubator could be used only for pre-conditioning media.
However, this is not always economically feasible.
Inside the incubator
To maintain stable conditions:
- open the incubator door as rarely as possible
- close it quickly
- avoid creating “cold spots”
- do not overcrowd shelves
- allow space between vessels for proper air circulation
Perforated shelves improve air circulation and heat distribution.
However, they may create local temperature differences between metal surfaces and open areas.
👉 If uniform growth is critical, vessels can be placed on solid trays to improve uniformity
(although this is not always necessary for routine cultures).
Minimizing exposure outside the incubator
The laboratory environment is:
- hypothermic
- hypocapnic
- hyperoxic
compared to optimal culture conditions.
👉 The longer cells remain outside the incubator, the more they are exposed to non-optimal conditions.
Best practices when cells are out of the incubator
- organize all materials before starting
- work quickly and efficiently
- familiarize yourself with protocols to minimize delays
- minimize exposure of liquids to air
- remove medium from the incubator only when necessary
Room temperature control
Room temperature often fluctuates significantly between seasons. This affects the magnitude of temperature changes during manipulation.
👉 While it is not practical to maintain the room at 37°C, it is important to keep it as stable as possible (e.g. ~25°C). This avoids introducing seasonal variability in experimental conditions.