Don’t Overlook Your Incubator: Cell Culture Optimization

Achieving reliable cell culture growth while avoiding contamination is instrumental in achieving results you can rely on when you carry out cell-based assays on a regular basis. It’s ingrained into our routine procedures to take great care when setting up and handling cell cultures, but when it comes to the incubation step it’s easy to take your incubator for granted.

There are a lot of factors that contribute to perfect cell growth, for example, homogeneous temperature, consistent gas mixture for optimal pH, and high steady-state humidity to minimize evaporation of the medium. To meet all these requirements, it is important to have the best-designed incubator for your application that is easy to operate and maintain.

In a recent expert-led SelectScience webinar, now available on demand, Dr. Jens Thielmann from BINDER explains the concepts of seamless cell culture, the factors that influence reliable cell growth, and how the incubators we use can affect both workflow efficiency and the integrity of cell lines and samples.

Read on for highlights from the webinar Q&A session or register to watch the webinar on demand.

Q: How do you prevent contamination from airborne contaminants?

JT: The BINDER incubator doesn’t employ a fan inside and doesn’t have a HEPA filter. When airborne contaminants enter the incubator through the door – how do you prevent them from entering your culture? In CB series incubators without fans, there’s only very gentle convection and diffusion that mixes up the gas. There’s no airstream to blow contaminants into your culture dishes. When you have a fan, you need a HEPA filter – when you don’t, you don’t need to have a filter because any airborne contaminants will gently settle on any surface. As you always have lids on any culture dishes, contaminants won't get into your cultures.

Q: Do you need to use higher CO₂ concentrations at higher altitudes?

JT: In literature, you always read that you need 5% CO₂ and consequently, everyone switches the incubator to 5% CO₂ regardless of whether they are working at sea level or elevated levels. pH is, in fact, driven by the partial pressure of the CO₂, not by the concentration. The partial pressure in high altitudes is naturally much lower. You will need to compensate for altitude with a higher percentage of CO₂. For example, in Bogota, you need roughly 7% CO2 to maintain the correct pH.

Q: How frequently should the incubator be sterilized even when there isn’t any contamination present?

JT: Many people have a different approach to this. Some do it every week. The CB incubator will stand up to the harsh procedure, but I don't think that it's necessary to sterilize the incubator so regularly. Once per month, and you're absolutely on the safe side. Some people do it only after they encounter any contamination. I wouldn't wait that long, but between once per month and once per three months should be sufficient.

Q: What are the advantages of the new CB 170 in comparison to its predecessor?

JT: There are many nice features in the new CB 170. The size has been increased from 150 to 170 L without enlarging the footprint or even the incubator’s height. Most incubators are stacked in the lab up to around 1.8 m, so you still have convenient operation even with the upper incubator. We really focused on the size inside and outside. The CB 170 doesn’t have a fan inside anymore, so it doesn't make any noise, vibrate or input heat. That is to avoid negatively influencing the air jacket as in the predecessor.

One feature I like a lot is the option for humidity control. This is a new anti-contamination feature. The weakest point of this concept is the water pan. If you don't take care of tidiness above the water pan, then you might be in trouble - so you have to take care of the water pan. But, if you don't want to do that and it's not convenient to clean or autoclave the water pan, the humidity control is available without a water pan in this model.

Q: Should CO₂ percentage differ with the cell type?

JT: It should not. Normally, mammalian cells all have an optimum pH around 7.4. I'm not aware of mammalian cell types which are grown under different pH. Other animals like insects or fish need will need a different pH because their cells have a different optimum pH. The CO₂ concentration makes sure that with the specific buffer you're using, you get the correct pH in your vessel.

Q: If you don’t use a humidification method as an anti-contamination measure, would just refreshing media and subculturing cells twice a week be sufficient to prevent any deleterious changes in osmotic pressure?

JT: Osmotic pressure really depends on what kind of vessels you're using. For people who use multiple plates and do 96 different experiments in one plate, it's absolutely necessary to have very high humidity so that the outer wells don't dry out quicker than the inner wells, because any drying out would have bad effects on the cells. Otherwise, if you have a relatively high volume of cell culture media, it's not so important. For those cell cultures, refreshing the media twice per week should avoid any unhealthy osmotic pressure.

Q: How can you counter check the CO₂ concentration in the incubator?

JT: The easiest way to do this is to have a pH indicator in the cell culture so you can tell from the color if there are any problems with the pH in the media. Normally, you should adjust the pH sensor once per year or at least once per two years to be on the safe side. With the sensor we employ, an optical system, with a very low drift, you can rely on that for a long time in contrast to other sensor types. One quick test you can do is to open the door and see if your sensor runs down to zero. If it doesn't, then you have a problem with the sensor.

Find out more on this topic by watching the full webinar on demand>>

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