Pioneered in the 20th century, cell culture has since been refined and has formed the basis for almost all major advances in medical science to date. From understanding cancer, Alzheimer’s and HIV, to the production of vaccines and gene therapies; being able to grow, manipulate, and study cells in the laboratory – although now often taken for granted – was once considered the holy grail of the medical sciences. Proper cell culture requires zero tolerance when it comes to contamination, and maintaining a strict level of sterility is pivotal both in the production of viable results and the reduction of time and resource inefficiencies.
Types of contaminants
Cells in culture are susceptible to almost every form of biological contaminant; including bacteria (and mycoplasmas), viruses and fungi. Following decades of R&D, commercial cell culture media have been optimized to facilitate the growth of desired cells, but they can also promote the growth of bacteria and fungi (i.e. molds and yeast), which are widespread in nature and certainly not absent from the laboratory. Bacterial contamination can be easily identified by the cloudy or yellowish (acidic) culture medium as well as the total destruction of the desired cells that can be observed under the microscope. Likewise, the presence of floating masses that appear furry is an indication of contamination with myceliated mold.
Mycoplasmas are the smallest known self-replicating organisms, and are technically bacteria, but lack a cell wall. They escape visual identification and are unaffected by most of the antibiotics used in culture media. Their presence is known to have a serious effect on the morphology and metabolism of the cell, and so their very presence can render months of hard work unfit for publication.
When it comes to viruses, a major concern is whether laboratory personnel could become infected. It is therefore recommended that all laboratories adhere to necessary safety regulations, including regular testing and certification of laboratory biological safety cabinets (BSCs).
When aiming to prevent biological contamination, the use of rigorous aseptic techniques is vital. For instance, working with a BSC, it is critical to protecting the integrity of cells and for lab workers’ safety. In addition, critical steps need to be made to ensure that designated areas and equipment are kept clean and disinfected before interacting with cell lines. The best approach is a clean laboratory where proper training is provided for personnel (including Good Pipetting Practice (GPP)), clean lab coats are worn, and records are properly kept.
The design/layout of a laboratory also needs to be considered; a dedicated area for cell culture is recommended, with access granted only to personnel who use the space. CO2 incubators should not be installed directly under HVAC units, as contaminants may be blown into the chamber.
In addition, good common culturing practices must be adhered to. The most basic being the avoidance of cross contamination between different cell lines by ensuring only one line is worked on at a time within the BSC (which should be properly cleaned and maintained between sessions). Avoiding the use of routine antibiotics is also recommended to help ensure that resistant microbial strains are not being selected for and underlying contamination is not being masked. Mycoplasma testing should be undertaken monthly, as it is the only way to ensure they are not hiding among cells.
Implementation of cleaning regimes on surfaces and equipment is another necessary step towards ensuring cleanliness and sterilized conditions throughout the cell culture process. Regular, scheduled cleaning of all surfaces is key, covering work and high, flat surfaces, such as the tops of refrigerators and incubators. Inner surfaces of the BSC should be cleaned between sessions with 70 percent ethanol, and monthly cleaning regimes should be undertaken on all surfaces within the BSC, including underneath the working surface tray. In addition, experiments undertaken within the BSC should be planned or designed to eliminate the removal of a user’s hands during use by loading the BSC with all necessary supplies before the experiment and removing waste only after the experiment is over. To advance protection against contamination, modern CO2 incubators require features that prevent the establishment of contaminants that may gain entry when the door is opened. It is important to ensure that all equipment in the cell culture lab is up to date.
Filtering systems as a last line of defense
Unfortunately, contamination may still occur despite all preventative procedures being undertaken effectively, simply due to the abundance of microorganisms in the environment. One cost effective and simple preventative method is regular filtering of culture media for the removal of unwanted particles that may have accumulated over time. Membrane filters have three mechanisms that provide efficient particle filtering (size-exclusion, physical entrapment and non-specific binding), and are often used for quality assurance of culture samples. Use of 0.2 µm filters is recommended for protection against bacteria and fungi; 0.1 µm filters will additionally remove mycoplasma. It is recommended that filtration options are explored, enabling the best membrane selection for the specific needs of the laboratory or research application.
Implementation of these straightforward practices can improve the reliability, quality, and publishability of research, while helping to reduce the loss of valuable time, as well as keep costs and waste to an absolute minimum. It is recommended that laboratories adhere to these simple practices in order to maintain the health, viability and reproducibility of their cultures.
Filed Under: Drug Discovery