Cell culture CO2 incubators: Overview and comparison

Of course, an incubator in cell culture should regulate temperature, humidity and CO₂ content. But is that really all? No! There are many things one should look at more closely, especially when purchasing a new cell culture incubator. Our overview of the criteria and comparison of different manufacturers is intended to help you with this.

How important the individual points are for your purchase decision also depends on whether you work in a research laboratory, a testing laboratory, or whether you want to produce, for example, an ATMP (advanced therapy medicinal product) or TEP (tissue engineered medicinal product). A ranking of the purchase criteria that we think should be considered when comparing manufacturers is given at the end of this page.

For cell culture incubators, two main principles are used to heat the chamber and to keep the temperature more or less constant:

+ Water jacket incubators

Advantages: better temperature stability, lower variation after door openings, longer temperature stability upon power failure

Disadvantages: the incubator is heavier and therefore harder to move, slower temperature recovery after long door openings, maintenance includes water exchange

- Air jacket incubators

Advantages: high temperature sterilization is possible, the incubator is lighter and easy to move, faster temperature recovery after very long door openings (>15min), maintenance without water exchange

Disadvantages: lower temperature stability, fast temperature drop upon door openings, short temperature stability upon power failure

Recovery after door opening - measurement methods

For the comparison of technical data from different cell culture incubator manufacturers, one can usually look up values collected during the mandatory normative type examination test (e.g. product characterization test, DIN Baumuster-Prüfung). Data is either given in the instruction manual, technical specification sheet or has to be inquired from the sales department. If you want to purchase an incubator, make sure you inquire all recovery times (temperature, humidity and CO₂) from the vendor for comparison.

German manufacturers and so some others validate the recovery of temperature after door opening (specification) of their incubators according to the DIN standard for incubators and list the recovery time in min. Here, it is important to know that this DIN standard (DIN 12880) was revised in 2007 and changes were made to the parameters and the analysis.

The new version is DIN 12880:2007-05. Important for the comparison is that due to the changes in the measurement method, values acquired according to the new method give longer recovery times. Additionally, in larger incubators like those for cell culture more readings are required: 27 instead of 9 readings. The values are therefore, more precise.

The different measurement set ups are depicted in the two schemes on the right. Top (old) and bottom (new).

According to the old version, the temperature recovery time after a 30s door opening was reached as soon as the temperature once rose above the lower temperature fluctuation limit. As shown this results in very short recovery times. 

According to the new standard, the temperature recovery after a 15s door opening is reached only when the temperature fluctuation does not any more leave the temperature range between the lower and upper fluctuation limit. So on the one hand, the door opening time was shortened and on the other hand, the recovery is defined much more strictly. As a consequence, values acquired with these two methods can not be compared.

In addition to this, one should check whether the incubator of interest has a separate door heating system (all six walls are heated). This reduces temperature gradients and condensation at the door. In comparison to the recovery of CO₂ after door opening, the temperature fluctuation is less important.

- Direct water fill onto the bottom plate (in our opinion a total no-go)

+ cheaper than other variants
+ large surface for evaporation
- water exchange is laborious and annoying
  (usually 1x per week)
- cleaning has to be performed while the incubator is open (long door opening)
- cleaning chemicals may evaporate and act on the cells
- highest contamination risk
- water has to be treated with additives to reduce germ growth
- refill with autoclaved, deinonized water

+ Indirect water fill into tub on bottom plate (state-of-the-art)

+ cheaper than the volatilization method below
+ medium surface for evaporation
+ much easier to clean than a bottom fill (1x per week)
+ tubs may be taken out for cleaning
+ cleaning outside the incubator allows
   free choice of disinfectant
- medium risk of contamination
- water has to be treated with additives to reduce germ growth
- refill with autoclaved water

+ Volatilization of water (steam injection) (new but systems have to be thoroughly checked)

+ at first sight safest variant
   as the water is decontaminated during volatilization
- the reservoir may be hosting algae after some time or water for injections has to be bought
+ less cleaning effort (only reservoir with hydrogen peroxide),
   only water refills
+ do not add additives to reduce germ growth!
- most expensive variant, higher costs for maintenance, check whether maintenance is possible without technical assistance
- cleaning of tubes and injectors not possible for lab personnel
- humidity sensor needs maintenance,
   condensation should be checked

The CO₂ content of the incubator regulates the resulting pH in the used cell culture media. This means the CO₂ content and the used medium are a system and work together to buffer the pH. When placing media in the incubator, an equilibrium between the sodium hydrogen carbonate solved in the medium and the CO₂ content (in %) inside the incubator develops. The higher the CO₂ content  in the incubator the lower the pH of a given cell culture medium. Normal CO₂ contents are 5%, 8,4% or 10%, depending on type of medium used. During the culturing period, the cells continuously secrete acidic waste products which make the medium become more and more acidic. The carbonate buffering system slows this acidification. The medium should be changed depending on the cells, culture conditions and the actual condition of the medium.

CO2 measurement types

The CO₂ content of an incubator is measured by a special sensor. This sensor may be either a TC (thermal conductivity) or IR (Infrared) sensor. When choosing a new incubator, you should pay attention that the incubator has an IR sensor of good quality (measurement time and precision). TC sensors are influenced by the fluctuation of temperature and humidity resulting from the door openings. Therefore, the TC measurement is much slower and the true values for the CO₂ content are not displayed in real-time. As a consequence, the display may already show a CO₂ content of 5% but the true percentage is still lower. For the cells, this results in a longer phase of pH basification which adds up especially when the doors are opened successively. IR sensors react much faster as the CO₂ content is measured directly and independently of humidity and temperature. They show true values in real-time, make the recovery faster and thereby regulate the medium pH more efficiently and precisely.

Another feature of the sensors is that some of them are built in such a way that they can remain in the incubator during a disinfection or sterilization routine. Other sensors have to be removed which takes more time and may be laborious.

Stability of CO₂ content and recovery after door opening

The stability of the CO₂ content and the recovery after door opening are usually measured according to the standard DIN 12880 and are given in % and min, respectively. In contrast to the temperature, the CO₂ content is measured at a single central reading point. It is important to note that the standard was revised in 2007. The actual version is now DIN 12880:2007-05. The revision changed the time of the door openings (15s vs. 30s) and way the recovery data has to be analyzed (see temperature). As a consequence, measurements according to the new version lead to a longer recovery time. So, to compare incubators always inquire the recovery time and measuring parameters.

General leakage of incubators

All incubators leak CO2. When the doors are closed, non-divided doors leak less than divided doors but in most labs this is counter-influenced by the number of doors openings per day. Compare to table below.

Door openings and leakage in incubators

With each door opening, CO₂ and humid, warm air are lost from the incubator chamber. The larger the door and the longer the door remains open, the larger is the loss and the longer does recovery take. Therefore, the door openings are crucial factors influencing the stability of growth conditions for the cells. They may become a problem, when many people share one incubator and the doors are opened successively with high frequency (e.g. university labs). In order to limit the extent of loss it is highly advisable to purchase an incubator with divided doors when several people share an incubator. Usually this results in a quite remarkable increase in price, but it's definitely worth it! You will later save money on CO₂ and time on gas bottle exchange. In addition, the stability of surrounding conditions will make your results less variable. Always invest in divided doors in such cases! Depending on the manufacturer and model, incubators are offered with 2, 3, 4, 6 or 8 doors. As the doors of course change or limit the accessibility of the cell culture vessels inside, one should check the size and positioning of doors. For some incubators, 4 doors are a much better compromise than 6 or 8 viewed from both perspectives. In the photo on the right you see an old New Brunswick incubator with 4 doors which is now produced in a new and optimized design (see next section) by Eppendorf.

As stated above, all incubators leak gas. In general, divided doors leak more. However, depending on the number of door openings, this may play a large or no role compared to the loss caused by door openings. Compare the values given in the below table. This table shows some examples of leakage values for Binder incubators and the break-even calculation indicates how many door openings per day cause an incubator with divided doors to be the choice. In most labs divided doors are definitely the choice. For hypoxia conditions, one has to carefully calculate before deciding, as the large amount of nitrogen needed to wash out oxygen may lead to decide for a non-divided door.

Another point where one can save money is the leak tightness of the door sealing and quality of the hinges of inner doors. Check the hinges for stability against vertical deformation and note where the doors are attached (only in the middle of the door is very instable). Compare the image on the right and the one above. The lower image shows the high quality door hinge of the new Eppendorf incubator. Also check whether the door closes well and whether its probable to not close the door correctly. Try to move the seals. Is it probable that they turn and create a leak? 

For the outer door, check how the door is closed. Is it probable to not close the door be mistake? Will it spring open?

This is a feature that initially is often ignored. However, efficient cleaning of the incubator is essential for contamination prevention and the easier this is achieved the smaller the risk of germ growing in the incubator. If we are honest, no one likes to clean the incubator. Especially not when racks and plates have to unscrewed, have corners and angles. So, the line of decision is clear - less is definitely more in this respect. When buying an incubator and when cleaning, think about two points:

• in corners, clefts and holes, dirt and dust can form a basis for germ growth that is hard to reach while cleaning
• cleaning and disinfection of inner racks, rods and ledgers are often not performed accurately because it is laborious and takes very long. Remember, sterile dirt is soon being repopulated after disinfection!

Another add-on in some incubators is the possibility to order the incubator with sockets or cable ports. This is a big advantage when you plan to perform new assays with equipment placed in the incubator like impedance measurements for cytotoxicity or migration or live-cell imaging (video microscopy) with small microscopes that fit in the incubator like the JuLI. If you don't have a socket or cable port, cables have to be squeezed through the inner and outer doors (image on the right). This results in CO₂ leakage and produces gradients inside the incubator with in turn may influence your assays or the other cells growing in the incubator and increases your use of CO₂ . Cable ports can be  sealed better than the doors and sockets circumvent the problem completely. Usually they are integrated in the chamber sides and have to be ordered with the incubator as they cannot be installed later on. Not all manufacturers offer this feature. Binder does and additionally, you can also decide where you want it to be positioned (surcharge).