Can I put a shaker into a CO₂-incubator?

This article was published first in "Inside Cell Culture" , the monthly newsletter for cell culture professionals. Find more interesting articles about CO₂ incubators on our page "FAQs and material on CO₂ incubators" .

• Introduction – Why putting a shaker into a CO2 incubator?
• Influence of the shaker on growth conditions
• Shaker impact on contamination prevention
• Impact on shaker lifetime
• Use of a shaker in CO2 incubator – Additional considerations
• Summary – Can I put a shaker into a CO2 incubator?

Introduction - Why putting a shaker into a CO₂ incubator?

Mammalian cells in suspension are mainly cultivated in shake flasks to improve aeration and the exchange of nutrients and metabolic waste products in the cell-surrounding medium. These cultures are often used, e.g. for the expression of recombinant proteins or the production of bioreactor starter-cultures (inoculum). To ensure optimized cell growth, the temperature and pH of the medium needs to be controlled. Furthermore, the volume of the medium needs to be maintained to avoid harmful impacts by significant shifts in concentration of metabolites, salts, etc. This is especially critical because shake flask agitation increases medium evaporation. Appropriate growth conditions are provided by a CO₂-enriched atmosphere with high relative humidity that requires specialized devices. Two systems are commonly used: a) CO₂ resistant shakers that are placed inside a traditional CO₂ incubator and b) CO₂ incubator with an integrated shaker (Read more about the advantages and disadvantages of both systems and proper selection in our White Paper ).

In this article, we focus on the challenges of putting a shaker into a CO₂ incubator.

Influence of the shaker on growth conditions

Reliable growth conditions inside the CO₂ incubator are crucial to achieve optimal and reproducible cultivation results. In addition to proper CO₂- and temperature control, a high relative humidity, the avoidance of vibrations is another factor to consider, especially if simultaneous cultivation of adherent cells in the same device is planned.

The use of a shaker in a CO₂incubator introduces additional heat to the chamber environment. As CO₂ incuba­tors are thermally insulated and usually lack active cooling, this additional heat may disrupt proper temperature control of the CO₂incubator. A warming above physiological temperature of only one degree can make a difference to cell growth¹ .

Therefore, it is recommended to use a shaker with low heat dissipation.

If the CO₂ incubator is also used for cultivation of adherent cells, two additional challenges of putting a shaker inside it may occur. As the weight of a shaker can easily exceed the maximum shelf load (usually in the range of 10 kg/22 lbs), shelf bending may occur. Even if this may occur unproblematic at first glance, slight bending leads to a cultivation of vessels at a slope, thus difference of medium above the growth surface. In addition to shelf bending, shaker-induced vibrations can become problematic for parallel cultivation if vibration-sensitive cells like stem or primary cells.

Ideally, shakers should be used that are specialized for a use inside CO₂ incubators and the performance should be tested to ensure it doesn´t interfere with temperature control and airflow. If shelf bending or vibrations for adherent cells are a concern, a CO₂ incubator with an integrated shaker should be considered.

Shaker impact on contamination prevention

The warm, humid atmosphere inside the CO₂ incubator and nutrient-rich media used for mammalian cell cultivation provide ideal growth conditions for contaminants. In addition, cross-contaminating eukaryotic cell lines that can be introduced by earlier or simultaneous projects inside the same device. Regardless of its type, contamination prevention is crucial to avoid additional expenses by loss of sample material, the need to repeat experiments, combatting the contamination itself, and possible downtimes imposed by these measures. In addi­tion to the use of aseptic techniques, it is worth thinking about the cleanability of a shaker as a structural defense, especially given the labor invested in cleaning over it´s lifetime.

Contaminants preferably grow on moist surfaces or in liquids. Therefore, the shaker inside the CO₂ incubator should facilitate the detection and fast, thorough removal of medium spills. To ensure the exclusion of moisture inside the shaker, electronics and drive-related mechanical parts should be sealed. In addition, the performance of the shaker inside the CO₂ incubator should be tested to ensure it doesn´t lead to an increased contamination risk by condensation.

High-temperature disinfection for several hours has become the gold standard in CO₂ incubator disinfection. Thus, ideally also the shaker should be resistant to the temperatures used (120 – 180 °C) to avoid contamination. Even though, parts of the shaker can be autoclaved, there is currently no shaker on the market that is fully resistant to these temperatures. Therefore, it needs to be removed before disinfection of the CO₂ incubator. In addition, reinstallation of the shaker poses a high risk of (re)introducing a contamination by the device itself, but mainly by airborne contaminations due to prolonged long door opening during the installation.

If a high degree of contamination prevention is necessary, a CO₂ incubator with an integrated shaker and a high temperature disinfection for all internal parts is advisable.

Impact on shaker lifetime

The warm, highly humid (~95 % relative humidity) and slightly acidic atmosphere inside a CO₂ incubator is a challenging environment for a shaker, especially regarding corrosion. Therefore, it is advisable to select a specialized shaker with sealed electronics and drive parts that protect the device from the corrosive atmosphere and spills. For added resistance, the exterior should be made of corrosion-resistant materials like stainless steel or specialized plastics like Acrylonitrile butadiene styrene (ABS).

Use of a shaker in CO₂ incubator – Additional considerations

If you are considering buying a new shaker and/or CO₂ incubator for the cultivation of mammalian cells in suspension, there are additional aspects to keep in mind: Capacity/Throughput, device flexibility for various applications, shaker control and initial invest vs. running costs. Read more about these aspects in our White Paper .

Summary – Can I put a shaker into a CO₂ incubator?

A standard open-air shaker should not be used inside a CO₂ incubator for several reasons described above (risk of temperature control and airflow interference, condensation formation, contamination risk, impact on shaker lifetime etc.). Instead, a shaker especially designed for this use or ideally a CO₂ incubator with an integrated shaker should be used. Read more about proper selection of a system in our White Paper .

References

1. J. Wagener, F. Stöhrer, A. Tacheny 2020 - CO₂ Incubator Temperature Control:
What Is the Best Place For Your Cell Culture Vessels?

• White Paper “CO₂ Resistant Orbital Shaker Selection and Comparison with Integrated Devices”
• Monthly Inside Cell Culture Newletter
• CO₂ incubator FAQs and materials