Cell-Culture Health

Cell culture is not rocket science (except maybe when it’s being performed in an orbiting space station). It’s pretty straightforward to keep your cells happy—or to tell when they’re not—largely by following common sense, good laboratory practices (GLPs) and a few tricks of the trade. Here we look at some best practices and how to integrate them into your workflow to culture and maintain healthy and happy cells.

First things first

Get to know your cells, suggests Yvonne Reid, manager/scientist at the American Type Culture Collection (ATCC). Documentation detailing the cell-line name, tissue of origin, the originator of the cell line and how it was derived should be available. If the cell line was purchased from a “reputable institution,” such as the ATCC, such information would typically accompany the cells and/or be available on the website; similarly, publications describing how it was derived can “provide some validity to that cell line.”

The documentation should include optimal conditions for cell growth. What basal medium, growth factors and other supplements are required in culture media? What is the growth profile (curve) of the cells? This information can help researchers obtain more reproducible data and provide troubleshooting guidance.

ATCC recommends establishing a growth profile for a cell line obtained from an unfamiliar source. This should include the inoculum to be used when plating the cells, the doubling time, the concentration or density range at which they should be subcultured (passaged or split) and the reagents and implements used for culturing cells. Plate the cells in duplicate or triplicate and then plot the cell number over time to determine when the culture is in log phase, and when it enters either stationary or plateau phase. Explains Reid: “We also photograph cells, so we know what the cells look like at all stages of the growth profile. For example, we will know what the cells look like during log phase, and we also know how many cells/cm² correspond to that confluence.” This enables the user to determine the doubling rate. And “because you’re counting your cells, you have total cell count and viability, so you know when your cells are at the most viable stage of the process.” Typically, during late log phase (at around 80% confluency) cells are harvested—for experimental purposes as well as passaging and cryopreservation. To obtain consistent experimental results, it is important to harvest cells at the same stage of the growth profile.

Now freeze

After the cells have acclimated in culture, it’s a good idea to freeze down aliquots to create an “early passage bank … because if something goes wrong with a culture, you want to go back and start with something you know for sure is good,” says Uma Lakshmipathy, principal scientist at Thermo Fisher Scientific.

Freezing down cells and then drawing from the master cell bank (as it’s more commonly known) for each experiment offers a variety of benefits. For one, primary cells can undergo a finite number of divisions before they reach the Hayflick limit and begin to senesce—so “you need to stay at a low passage number—define what that passage number or population doubling should be, and stay within that metric,” Lakshmipathy says.

Because cells in culture can undergo generic drift, drawing from a master bank helps assure consistency in experiments even for stem cells and immortalized cells. And cryopreservation enables long-term storage as well as transport.

Cryopreservation is carried out in the liquid or vapor phase of liquid nitrogen. To minimize the osmotic shock and other potential damage that may occur during both freezing and thawing, researchers use a cryoprotectant. These can be homebrew formulations, containing DMSO or glycerol, and serum or perhaps serum alternative. Commercial cryopreservation media formulated specifically to protect and preserve cells during the freeze/thaw cycles—often proprietary—are also available, and many of them are optimized for specific cell types, says Kiran Chin, associate director of marketing for cell culture at GE Healthcare Life Sciences.

After making a cell bank, it’s a good idea to thaw out a vial from the liquid-nitrogen tank “to test the success of the freeze,” advises Mark Rothenberg, manager, scientific training and education, at Corning Incorporated Life Sciences.

Watch for clues along the way

Part of getting to know your cultures is learning how they normally look and watching for any changes. The media color change (if it contains a pH indicator like phenol red) should by gradual, and the cells should maintain a consistent doubling time. Changes in morphology could indicate genetic drift or contamination, and “there are features of the cells that can reveal whether or not the cells are senescing—they become highly vacuolated, very heterogeneous, and they may stop growing—all of which could impact your results,” explains Reid.

Rothenberg recommends weekly examinations to evaluate changes in cellular morphology “and to verify that the lines are free from any biological contamination, such as bacteria or fungi.”

If gross contamination is not obvious to the nose (strong bacterial odor) or naked eye—cloudy, turbid or quickly yellowing media, for example—the offending organisms (or their effects) often can be seen under the microscope. Paradoxically, the use of antibiotics can mask low-level contamination, because “you don’t know for sure whether you are keeping your cells completely aseptic,” says Lakshmipathy, whose facility does not use antibiotics.

Insidious organisms such as mycoplasma – which can lead to false data and irreproducible results -- are a different story. Mycoplasma-contaminated cells can look perfectly healthy, even at high titer, “but the physiology of the cells can significantly change,” Lakshmipathy explains. Kits to rid mycoplasma from culture are available from various vendors, with the gold standard being culturing under permissive conditions for several weeks (either in-house or by a third-party lab). When and how often to test—every culture once or twice yearly, like Rothenberg, or a monthly facility-wide sampling, like Lakshmipathy—varies, but mycoplasma testing is a recommended, routine, periodic procedure. It’s also recommended that cell lines be quarantined until they have been tested.

It is what it is … isn’t it?

Whether because of genetic drift, misidentification, cross-contamination with other cell lines or some other reason, what’s in the flask may surprise you. It’s a good idea to do cell-specific tests to make sure your cells are what you think they are, and that they do what they’re supposed to do. For example, when Lakshmipathy grows pluripotent stem cells, she may look at the expression of cell-surface markers by flow cytometry and also perform “spontaneous differentiation, and see whether this results in cell types representative of the three germ layers.”

In general, “you want to be sure that the functional profile you’re looking for—whether epigenetic or phenotypic or genomic—that those characteristics are maintained during that period you are propagating your cells,” says Reid. “It may not be just visible observation. You may have to perform the functional test that is unique to your cell line.”

The good news is that tests generally can be conducted concurrently with whatever else you’re doing with the cells, with the caveat that an unfavorable test may invalidate your experiments.

Adequate training and maintaining GLPs—along with getting to know and keeping an eye on your cultures, growth optimization, periodic testing and drawing from a well-stocked cell bank—should go a long way toward keeping your cells happy and healthy. And just to make sure, cautions Reid, when you’re ready to publish, test your cells again—at least to assure that there’s no mycoplasma and to confirm the identity of the cells.

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