Shakers in CO2 incubators: The key points to consider

The main benefits of using shaken cultures versus static are related to what you can get in a given time i.e.

• Better aeration and mixing leads to improved cell growth
• A move to suspension culture aids subsequent growth in bioreactors for scale up and makes physical transfer easier

This is fine in principle, but incubator shakers can be large, expensive capital items. If you have a limited budget or want to prove the benefits for your applications, an option is to use an existing laboratory incubator and add a shaker as a free-standing extra.

The first reaction is often to grab any simple laboratory shaker and just place it into the incubator. While this will appear to work at first, several factors will already be working against a successful outcome:

• The physical properties of the shaker define how useful it can be for specific tasks.
• What happens to the stability of the temperature control when the shaker is in operation?
• Do you have to open the door to adjust, losing heat and gas (for CO₂ incubators)?
• What happens to the shaker over time in terms of corrosion and damage to the controller?
• Does the design of shaker help prevent contamination?

A simple idea has now turned out to have a lot of factors for consideration. This brief overview will consider these factors in more detail and explain why a modest investment in the correct equipment will give returns in terms of capability, durability, and validity.

Take a look at the physical properties of the shaker

It goes without saying, that the shaker must fit into the incubation chamber. Next to the correct dimensions and capacity, a suitable small shaker should also provide the throws and shaking speeds typically used for larger incubator shakers, so that you can compare like with like.

Shaker capacity:

• A shaker with a large capacity e.g. a dozen or more 250 mL Erlenmeyer flasks, will allow for multiples of flasks per experiment, so allowing for statistical validity.
• Depending on flask choices, enough material may be produced for further processing without the need to move to a small bioreactor.
• A suitable volume of culture could supply the seed for inoculating a bioreactor.
• A large capacity could be used for tubes, micro-well/deep-well plate or even small bags.

Shaking throw:

This is not so critical with laboratory shakers used for chemical work. For biological cultures, certain throw diameters are used from the smallest to the largest shakers. A shaker with a standard throw will allow easier scale-up. Common throws are 19 mm, 25 mm and 50 mm diameter.

Speed range:

To cover cell culture, a range from 50 min-1 to 150 min-1 is optimal, depending on the shaker throw.

Beware of additional heat

Adding heat to the chamber may de-stabilize temperature control and lead to poor growth. This is because the motor driving the shaker platform can get warm or even hot in an incubator. The incubator chamber is usually gas tight and that also restricts air movement, which could cool the motor. As temperature control in the incubation chamber does not account for this extra heat, it can lead to problems with the stability of temperature control. For cell culture, even a slight warming of a degree Celsius can lead to poor growth or cell death. Use of a drive system with almost no heat output can reduce this effect significantly

You need to have clear access to the controls

This point has three elements:

• If the electronics can be mounted outside the incubator, they cannot either add heat or be affected by it.
• Adjustments in speed or setting timers do not involve opening the chamber door, so helping to keep a constant temperature and reduce losses of gas, if relevant.
• An external display can display current speed and warn of any events such as power loss or expiration of a timer immediately.

A bonus is if the controller outside the incubator is also the connection for mains power. If so, a lower voltage can be passed to the shaker mechanism. This also puts heat generating from the transformer outside as well.

Having control from outside is an advantage. Example of an shaker operating panel magnetically connected to the incubator door.

Corrosive resistant materials ensure a long lifetime

The inside of a static incubator is not a friendliest environment for equipment with moving parts and electronics. The prime factors are heat, humidity and CO₂. None of these conditions apply in the main laboratory environment. All then can influence the operation and durability of a shaker placed inside the incubation chamber. This is the key reason for selecting a shaker made explicitly for the job rather than a general laboratory unit.

Heat (up to 60°C)

• May dry out any thin lubricants used on moving components such as bearings over time.
• Could contribute to a shortened life for electronic components.
• Cause the shaker to run hot and so reduce its useful life.

Humidity (close to 100 % RH)

• May influence the electronic, especially if moisture can condense into liquid.
• Cause corrosion to any mild steel components, such as counterweights or housings.
• With high humidity and a plug/socket combination at the shaker, the use of 240V units may be considered a safety hazard.

CO₂ (up to 20 %)

• Can lead to corrosion of mild steel components when dissolved in water.
• CO₂ gas can increase the risk of corrosion for some shakers over time, when high levels on humidity are also present. Resistance to all these factors should be “must haves” when choosing a suitable shaker.

A good construction will have the following elements:

• A composite housing which is smooth enough for easy cleaning. Incorporation of an anti-microbial coating would be useful with respect to reducing the risks of contamination.
• Internal construction using non-corrosive parts, including stainless steel for components such as bearings.
• A water-tight construction, so any condensed water, culture and cleaning agent cannot get into the moving parts.
• A maintenance-free shaking mechanism, such as an inductive drive.
• Removable shaking table and trays, which can be autoclaved, if necessary.

A crucial point for constructions which use composite materials for the housing and adhesive matting is not to expose them to ultra-violet radiation.

Hygienic design is a key factor against contaminants

Cell cultures are especially prone to contamination from microbes in the environment. The walls of the incubator are usually made of polished stainless steel, to prove a smooth surface which can easily be wiped down. A shaker which is difficult to clean thoroughly would provide a “magnet” for contaminants in places difficult to reach or prone to corrosion. A smooth housing, internal components sealed away from ingress of liquid or dirt, and overall cleanability are vital.

Summary

This basic overview has covered the key properties, functionality and applications of a shaker placed in an existing static incubator. The need for a dedicated unit and the benefits this brings cannot be stressed highly enough. Compared to the step change of a dedicated incubator shaker it’s a cost-effective, simple way to “put your toe in the water” before jumping in.

Specifics covered include:

• The economic case for this approach.
• Making sure the incubator and shaker are paired in terms of size.
• Environmental effects (both ways) are important for good growth and reliable operation.
• A dedicated shaker will have features which will make it easier to transition to a larger scale.
• Usability and access are important for the user and can influence the quality of results.
• Cleaning and decontamination are important considerations.