Culturing mammalian cells in a lab is important for biomedical research. Mammalian cellular cultures serve as important in vitro models where lab experiments and tests are conducted on cellular tissue outside a living organism, thereby foregoing the need for in vitro studies to be conducted on living organisms until any potential side effects are known. While mammalian cells are extensively used in biomedical research, they tend to be rather delicate, and therefore a specialized environment, typically within a CO2 incubator, is required to grow the cells. However, in order to successfully culture mammalian cells, the CO2 incubator needs to be functioning optimally and special care needs to be taken to prevent contamination of the cellular culture. Follow the tips outlined below to optimize the environment within your CO2 incubator.
How to prevent contamination of your CO2 incubator
Since mammalian cells are grown in nutrient-rich media at temperatures that will stimulate the growth of most organisms, including unwanted microorganisms, they are vulnerable to becoming contaminated. While researchers often add antibiotics and antimycotics to their culture media in an effort to prevent contamination, this is not effective at preventing errors in the methodology and may even prevent researchers from detecting a problem. The use of antimicrobial agents is therefore not recommended; the most effective way to prevent contamination is to maintain a clean and sterile environment, including the environment within your incubator.
Tips for keeping your CO2 incubator clean
Incubators typically become contaminated when microorganisms enter via the access door. While this may seem obvious, bearing this in mind when accessing the incubator will help you to focus on improving cleanliness and ultimately limit contamination of your cell culture. The following habits will help you avoid any contamination issues:
- Always wear gloves when accessing the incubator, and keep the inner glass door open for as short a time as possible.
- Periodically clean the door handle with a solution of 70% ethanol.
- Replace the water in the humidity pan weekly to ensure it remains clean. Periodically wash the pan with soapy water and wipe down with ethanol, or steam clean in an autoclave.
- Should you spill any culture media inside the incubator, remove the affected shelf and clean thoroughly before placing it back inside the incubator.
How often you need to clean your incubator will largely depend on how much in demand your incubator is. If you are in a large lab and the incubator gets lots of traffic, it needs to be cleaned more frequently than an incubator in a smaller, less busy lab. Your best bet is to start off with a clean, sterile incubator, and carefully manage what goes in and out of it. If you do this and maintain a sterile working environment, you shouldn’t have to clean the incubator again for a few weeks, if not months.
Check CO2 levels
Mammalian cell culture media typically consists of a buffer system composed of bicarbonate. CO2 forms a key part of the buffer system and needs to be kept at 5% in order for the medium to maintain a pH similar to the pH that normally prevails in the human body. While most incubators are fitted with a CO2 sensor to control CO2 levels, over time, it may need to be recalibrated to ensure it functions optimally. This requires the user to determine the CO2 level in the incubator. The simplest and most accurate way to measure CO2 levels in an incubator is with a gas analyzer. The gas analyzer is zeroed using room air (CO2 level of 0.04%) or according to the manufacturer’s instructions, and the air within the incubator is analyzed through an opening in the incubator. Older incubators may need to be recalibrated more frequently than newer models, many of which are automatically recalibrated on a monthly basis. Even so, it is a good idea to check CO levels within the incubator using a gas analyzer.
Maintain a temperature of 37°C
Cultured mammalian cells do best at a temperature of 37°C, which is not too surprising given that this is the optimal body temperature of humans and other mammals. CO2 incubators typically display the temperature inside the interior chamber, however, we recommend double-checking it from time to time, particularly if your culture starts showing signs of suboptimal or unusual cell growth. To do this, you will need a good-quality thermometer that has been correctly calibrated. Open both the outer and inner doors of the incubator and fix the thermometer to the inside surface of the inner glass door with adhesive tape. Make sure that you will be able to read the temperature once the door has been closed. It is advisable to do this before leaving the laboratory at the end of the day. When you arrive back at the lab the following morning, open the outer door of the incubator and take a temperature reading. If the temperature recorded is not 37°C (assuming you are using a calibrated thermometer that is accurate), you will need to recalibrate the incubator’s built-in temperature sensor.
Maintain humid conditions
Because the temperature within the incubator is relatively high at 37°C, it is important to maintain high relative humidity (between 90-95%) to prevent the culture medium from evaporating. This is achieved by ensuring there is always water in the bottom tray of the incubator. However, you need to bear in mind that this water-filled tray can also harbor microorganisms that can potentially contaminate your cell culture, and therefore cleanliness is paramount (as outlined above).
To prevent contamination of your cell culture and to ensure you CO2 incubator keeps functioning optimally, adhering to a regular cleaning and maintenance schedule is recommended.
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