Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Mammalian cell cultures have been around for decades—but they definitely aren’t going anywhere. They are increasingly valuable in drug development, for which the pharma industry is turning to cell-based assays for drug-safety testing because animal models are sometimes unreliable, or even inhumane. “For example, benzene will cause leukemia in people, but it doesn’t cause leukemia in mice,” says Don Finley, market segment manager for research cell culture at Sigma-Aldrich. “Because you can’t test experimental drugs in their early phases directly on humans, and animals alone aren’t perfect models, people turn to human cell-based assays as a way of determining whether a drug candidate is toxic, for example.” Cell-culture tools and expertise abound today, and it has never been a better time to learn the tricks of the trade. Perhaps you’ve helped with maintaining cultures, but now you’d like to start your own. Here are some main considerations to keep in mind when setting up your own cultures.
Choosing the right media and supplements
There is no single answer to the question, “What’s the best medium to use?” because the needs of cultured cells depend on cell type and the culture’s purpose. A common type of medium used for general maintenance of adherent mammalian cell cultures is Dulbecco's Modified Eagle Medium (DMEM), but nonadherent cultures may do better with media formulated for cultures in suspension.
Educating yourself about the origin of your cell lines can guide you toward the right media. This information should be available from either the cells’ vendor or from the literature, if the cells came from another lab. The European Collection of Cell Cultures (ECACC), for which Sigma-Aldrich is a distributor, also can recommend media for particular cell lines. Specialized cell lines—like hybridomas, whose sole purpose is to produce an expressed protein—may require extra support from specially formulated media. “The media become more specialized and customized according to the needs of that particular cell line, and even the particular protein they’re expressing,” says Finley.
For less specialized cultures, a common practice is to use a general medium, such as DMEM, and supplement it with factors that support the cells. For example, many researchers supplement their basic DMEM with fetal bovine serum (FBS). “Serum provides a lot of nutrients as well as certain attachment factors,” says George Sitterley, a market segment manager for research cell culture at Sigma-Aldrich. Experimenting with serum levels is smart, because some cells prefer more serum than others. “We recommend a starting percentage of 10% FBS, but you can increase or decrease the percentage based on cell viability,” says Tabitha Eckert, custom monoclonal team leader at Rockland Immunochemicals. “At Rockland, we look for media that are formulated to contain high glucose and also L-glutamine, but not all DMEMs contain these ingredients at the same amount.” Cells use glucose and glutamine as energy sources, but their preference for amounts can vary by cell type. “Some cells prefer more glucose than others,” says Sitterley. “Glutamine is usually very important, so we offer media with and without it to allow people to modify the glutamine concentrations. Even within a normal glutamine range, certain cells will like more glutamine than others.”
Sometimes cultures are supplemented with antibiotics to prevent bacterial contamination, but it’s better to use them sparingly. “A cell culture purist would say that you never have to use antibiotics,” says Sitterley. “However, for the rest of us, a certain use of antibiotics in short-term applications is okay. But you shouldn’t use them on a continuous basis, or you run the risk of establishing resistant bacterial cultures. And that does no one any favors. And then you have to use more exotic antibiotics that may kill the cells.”
T-flasks, plates or a more specialized solution?
Deciding what type of culture vessel to use (such as cell-culture plates, dishes or flasks) strongly depends on what you are planning for the cells as well as how large the cultures will be. T-flasks are the standard for simply growing up a bunch of cells. “If you’re going to do studies on the cells while they’re growing,” notes Sitterley, “you might choose multiwell plates designed for optical studies, so you can tell what the cells are doing.” Cell-based assays might require high-density microwell plates.
As an antibody company, Rockland Immunochemicals chooses cell-culture conditions that optimize antibody production. The company considers the volume of antibodies to be produced (e.g., a small amount of less than 5 mg or a larger amount of more than 100 mg), starts growing cells in a T-flask and then expands the cultures up to the most appropriate vessel size. “Don’t always assume the bigger the vessel, the larger the yield,” cautions Eckert. “Sometimes the smaller flasks on the market do just as well or better than large flasks. We prefer flasks that are specially treated for tissue culture with vented caps for maximum cell adhesion and easy CO2 exchange for the cells.” Indeed, different volumes or types of vessels have different venting characteristics. For example, larger quantities of cells grown in suspension might grow better in roller bottles, which allow more gas exchange. “You can also perform roller bottle production with non-vented caps in which the cells produce their own CO2 within the bottle,” says Eckert. “Therefore they can be in culture without the use of CO2 and can culture upwards of 30 days in culture.”
The cell line’s growth characteristics are another consideration, notes Mark Rothenberg, applications manager at Corning, because the required vessel volume depends on the number of cells you require, the cells’ doubling times and the degree of proximity the cells require for ideal growth. “A culture plan should be developed for scaling up the cells, taking into account the [cells’] characteristics and time line,” Rothenberg says. “Utilizing vessels that range in size from T25cm2 to T225cm2 will get to the goal, but there are newer alternatives that may be more beneficial. These include multilayer flasks or HYPERFlasks® from Corning®.” Sigma-Aldrich is also in the process of launching a line of cultureware, including tissue-culture treated flasks, serological pipettes, conical tubes and cryovials.
Larger cell-culture systems or bioreactors often are used for growing larger amounts of cultures over extended periods of time. These can be beneficial, because they let you control different parameters, such as automated media exchange, feeding rates and oxygen or CO2 levels. Hybridoma cell lines used for antibody production particularly appreciate the extra attention to their health. “If you are looking at producing large quantities of antibodies over a period of time, I would recommend moving your cultures into a cell-culture system,” says Eckert. “We currently are using Wheaton’s CELLine™ bioreactors, [which] fit well into a standard incubator and are as easy to use a T-flask. The advantage of this system is that it allows for production without the requirement for special incubators, and in the long run may cut down your culturing time vs. other culture systems.”
Recommended general practices
General techniques
One of the most important skills to acquire or refresh is how to work with cell lines under the hood with basic sterile technique. Rothenberg also recommends developing “the necessary skills to manage a cell line, including understand[ing] your cell-culture vessel options [and] understand[ing] techniques such as cell seeding, passaging/harvesting, cryopreservation and recovery.” He also advocates keeping a cell-culture log that includes information to help you and others care for your cell lines. “The log should include the cell-line name and derivation, culture conditions, medium requirements, images of the cell line, genetic confirmation of the cell line and verification of mycoplasma analysis,” Rothenberg says.
Sigma-Aldrich and the ECACC have put together a handbook of fundamental cell-culture protocols. “It contains a lot of different basic ‘dos’ and ‘don’ts’ of cell culture,” says Sitterley. “For somebody who’s been doing cell culture for a long time, it’s a really basic primer, but for somebody who’s new to cell culture, it gives you a lot of good tips and techniques on things that you should and shouldn’t be doing.”
Work with one cell type at a time
Although it may seem obvious, many people do not follow this tenet in their haste to save time—understandable, but imprudent. “There are things that experienced cell culturists do that they shouldn’t necessarily be doing, like working with more than one cell line under the hood at the same time,” says Sitterley. “You really shouldn’t, because you’re running the risk of cross-contamination between the cell lines.” And that can significantly complicate matters (see below).
Verify your cell lines
Contamination between cell lines can confuse the origins of cell lines, which is very important to know. “About 35% of the cells out there in research are contaminated, or they are just not authentic,” says Finley. “The best thing to do is to get your cell lines from a repository like the ECACC. Each and every cell line there is determined to be authentic, that is to say it’s the right cell line, which is huge. And also they’re determined to be mycoplasma-free.”
In fact, knowing the correct identity of cell lines is a problem that’s been growing for decades. “More and more people are starting to raise the issue,” says Sitterley. “All of the AACR [American Association of Cancer Research] journals are now requiring that if you’re publishing in them, then the cell lines that you’re using have to be verified. It’s very important.” He gives an example of Chang liver cells: “People still publish liver studies using Chang liver cells, yet it’s been known for decades that, in fact, they are HeLa cells.”
This underscores the importance of using only one cell line under the hood to avoid cross-contamination. “HeLa cells are kind of like weeds,” says Finley. “They can actually survive drying out, and then if you add media to them later, they’ll grow. They get into everything.” Human error can compound problems, especially if you don’t take the time and trouble to verify cells. “People get things mixed up or mislabel vials,” says Finley. “And then suddenly you think you’re growing human cells, and they’re actually hamster cells. It’s just human nature.”
Cryopreserve your cells
That being the case, it’s a good idea to protect your research from the ever-present threat of human error. Do yourself a favor, and put some cells in the bank for a rainy day. It’s wise to cryopreserve them for two reasons. One is simply as a backup. “Even with the best plans, contamination still happens,” says Sitterley. “You could lose everything you have if your current cell cultures are all contaminated. But if you have a backup that was cryopreserved before they got contaminated, you’re saved.”
A second reason to cryopreserve is if you plan to use the cells over a long period of time. A common and smart practice is to establish a master cell bank in your lab. “You grow up cells, aliquot them and then freeze away that master cell bank,” says Sitterley. “And then from the master cell bank, you establish working cell banks, where you pull them out, expand them out, and work with those.” This ensures that you will always be studying cells at the same stage, such that they’ve divided a similar number of times. After some time has passed, you begin a new batch from the working cell bank. But make sure to store your cells in liquid nitrogen, as even cells in the freezer change with age. “If cells are stored at liquid-nitrogen temperatures, then essentially they should be good forever,” says Sitterley. “If they are stored at -80°C, they slowly will change.”
After acquiring some basic training in cell-culture technique … practice, practice, practice. “Experience in cell-culture work is very important,” says Eckert. “The best way for training and learning is continued hands-on experience.” And investigate the wide variety of cell-culture tools and technologies available today that can help cell biologists with any level of experience. “Be willing to try out new technology on the market,” Eckert advises. “You will continue to improve as the technologies continue to improve throughout the years.”