For those who perform cell culture experiments, it pays to be meticulous. Even seemingly minor handling techniques and lab equipment features can make a difference in achieving success.
It can be challenging to maintain pure cell cultures and keep them healthy and growing properly. By their very nature, these cell cultures are extremely fragile. Any researcher who has had problems with cell culture health, longevity or contamination knows how time-consuming, costly and frustrating this can be.
Some researchers are almost “superstitious” about trying new cell culture vessels or media, or even altering their techniques. But not all cells types respond to growth conditions in the same way. What’s more, products are continually being developed that increase convenience and efficiency in the research lab. The trick is to find ways to maximize experiment results while minimizing wasted time and money.
The following tips, compiled from interviews with experienced researchers and equipment manufacturers, can help you increase your success rate without relying on luck.
Choose the right growth surface
There is no universal growth surface on which all cells grow well. For non-anchorage cells, be sure to select a vessel surface that is hydrophobic in order to minimize attachment.
Finding a surface for cells that you wish to attach can be a bit trickier. For example, if your cells grow poorly on a traditional, negatively charged cell culture surface, a specialty surface with a positive charge, such as Cell+ from Sarstedt may be a viable alternative to achieving necessary adhesion without laborious coatings.
“The transfection of adherent cells is good with Cell+ and it gives better results than traditional growth surfaces,” notes Dorain Thompson, research associate at Ferring Research Institute (San Diego, CA).
Lee Fuller, president, Fuller Labs (Fullerton, CA) concurs: “Cell+ is very useful; my DHP and insect cells attach more quickly and growth performance is better.”
Look for a vendor that offers flasks with colour-coded surface options so that it is easy to differentiate them in the lab. Not only will colour coding help to prevent mix-ups among various cell cultures, it also serves as a visual aid for quick and reliable identification of different growth surfaces.
“Colour coding on Sarstedt flasks makes it easy to quickly discern between suspension and adherent cells, for instance,” according to Dr Patrick McDonald, associate professor of immunology at Université de Sherbrooke (Sherbrooke, QC). “This is a nearly fool-proof system that benefits everyone.”
Be sure that your vendor uses high-grade materials and products are manufactured under stringent purity conditions, free of pyrogens and cytotoxins. This is essential to achieving reliability and consistency for your experiments.
Also, make sure that vessel surfaces are as flat as possible. This can provide better adherence where needed and minimize “patchy” cell growth that could compromise your results.
Filter out potential contaminants
Contaminants corrupt cell cultures and the results of experiments. If proper care is not taken, cell lines could be lost. Take the time and steps necessary to filter and purify everything that will come into contact with your cells.
For proper filtration, choose a membrane such as PES (polyethersulfone), which is low in protein binding, low in extractables, and offers a high flow rate. To make certain that media and any nutrients you might use are free of mycoplasma, use a filter with a 0.1 micron pore size.
According to Dr Aaron Haubner from Sarstedt, “At Sarstedt, we have put our new low-binding, 0.1 micron PES filters through extensive testing and found that they are incredibly effective at clearing mycoplasma from cell culture reagents. Our new 0.1 micron PES bottle-top filters are able to effectively clear mycoplasma even from densely spiked large volumes, while providing similar flow rate and performance as the traditional 0.22 micron bottle top filters.”
Flasks with standard non-vented membrane caps are designed for use in closed systems (eg, Leibovitz L15 media), providing a liquid- and gas-tight seal. Non-vented caps may be used in the vented position when gas exchange is desired and in the closed position when gas exchange is not desired.
When there is no requirement for anaerobic conditions, vented caps with a non-wettable hydrophobic membrane can be closed tightly to allow regular gas exchange while keeping potential contaminants out.
Using a vented cap also eliminates the step of setting the cap to a vented position, and prevents the liquid buildup that sometimes forms around the inside of plug seal caps, partially sealing them and inhibiting gas exchange.
Look for quick-release caps for one-handed attachment or removal. Generally, cell culture flasks with traditional screw caps require one or more turns to remove or replace the cap. A quick-release cap design should require less than a full turn.
Do not disturb
Just like an infant, your cell culture does not like excessive noise or movement. Make certain that your incubator is level, stable and in a location away from heavy foot traffic or vibrating, motorized instrumentation, such as compressors, laminar flow hoods, or centrifuges. Also make sure it has a rattle-free fan and that the shelving allows for a uniform temperature profile. Bear in mind that temperature fluctuations will more likely occur in the front of the incubator, so keep your more critical experiments toward the back of the storage compartment.
“I believe that researchers experience this problem more often than you might think,” says Dr Pillari Ratnakar, veteran cell culture researcher. “I have seen instances where an incubator was placed next to a centrifuge and a staff member thought there was a problem with the culture vessel, incubator or culture medium, for example. The real problem was the agitation caused by the centrifuge equipment, and it ruined the experiment by causing cells to form different circular growth patterns on dishes.”
Cell scrapers are useful tools that allow you to remove cells without using chemicals to dislodge them in preparation for experiments.
However, choosing the proper scraper design is critical. For example, many cell scrapers have hard, inflexible blades that can cause more cell damage than successful removal. To avoid such unnecessary or excessive damage, consider using a cell scraper with a thin, flexible, non-toxic blade material.
More advanced cell scraper designs can also include adjustable, swiveling blades that are easier to use even in culture containers that are difficult to access. This type of scraper blade can be aseptically turned 90° from the ‘scraper’ position, ideal for use in flasks, to the ‘lifter’ position, which is useful for harvesting cells in dishes.
Once you have grown your cell culture, remember that freezing and thawing are critical for successful preservation and recovery. Follow an established freezing protocol and always use storage tubes that are specifically designed for cryogenic use. Also, be sure to use cryo tubes that are tested and certified to be completely free of potential contaminants that would harm cell materials and cellular components during preservation.
Freeze your cells in a container at -80°C with a cooling rate of 1°C/minute for several hours before transferring them to a liquid nitrogen tank. Store your samples in the gas phase of the tank to prevent contamination from frozen organisms in the liquid nitrogen, and to avoid potential tube breakage due to liquid nitrogen entry and expansion during thawing.
When thawing, use a 37°C water bath with cover. Disinfect the tube before opening with 70% alcohol.
To avoid confusion or mixing of frozen samples, consider using a tube system
with colour-coding. In its CryoPure system, Sarstedt offers a total of 25 different colour combinations for its tubes. These also have uniquely shaped bases that interlock with cryogenic racks, allowing easy, one-handed operation of tubes and caps.
“The main point is, once you have successfully grown your cells and are ready to store them long term, you don’t want to risk losing them during freezing,” advises Dr Ratnakar.
Attention to detail is the name of the game in preparing and storing cell cultures. Make careful decisions about techniques and equipment, continue to look for ways to increase efficiency, and you’ll avoid the headache of lost cell lines and ruined experiments.