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Maintaining the Optimal Tissue Culture Environments for Microbiological Research


Cell Culture ProcessThe dynamics of a cell culture process are very fluid in nature and its success relies on many different factors. When you have everything set up optimally for your specific requirements the process is more productive, allowing you to focus more on your research. In this article you’ll learn about the factors and conditions that affect the cell culturing process and how you can optimize the parameters to enhance your research. You’ll find it useful whether you’re still in the process of setting up the cell culturing process or you’re up and running already but need to optimize the process or resolve a problem you’re having with it.

Factors That Can Influence the Culturing Process

Like any scientific research, optimization of specific parameters is paramount to effective research. There are many factors that govern a tissue culturing process. If any of these parameters are not optimized correctly, then you may find that your cell lines do not culture properly. The most important conditions to be considered are the gaseous environment (% of CO2 added to the atmospheric air) and temperature. Other factors include nutrient concentration, hormone concentration, pH, osmotic pressure, glucose concentration, plating factors, anchorage dependence and growth factors.

If you don’t optimize your conditions correctly, then your samples can incur a variety of problems such as: - nutrient depletion, pH changes, accumulation of dead cells, contact inhibition, cellular differentiation, cell line misidentification, contamination with mycoplasma, genotypic and phenotypic instability and natural selection of cells.

Optimising the Parameters

CO2 IncubatorAs with anything that requires the handling of biological material, maintaining an aseptic environment is the easiest way to control the levels of bacteria in your workstation. However, the gaseous environment is your most important consideration when looking at a parameter to be optimized. CO2 Incubators are the most effective way (without using hypoxic conditions) to control the CO2 concentration, humidity and the temperature in a cell culturing environment.

CO2 interacts with the buffering media which can have an effect on the pH. A drop in the pH can cause a build-up of lactic acid in the growth media, which can be toxic to cells. So by optimizing the CO22 environment, you can optimize the pH environment also. If you experience a significant drop in the pH (0.1-0.2 pH units), then sub-culturing of the cells should take place. CO2 Incubators control the level of carbon dioxide in the culturing environment by two different types of sensors- Infra-red (IR) and thermal conductivity (TC). Although IR sensors are now more popular than TC sensors, because TC sensors are prone to humidity and temperature fluctuations.

Temperature control can pose a problem in many environments. When incubator doors are opened, currents of cooler air can enter the incubation area and disrupt the internal temperature. CO2 incubators regulate the temperature via the use of jackets. They come in many types with water and air jackets being the most common. A water jacket is particularly useful when there is a power failure but requires high maintenance. An air jacket provides a more uniform temperature, controls air fluctuations effectively and is cost-effective. Electric coils and refrigeration units can also be used.

Maintaining a consistent humidity is key to preventing your cell lines from drying out. A water pan is used to induce moisture into the culturing environment, coupled with humidity sensors. This allows for easy inspection and refilling of the water when necessary. However, opening the incubator doors can disrupt the humidity levels. Atomizer systems can also be used.

Cell Culture incubators can also be fitted with HEPA filters, copper surfaces and sterilization (heat) cycles to improve the air quality, increase the antimicrobial activity and to disinfect the incubation zone, respectively. Hypoxic incubators such as the tri-gas chamber, monitor the level of O2 instead of CO2.

There are also many different types of chemicals that can be added into the growth media to enhance the stability, optimize the conditions or help to remove some of the complications associated with cell culturing processes. They are detailed in the table below:

Media that can be added to optimize the conditions

Effect on the media

Inorganic Salts

- Retains the osmotic balance

- Regulates the membrane potential

Buffering Systems

- Regulates the pH


- Introduces sugars into growth medium

- Supports growth

Amino Acids

- Optimizes cell density

- Introduces essential amino acids required for growth

- Stimulates growth

-Prolongs the viability of the cells


- Promote cell growth

- Promote cell proliferation

Proteins and Peptides

- Used in serum-free media to replace essential proteins found in serum

Fatty acids and Lipids

Used in serum-free media to replace essential lipids found in serum

Trace Elements (Zn, Cu, Se)

- Detoxifier

- Remove oxygen radicals

Serum (Foetal Bovine Serum is the most common)

- Increase cell growth

- Increase the cloning efficiency

- Increase the plating efficiency

- Preserves cell characteristics

- Increases the buffering capacity

- Protects against mechanical damage

- Neutralises toxins

The optimized environment for mammalian cell cultures is 37 °C, pH of 7.4 with a 5% CO2 gas concentration.


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