The pH value is a key factor in cell and tissue cultures

Cell cultivation

The term “pH” is derived from either “pondus hydrogenii” or “potentia hydrogenii”. Both are originally Latin phrases, with “pondus hydrogenii” meaning “weight of hydrogen” and “potentia hydrogenii” meaning “power of hydrogen”.  The pH value is a non-dimensional number that measures how acidic or alkaline an aqueous solution is.  It is defined as the negative decimal logarithm of the concentration of hydrogen ions.  The strength of the acid or alkali is identified by color indicators.  

Every cellular process has an ideal pH at which it functions best. If the process deviates from this ideal pH value, it can lead to conformational enzyme changes that impair function, resulting in effects that are as severe as those caused by extreme temperature changes. If the process deviates drastically from the ideal pH value, it can even cause damage to the DNA’s secondary structure, α-helix, and β-pleated sheet, leading to irreversible denaturation. 

In order for cells to grow well, it is therefore vital that the culture medium has a stable pH value. Most mammalian cells feel right at home at a pH value of 7.4. Transformed cell lines prefer slightly lower pH values of 7.0 to 7.4 while normal fibroblast cell lines have a preference for slightly alkaline pH values of between 7.4 and 7.7. 

Many culture media have a phenol red pH indicator added to them. If the pH value is 7.4, the culture medium will be red. With 40 µM of phenol red in DMEM, the medium changes color when the pH value rises, turning pink at a pH value of 7.6 and purple at 7.8. This might mean that the medium is contaminated with yeast or a fungus. As pH values become increasingly acidic, the indicator turns from orange to yellow – potentially a sign of bacterial contamination. (See below for more information about hot-air sterilization.)

A CO2 incubator stabilizes the set pH value in the culture medium by controlling the CO2 concentration. CO2 incubators play a key role in the activities of every cell and tissue culture laboratory – from growing adherent CHO cell cultures to growing cultures used in genome editing with the help of a CRISPR Cas9 system. 

Most of the time, the open hydrogen carbonate buffer system is used in the culture medium.
This buffer system maintains a balance based on an exchange between the CO2 contained in the incubator atmosphere and the CO2 released in the culture medium.

Key factors for the cell:

  • The cell requires an ideal pH value within very narrow bounds
  • Most of the time, the natural hydrogen carbonate buffer system is used
  • CO2 is a component of this open buffer system
  • The CO2 partial pressure shifts the balance of the buffer

Carbon dioxide


BINDER operating manuals provide useful information explaining how the pH values in media depend on the concentration of CO2 in the incubator.

ph valueImage: pH value of commercially available media buffered with NaHCO3 as a function of the CO2 concentration

Example: If a pH of 7.2 is measured in a medium buffered with 2.20 g NaHCO3 per liter, the concentration of CO2 in the medium environment is 8 vol. %

To ensure the culture media are supplied with the ideal amount of carbon dioxide and thus create optimum conditions in which the cells can grow, the way in which the gas is supplied to the CO2 incubator is crucial – this can be done either via a CO2 tank or an on-site supply.  There are three principles available to choose from.

gas mixing jetIn the Venturi gas mixing jet (A), the CO2 flows out of the gas tank through a narrow tube before it is supplied to the CO2 incubator interior. Suction is created inside the tube. The incoming carbon dioxide (gray arrows) is mixed with air containing carbon dioxide in the CO2 incubator; this air enters via side openings in the tube. As a result, the interior is supplied with gas that is practically pre-mixed, ensuring that there is an even atmospheric blend throughout the incubator.

In CO2 incubators with interior fans (B), the incoming carbon dioxide reaches the interior before it is mixed, which means atmospheric mixing happens later. Using fans increases the quantity of plastic – and therefore the number of surfaces that can be contaminated – in the interior, as well as the costs of spare parts, which is why BINDER completely avoids fans in the interior.

In CO2 incubators with a direct injector (C), the incoming carbon dioxide is fed directly into the interior. The carbon dioxide is spread via diffusion and convection, which is quite a slow process in comparison to the other methods.


Combining the gas mixing jet based on the Venturi principle with the benefits of an IR CO2 sensor provides the perfect solution for CO2 incubators. 

Features and equipment you might be interested in: 

  • BINDER’s 180°C hot air sterilization method, which has become the gold standard in this area, is the most effective way to eliminate contamination in CO2 incubators. It is extremely effective and straightforward to use, and it always delivers reproducible results. Our whitepaper on “Cell cultivation without contamination” explores this topic in detail from the perspective of CO2 incubators.
  • Divided inner glass doors – split glass doors that significantly reduce the access cross section can have a positive impact on the humidity recovery time. The new C 170 is in fact the first ever product to feature the innovative “quick sample access” facility.
  • CO2 gas tank changer – to ensure that CO2 continues to be supplied for a prolonged period,
    e.g., over a long weekend, we recommend connecting two gas tanks to one CO2 gas tank changer. When one of the CO2 tanks runs out, the changer automatically switches over
    to the full one, thereby ensuring a continuous supply of CO2.
  • Roller bottles – CO2 incubators are used to scale up suspension cultures, e.g., in spinner flasks.
  • The customer can select a CO2 concentration to be calibrated, e.g., 5% CO2 at 37°C, including certificate.

Conclusion


Good cell growth depends on many factors, such as the vitality of the source material, the right culture medium and growth factors, and passaging cells at the right time. However, it also depends on the individual technical solutions coming together in harmony to create the perfect environment for good cell and tissue growth in CO2 incubators. One of these elements is the pH value:

  • Venturi gas mixing jet for optimum CO2 distribution.
  • CO2 IR sensor for quick CO2 recovery times once the door has been opened.
  • Calibration for certified CO2 concentrations.

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Whitepaper Cell cultivation in the CO2 incubator

The aim of cell culture is to achieve and maintain good cell growth and, depending on the task, to attain appropriate cell differentiation. This involves taking numerous parameters and
requirements into account and technically implementing them.

The CO2 incubator or CO2/O2 incubator is a key element in cell culture. Various technical solutions are used to achieve the right temperature, humidity, CO2 and/or O2 concentration,
and interior design. This white paper compares various commercially available solutions for the four parameters and interior design. Always make sure you are comparing like with like.

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