Mimicking cell growth conditions in incubators – 5 crucial factors to maintain cell culture in vitro

Every researcher knows that an appropriate extent of gas exchange, temperature, and humidity are non-negotiable to keep a cell culture alive. However, do you remember why and how a carbon dioxide incubator maintains these conditions or what we can do better when maintaining cells? Time for a recap!

Maintaining the cell growth conditions, asepsis, and nutrient availability are central to expanding mammalian cells in an incubator. © Green Elephant Biotech.

Both fast-growing suspension cultures and adherent cell lines depend on carefully balanced growth conditions to reach their full expansion potential. As the use of induced pluripotent stem cells (iPSCs) and mesenchymal stem cells (MSCs) grows in regenerative medicine and Cell and Gene Therapies (CGTs), thoroughly understanding the conditions that mimic the human body has become more than a goal.

Although in vitro data can never fully capture the complexity of in vivo systems, cell culture remains the most powerful tool for studying and manipulating intricate biological processes under controlled circumstances. To cultivate cells in a way that reflects physiological reality, researchers must manage a combination of physical and biochemical growth conditions, including oxygen transfer rate, temperature, and humidity, while also ensuring consistent nutrient supply and sterility (1).

The ability to maintain this delicate balance determines not only experimental success but also economic efficiency, as culture quality directly affects yields, reproducibility, and cost per batch. Among the technologies that make this possible, carbon dioxide (CO₂) incubators have long stood as the center of laboratory infrastructure. These systems recreate the microenvironment cells need to thrive, providing the ideal atmosphere for seed expansion and consistent performance across experiments, when operated according to best practices.

In this article, we address how incubators sustain the fundamental growth conditions for mammalian cell growth, why those parameters matter for long-term culture success, and how attention to details shapes operational outcomes.

Incubators have been around since ancient times

The concept of incubation traces its roots back to ancient Egypt, where clay ovens were used to nurture chicken eggs long before the idea of controlled environments existed. Fast-forward a few millennia, and the modern CO₂ incubator has become one of the commonest practices in mammalian cell culture. Since their first commercial appearance in the mid-20th century, incubators have transformed from simple warming boxes into precise microclimate systems that recreate the key physiological conditions of the human body – namely, CO₂, temperature, and humidity.

Inside, non-dispersive infrared sensors monitor CO₂ by measuring how the gas absorbs infrared light, while heat dissipation systems or electrical heating maintain the steady warmth of 37 °C. A water reservoir ensures high humidity, preventing the evaporation that could otherwise concentrate salts and stress the cells (2, 3). Yet, even the most advanced incubator cannot guarantee perfect sterility or nutrient consistency. Regular cleaning, monitoring, and maintenance are not only good practice but a necessity to prevent microbial contamination, which can quietly alter experimental outcomes.

Backup systems represent another instance of good practices in life science laboratories, meant to protect long-term experiments from power fluctuations, affecting the pre-programmed growth conditions (4). Lastly, current protocols increasingly consider the incubator’s size, energy efficiency, and sustainable use that balances performance with lower carbon footprints.

Cells know the difference between “mountain air” and “urban smog”

Among all growth parameters, gas balance is perhaps the most delicate. Inside the incubator, the air is carefully tuned to 5% CO₂ to buffer the medium’s pH and roughly 18–20% oxygen (O₂), depending on the cell type and experimental aim. This mimicry of physiological “breathing” is what keeps mammalian cells stable and ensures a sufficient extent of metabolic activity (e.g., glycolysis, Krebs Cycle, oxidative phosphorylation).

The equilibrium between CO₂ and bicarbonate (HCO₃⁻) underlines the importance of a proper gas exchange, ensuring the maintenance of intracellular pH and mitochondrial respiration. Low oxygen triggers hypoxia pathways, while excess oxygen or poor regulation leads to oxidative stress, causing fundamental damage through Reactive Oxygen Species (ROS) and Nitrogen Species (RNS).

Studies in human embryonic stem cells (hESCs) reveal that a relatively small amount of ROS is not always consequential. Under certain circumstances, the process can even promote differentiation and adaptation. But once the threshold is crossed, the same molecules accelerate senescence and metabolic exhaustion (5). Like with most substances, the composition of air supplied to the cells must be tightly regulated, as even the most essential resource can become toxic.

Temperature decides fate, not just comfort

In mammalian cells, temperature is not just about comfort – this growth condition often modulates identity, function, and survival. Most cells thrive between 36–37 °C, a range that mirrors the human body, but even minor deviations can alter O₂ solubility, enzyme kinetics, and cell behavior. Excessive heat (≥ 56 °C) leads to protein denaturation, while prolonged mild stress can trigger apoptosis or necrosis. In primary cultures, subtle temperature differences have also been linked to shifts in differentiation potential (7).

Relative humidity, typically maintained at approximately 95%, plays a quieter but equally critical role (8). Generated by evaporation from the incubator’s water pan, humidity prevents the culture medium from drying out. When this parameter drops, evaporation concentrates salts and nutrients, changing osmotic balance of the culture and leading to toxicity – a small oversight that can cost weeks of lab work (9).

Healthy cells are hungry cells

An incubator can regulate the growth conditions, but sterility and nutrition remain human responsibilities. Every researcher’s aseptic practices are meant to protect from toxicity and physiological impact carried by the infectious agents. One of the stealthiest threats is represented by the Mycoplasma genus, whose members can go unnoticed for long periods and result in the cell quality decline and apoptosis (1).

Feeding the cells is equally critical. Mammalian cells depend on a balanced medium of carbohydrates, amino acids, vitamins, hormones, and trace elements, which must be consistently replenished with growth. In large-scale or long-term cultures, a fed-batch system is often employed, supplying nutrients gradually and preventig nutrient depletion (10).

Mixing and aeration, whether through gentle rocking in lab-scale flasks or dynamic stirring in bioreactors, help prevent persistent gas or nutrient gradients within the medium. In poorly mixed colony, some cells may experience excess of resources while others – starve, creating metabolic inequality with the potential to skew data and reduce yield (10).

Continuous research efforts are pushing the boundaries of how we design and maintain the cellular microenvironment, striving to close the gap between incubated cultures and the complex in vivo niches they aim to represent. Providing cells with the right balance of growth conditions, namely – temperature, gas exchange, and humidity through automated systems, alongside assurance of sterility and nutrient availability, remains pivotal to that goal.

Modern incubators come in a variety of configurations, from compact benchtop units to advanced CO₂-controlled systems capable of real-time monitoring and fine environmental regulation. Yet the principle remains unchanged since the earliest days of cell culture: cells flourish when their surroundings are tuned, clean, and carefully monitored. At Green Elephant Biotech, we are committed to addressing analytical challenges in life science laboratories by incorporating these core principles in everything we do.

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References:

1. Segeritz, CP and Vallier, L, Basic Science Methods for Clinical Researchers (2017), pp. 151-172, Academic Press. doi: 10.1016/B978-0-12-803077-6.00009-6.
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3. ALL YOU NEED TO KNOW ABOUT CO2 INCUBATORS (2024). MrcLab. Available https://www.mrclab.com/all-you-need-to-know-about-co2-incubators#:~:text=Working Principle of Co2 Incubators,optimal conditions for your samples (Accessed 09 September 2025).
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7. Reissis, Y, Garcia-Gareta, E, Korda, M, Blunn, GW, Hua, J, The effect of temperature on the viability of human mesenchymal stem cells (2013), Stem Cell Res Ther, 14; 6: pp. 1-11. doi: 10.1186/scrt350.
8. Triaud, F, Clenet, DH, Cariou, Y, Le Neel, T, Morin, D, Truchaud, A, Evaluation of Automated Cell Culture Incubators (2003), SLAS Tech, 8; 6: 82-86. doi: 10.1016/S1535-5535(03)00018-2.
9. Consider Humidity in Your CO2 Incubator for Excellent Cell Growth (2024). Thermo Fisher Scientific. Available https://www.thermofisher.com/blog/life-in-the-lab/consider-humidity-for-excellent-cell-growth/ (Accessed 05 September 2025).
10. Bellani, CF, Ajeian, J, Duffy, L, Miotto, M, Groenewegen, L, Connon, CJ, Scale-Up Technologies for the Manufacture of Adherent Cells (2020), Front Nutr, 7; 575146: 1-14. doi: 10.3389/fnut.2020.575146.