Cryopreservation is designed to minimize damage to cells. For most eukaryotic samples—whether bovine sperm, heirloom beer yeast, umbilical cord blood, or genetically engineered CAR-T cells—this means maintaining them cold enough so their biochemical and biophysical processes are effectively halted.
But just as important for healthy, viable cells is how they get from 37°C to below about -130°C, as well as how they’re brought out of stasis and reconstituted back to a functional state. Here we look at some of the current best practices in biobanking and cryopreservation science.
Get ready to freeze
For most cells, simply freezing them down in cell culture medium will likely kill them, as water crystalizes and punctures the membrane. Thus the freezing convention today is to add a low-molecular weight cryoprotectant solution to penetrate the cells and prevent ice crystal formation, bringing down the temperature slowly enough for the cells to adjust biochemically and osmotically, “and allowing the protective agents to do their job,” notes John M. Baust, president and CEO of CPSI Biotech.
The most common preservation medium today is still a home brew—basically culture medium, serum, or a mixture of these, to which DMSO has been added. But changing to a commercial preservation solution like Unisol, CryoStor, or Viaspan—can have a big impact on overall survival of the samples, Baust explains. While they still use a DMSO base, “they’re specially formulated to meet the needs of a cell at low temperature—because what a cell needs at 37°C is very different from what a cell needs at 4°C.” For example, sodium and potassium pumps don’t operate at 4°C, meaning that the cell’s ion gradient will be affected by hypothermia.
Of course, there are cryopreservatives specifically designed for certain cell types or applications, and not all cryoprotectants contain DMSO—some may be glycerol- or propylene glycol-based, for example, or take advantage of the properties of sugars such as trehalose or sucrose. Yet a large-scale biobank like Coriell prefers to “go with the cheap and cheerful DMSO reagent” that they produce themselves from the raw chemicals, says its chief laboratory officer Nahid Turan. “The DMSO may actually be a bit more toxic, but it can be slightly more protective in some regards as well.”
“You can reduce the toxicity by reducing the temperature,” explains John Morris, CEO of Asymptote, part of GE Healthcare Life Sciences. Standard procedure is to cool the cells to 4°C (or 0°C on ice) before adding cold cryoprotectant.
Go down
Then cool them down to somewhere around -60 to -80°C at about 1°C per minute, after which cells can be transferred to long-term storage.
There are several ways to control the freezing rate. On the DIY end, is simply placing the vials in a Styrofoam box and placing the box in a -80°C freezer—a favorite of many cash-strapped academic labs. A step up are inexpensive devices such as Mr. Frosty and CoolCell freezing containers, which also provide a gradual ramp using the freezing power of a -80°C freezer. But such solutions are “difficult to validate and they’re relatively low capacity,” Morris remarks.
Another option is a programmable controlled rate freezer. These give “very accurate cooling and very good sample-to-sample uniformity,” as well as enable the user to customize the ramping protocol, points out Morris. Cooling is achieved either by liquid nitrogen vapor or, in the case of GE’s Via Freeze, mechanical means.
Image: The VIA Freeze range of controlled-rate freezers are liquid nitrogen-free. They combine customizable freezing profiles, electronic control systems, and a conduction cooling method to optimize GMP-compliant cryopreservation. Image courtesy of Asymptote, part of GE Healthcare Life Sciences.
Keep cool
“The most common method for cryopreservation is liquid nitrogen,” states Joe LaPorte, director of PHC Corporation of North America (formerly Panasonic Healthcare Corporation of North America)’s product group. However, from a safety and material compatibility standpoint, it’s preferable to store the cryovials in the vapor phase rather than submerging them in the actual liquid, points out Baust.
Another option is an ultra-low temperature mechanical freezer that avoids liquid nitrogen altogether, although many can be fitted with a nitrogen backup for added redundancy in case of power outage or other failure.
The arguments in favor of either a liquid nitrogen or mechanical cryogenic freezer are beyond the scope of this brief article, but revolve mainly around infrastructure, ongoing costs, energy consumption, health and safety, and reliability. From an end user’s perspective though, “once you get below -130°C that’s considered infinite storage,” LaPorte says. “It really doesn’t matter, as long as you’re keeping it below those temperatures.”
Come back
Probably the most standard way to thaw cryopreserved cells is to place them in a 37°C water bath, but “it is shifting quickly, in the clinical area, to a dry thawing system,” notes Baust. “Because water baths are dirty, and there’s a risk of contamination.”
Morris points out other reasons for the shift: dry thaw systems are better controlled, and trackable for documentation purposes.
Regardless of the type of system, it’s best to gently mix or agitate the cells as they thaw to prevent overheating in spots, stopping as the last bit of ice disappears—about 3 minutes for a standard cryovial. “Then you dilute it with your culture medium, or do whatever processing you’re going to do,” Baust adds.
The cells may initially look fine, but be undergoing apoptosis or other stress responses that may take 12–48 hours to see. So Baust recommends assaying the cells after they have been back in culture for a while, ideally testing not just for viability but functionality as well.
Cryopreservation is still a mix of art and science. While equipment is being refined, protocols are somewhat generic, behooving the practitioner to consult with colleagues and the literature, as well as to experiment and optimize. What works well for reproductive medicine, after all, may not be ideal for cell therapy.