Laboratory contamination: The final defence mechanism
23 Jun 2014
In the final part of our exclusive laboratory contamination series, Thermo Fisher Scientific’s global product manager Dennis Gorlick provides useful advice on how to choose a membrane and filtration product.
Throughout the cell culture process, no matter how much has been done to prevent contamination, there is always a risk of microorganisms or unwanted particles polluting the sample.
An important prevention mechanism is to filter regularly to maintain the cleanliness of your culture and remove unwanted particles and microorganisms.
Filtration is the last line of defence against contamination and provides cost-effective insurance to protect laboratory work and ensure sterile fluids.
Determining which membranes and filtration devices are the most appropriate and reliable for your application can be a challenge.
Filtration is a process used to separate substances based on particle size. The three basic types of filters are depth filters, screen filters and membrane filters.
Depth filters are used for filtering fluids with a high load of particulate to trap the particles within the filter structure.
These filters are manufactured from plastic, glass or cellulose and have pores or pore-like constructs that vary in size and geometry.
Depth filters can shed particles so are usually not used for cell culture work.
Screen filters are manufactured from stainless steel mesh, polypropylene or nylon and rely on size exclusion to remove particles.
Screen filters can become plugged and are therefore also not usually used for cell culture work.
Filtration membranes work in a variety of ways, including size exclusion, physical entrapment and non-specific binding.
In size exclusion, the membrane pores are small enough to block unwanted material so that these materials cannot pass the membrane barrier.
With physical entrapment, particles become caught in “dead-end” membrane pores, which can capture unwanted particles that are smaller than the pore size.
Non-specific binding occurs at molecular and electrostatic levels causing particles to adhere to each other and to the filter material.
All three of these mechanisms work together in membrane filters to provide efficient particle removal with relatively high flow rates and throughput, making membrane filters the best choice for filtration of cell culture fluids.
Choosing the right filter for fluids used in cell culture is important for maintaining cultures free from contamination of bacteria, fungi and mycoplasma.
It is highly recommended to routinely use 0.2-micron filters to protect against bacterial and fungal contamination for a trouble-free cell culture environment.
Mycoplasmas are detrimental to cell lines because they affect the host cells’ metabolism and morphology, causing chromosomal aberrations and damage.
Mycoplasma contamination is difficult to detect under a microscope, but can be removed by 0.1-micron membrane filtration.
If mycoplasma is a concern, filtration systems with 0.1-micron filters are a critical lab resource. It is also important to purchase media and sera from reputable sources that sterilise with filtration levels of 0.1 µm or smaller.
The majority of media and sera cannot be heat-sterilised and require membrane filtration in order to remove biological contaminants.
In addition to 0.2-micron sterile filtering, media should be checked before use for correct pH, combined with a visual assessment of colour and for other signs of contamination.
For example, if a bottle of media has been unused for a long period of time, low-grade contamination may result in fine sediment forming on the base of the bottle.
If, after a gentle swirling, a spiral of sediment rises from the bottom of the bottle, then the media may be contaminated and should be discarded.
Media should also be discarded if it looks cloudy as this can indicate bacterial contamination.
A well-designed filtration system begins with choosing a filtration membrane that is appropriate for your fluid.
Differences in how a filter is manufactured, material components and how it performs will have a direct impact on your work.
While most membranes have inconsequential levels of leachable (extractable) substances, some membranes, especially some cellulose-based filters, contain wetting agents that, at higher concentrations, could impact cell growth.
Also, consider pore size and volume of the fluid being moved through the filter.
The following provides some recommendations for choosing between membrane types:
PES (Polyethersulfone) membranes are the best membranes for cell culture fluids because they are low protein binding and thus unlikely to remove critical protein from media. The membrane is hydrophilic, so no external wetting agents or surfactants are needed, resulting in low levels of extractables that could affect media purity.
SFCA (Surfactant-free Cellulose Acetate) membranes are acceptable for culture media and are a better choice than standard CA (cellulose acetate) as they contain none of the wetting agents found to be toxic to specific cell lines that are present in standard CA.
Hydrophilic PVDF (Polyvinylidene Fluoride) membranes are used for harvesting cell cultures and removing contamination, but they typically have slow flow rates.
NYL (Nylon) membranes are tough, alcohol-resistant, and have low levels of extractables. These membrane features make it the best choice for special applications with alcohols and weak solvents.
CN (Cellulose Nitrate) membranes are high protein binding and contain surfactants that may be unsuitable for most cell culture work. They should be used for water and buffers where protein binding is not a concern.
Always double-check the type of containers you entrust with valuable samples and solutions.
Trouble-free cell culture work requires storing media in bottles that are biologically tested, leak-proof and sterile.
Storage bottle and cap system designs vary and can greatly impact how long the media will last.
For example, Thermo Scientific Nalgene bottles and caps are constructed to maintain better pH over time and are ideal for storing filtered media.
Filtration systems, types of filters that remove contaminants from cell culture fluids and media sources vary and should be selected carefully.
For optimal results, researchers need a filter that is free of chemicals harmful to the filtrate and will provide consistent, rapid results.
It is important to purchase filters with media of the highest purity to avoid trace biological contaminants.
Recent technological advances in filtration systems and membranes are designed to perform at a rapid rate and also safely improve lab performance metrics.
