Post-doc Mario Malfavon, new to his lab at the University of California, San Diego, is the only one in the group working with primary brain cells from rats, instead of mice. In preserving cells for his studies on the blood-brain barrier, he had to guess at what kind of medium would best support the cells in the freezer.
On his first attempt, he guessed wrong—only about a quarter of the cells survived after thawing. However, Malfavon had ordered some cells from Cell Applications, Inc., that came with a special freezing medium. That material was a winner. Malfavon can now thaw enough cells to run his large-scale experiments all at once.
Scientists often freeze cells to save them for later experimental or clinical use. The basic procedure is fairly straightforward: Freeze the cells slowly, store the tubes above liquid nitrogen and thaw them quickly when needed. But as Malfavon’s experience shows, having the right cryopreservation fluid makes a big difference.
Cryopreservation media support cells and prevent ice crystals, which would puncture membranes, from forming.
Some researchers make their own. Others—particularly those working with increasingly popular but finicky cell types, such as primary or pluripotent cells—prefer to purchase standardized commercial products. Very sensitive cell lines may also benefit from supplements on the thawing end, to prevent apoptosis as the cells come back to life.
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“I always recommend that folks test a variety of different [media] to see which one works best for their particular cell line,” says Mark Rothenberg, manager of scientific training and education at Corning Life Sciences, which distributes CoolCell containers to control the rate of cell freezing. “Every cell line is different, each line has its own requirements, and each line has its own sensitivities.”
Homebrew recipes
Immortalized cell lines tend to be fairly sturdy; therefore, using an inexpensive lab-made freezing medium may work fine. Typical recipes include 5% to 10% DMSO and 5% to 40% serum in growth media, or 10% DMSO in just serum.
DMSO replaces some of the water in the cells, preventing formation of ice crystals that would otherwise pierce cells.
It also prevents some of the cell shrinkage that occurs during freezing, followed by swelling at the thaw time, which can damage cells, says Rhonda Newman, senior staff scientist at Thermo Fisher Scientific.
Serum, replete with nutrients, supports the cells directly. “It kind of cocoons the cells in a rich environment that allows them to recover much better when they come out the other side,” explains Daniel Schroen, vice president for sales and marketing at Cell Applications. Albumin is a key component that can form a protective coating around the cells. Serum may also bind up toxins released when the cells begin to thaw, says Newman.
Commercial options
But serum has a downside, in lot-to-lot variation that could alter recovery rates after thawing. That’s one reason many scientists prefer to purchase freezing media. These often contain serum alternatives, such as purified albumin at a set concentration.
“A lot of it depends on the risk that you’re willing to take with your cell types,” says Newman. “When you’re working with critical cell types … it’s in your best interest to use one of these commercial solutions.”
Primary cells tend to be sensitive, and with pluripotent stem cells (PSCs), serum components can cause them to differentiate—and often not in the desired direction.
For example, Thermo Fisher’s PSC Cryopreservation Kit comes with a medium that is chemically defined and lacking all animal materials, aside from human-derived components. Avoiding bovine serum means better reproducibility, says Newman, and the medium also works well with other cell types, such as neural stem cells.
Although chemically defined, quality-checked commercial media are not as economical as mixing your own freezing solutions, they save money in the long run for scientists working with stem cells, says Duncan Liew, product manager for cell therapy at Irvine Scientific. The company’s FreezIS medium, at $35 per 10 ml, is relatively inexpensive—and it’s worth what it costs, if more cells survive the freezing process, he posits.
As each cell type has its own requirements, Cell Applications makes freezing media specific for certain types, including blood cells, pluripotent cells and neurons. The company will release a version for cardiomyocytes, which are difficult to freeze successfully, later this year, says Schroen.
Warm-up support
Although DMSO protects cells from ice crystals, it’s also toxic.
There are alternatives, such as glycerol and proprietary formulations developed by vendors. For example, Irvine Scientific makes a DMSO-free version of FreezIS. But Newman says so far, no alternative cryoprotectant works as well as DMSO.
The toxicity makes it important to remove DMSO from thawed cells as soon as possible. Some researchers spin the thawed cells down and re-suspend them in a DMSO-free growth medium; others prefer to plate the cells and change the medium once they’ve settled.
Those first 24 hours are crucial. Free radicals build up as cells recover, and many that were alive upon thawing may still die. Human embryonic stem cells or induced pluripotent stem cells are particularly fragile at this time, says Liew.
For sensitive cell lines, using media supplements during the first day after thawing may help. ROCK inhibitors such as Y-27632 block the Rho kinase pathway that leads to apoptosis, so they can pull struggling cells back from the brink. Thermo Fisher Scientific’s PSC kit comes with the RevitaCell supplement, which contains a ROCK inhibitor with higher specificity than traditional ROCK inhibitors plus antioxidants and free radical scavengers, says Newman.
The key with any cell medium is to match the recipe to the cells. “It’s not always ‘one size fits all’ for cryomedia,” says Newman.
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