Examining the CO2 Incubator Myths

Cell culture is integral to the biological, biomedical, and biopharmaceutical sciences. CO₂ incubators, in turn, are integral to cell culturing.

Incubators, at their core, maintain optimal growth conditions. They offer carbon dioxide control (5 to 10 percent helps regulate pH using standard buffers), constant temperatures (37°C for most mammalian cells), and humidity control (95 percent prevents evaporation). They may also come with optional features around contamination prevention, space-savings, and establishing hypoxic or other extreme physiologic conditions.

When choosing a CO₂ incubator, certain myths and misinformation surface. A clear look at the technology behind incubators helps distinguish fact from fiction and better informs purchasing decisions. What features does an incubator actually need? Here we explore the three most common myths alongside familiar lab scenarios.

MYTH: FAN-ASSISTED INCUBATORS PROVIDE MORE UNIFORM GROWTH CONDITIONS

Maia was noticing a pattern. There had been clear variation in the performance of different cell cultures, but she was beginning to nail down the culprit. Performance seemed to depend on where her vessels were kept in the incubator, despite it being a recent purchase. Those on the bottom back left of the incubator were slower to hit confluency, while most of those in the top were unstable. Those along the back of the chamber seemed patchy in distribution as well.

Her first thought was that the door must be getting left slightly ajar, though that didn’t correspond to the pattern she saw. Yet, it had to be external. Didn’t it? She’d already confirmed that everything was still working. This model was supposed to be the one of the best for maintaining fully homogenous conditions within the incubator, advertising rapid recoveries with gentle, fan-assisted airflow. So how could this be happening?

Temperature uniformity across and between shelves within the incubator is critical to producing consistent, reproducible results. A difference of just one degree Celsius can impact cell morphology and colony density¹. Like Maia, you may have heard that you need a fan to achieve that level of uniformity, including rapid recovery following door openings, from top to bottom and side to side in your incubator.

Strong, directional airflow, however, can disturb cell cultures, increase evaporation of media, and aid the spread of contaminants. Fans also introduce vibrations that may affect cell distribution and attachment. Published guidelines for best practice in cell culture maintenance recommend fanless incubators for these reasons².

So, how does one achieve even conditions throughout the incubator chamber without a fan? It comes down to heating element and sensor design. Well-designed fanless units are capable of circulating air gently through natural convection currents. In a comparison of the fanless CellXpert CO₂ incubator and fan-assisted incubators using 27 temperature sensors, the fanless unit offered the greatest temperature uniformity across the incubator¹ . It also demonstrated recovery within five minutes after door openings without overshooting the target temperature.

MYTH: HEPA FILTERS ARE THE BEST OPTION FOR PREVENTING CONTAMINATION WITHIN INCUBATORS

The turbidity was a dead giveaway—the cultures were contaminated once again. Hector swallowed his annoyance, tossed the cultures and reagents, and scrubbed everything down. Again. He was fairly certain this was a technique issue, with two new students. His incubator was top-of-the-line for contamination control with copper interior, a HEPA filter, and built-in decontamination features. This should be easy. Additional training and reminders would be needed.

A few months later, things were sailing smoothly in Hector’s lab with no signs of contamination since the previous incident. Something was bothering him though. Had the cultures been slowing down? The latest results seemed slightly off. Diluted, compared to earlier work. The cells looked fine, though. He searched back through the archives. Yes, definitely a gradual decline in signal. It was difficult to tell for sure, but he thought certain conditions started weakening later. Almost like it was catching. With a sinking stomach, he dug out a mycoplasma PCR test kit. Positive. Another reset. Another delay. He’d seen huge improvements in technique, so where was this coming from?

Mycoplasma contamination is a concern in cell culture labs around the world. A suite of assessments in the 1990’s estimated its prevalence to 10 to 80 percent of cell line cultures in various countries. While it’s expected that infection rates have trended down over the years with increased testing and awareness, one 2015 study found mycoplasma sequences present in 11 percent of 884 published RNA-seq datasets³. Further, they found the expression of 61 genes was associated with mycoplasma contamination levels.

Mycoplasma is an insidious contaminant. It’s frequently spread through cross contamination between cultures but can come from almost anywhere. It is difficult to get rid of. It’s resistant to antibiotics and, at the 0.1 to 0.3 micron size range, flows right through HEPA filters⁴.

This brings us to our second myth, that HEPA filters clean the airstream in the incubator of all sizes of particles and microorganisms, offering the best protection against contamination within incubators. HEPA filters are only effective at filtering particles and organisms down to 0.3 microns in size. While HEPA filters are great at filtering out most contaminants—assuming the filters are changed regularly—the fan rapidly circulates the smallest contaminants throughout the chamber. 

The addition of the fan and ducts required for air filtration also increase surface complexity within the chamber adding seams, corners, and nooks that are more difficult to thoroughly clean and disinfect.

Recommended contamination controls—in addition to using proper technique combined with regular cleaning and disinfection protocols—include seamless copper and copper alloy interiors with removable shelving and racking to help with cleaning and disinfection efforts².

Additional features can improve contamination control. UV can be very effective at disinfecting airflow, but its disinfection area is limited to where the light touches. Some incubators also have built-in decontamination capabilities using H₂ O₂ or heat disinfection to facilitate cleaning.

MYTH: WATER-JACKETS PROVIDE THE BEST TEMPERATURE REGULATION

Anna was ecstatic to be inheriting a water jacket CO₂ incubator. She’d only used direct heat and air jacket models in the past, but had heard these were the best at temperature regulation, even offering peace-of-mind for power outages. She hadn’t thought about how long it would take to reach set temp though. Well, tomorrow would be as good a day as any to move samples.

After a few weeks passed, Anna was not impressed. She’d taken to checking the temperature readouts on the two incubators every time she passed them. The direct-heating incubator seemed to be more consistent, recovering more quickly from openings. She added additional sensors to check accuracy on the readouts. The same. Now, needing to move the incubator just a few feet to the left, she was realizing she’d have to drain the jacket. Anna tried to remember why she’d wanted a water jacket. Squatting in front of the incubator, she opened it to find moisture. Surely not … yes. It was leaking.

Water-jacketed CO₂ incubators may have been the best option at one time, but current technology enables excellent uniformity using 3-dimensional direct heating. The key to temperature uniformity lies with six-sided heating using individual heating circuits and multiple, fast-feedback temperature sensors⁵. Fast recovery is achieved through fine temperature control that generates gentle convection circulation.

Most models now come with air jacket heating, direct heating, or a combination. Both air jacket and direct heating allow for a slimmer, lighter-weight incubator requiring less maintenance, and both can be self-sterilizing through high heat.

Ultimately, the myths purporting that the best contamination control and temperature uniformity and consistency are provided by fan-assisted, HEPA-filtered incubators with water jackets are just that—myths. Misleading advertising claims have encouraged their persistence, sometimes to the detriment of the purchaser.

When combined with best practices in cell culturing, well-designed, fanless incubators with direct heating, seamless interiors, and high temperature disinfection can offer the best temperature and contamination control.

REFERENCES 

1. Wagener, J., Stöhrer, F. & Tacheny, A. (2007) CO₂ Incubator Temperature Control: What Is the Best Place For Your Cell Culture Vessels? 

2. Geraghty, R. J. et al. (2014) Guidelines for the use of cell lines in biomedical research. Br. J. Cancer 111, 1021–1046. DOI: 10.1038/bjc.2014.166. 

3. Olarerin-George, A. O. & Hogenesch, J. B. (2015) Assessing the prevalence of mycoplasma contamination in cell culture via a survey of NCBI’s RNA-seq archive. Nucleic Acids Res. 43, 2535. DOI: 10.1093/NAR/GKV136. /pmc/articles/PMC4357728/. 

4. Hartmann, I. K. & Jarvis, J. (2015) Effective Contamination Control with CO₂ Incubators. 

5. Hartmann, I. K. & Wagener, J. CO₂ Incubators – Making the Best Choice for Your Lab.