Advice for Successful Cryopreservation

Cryopreservation is an essential tool for preserving important cells used in molecular and cell biology and therapeutics. Without attention to detail, the process of cryopreservation can tear cells apart, literally—sharp ice crystals forming within cells can shred plasma membranes. But with careful adherence to best practices, a successful cryopreservation protocol can minimize such damage and shepherd your cells through the thawing process relatively unscathed. Here is some expert advice for preserving your valuable cells for future experiments.

Start with healthy cells

The oft-repeated advice is worth saying again: start with the healthiest cells possible. “Before freezing, the cells should have a high viability of greater than 90%, and should be free of mycoplasma contamination,” says Jonathan Steubing, Project Manager at CLS Cell Lines Service.

Even healthy cultures will incur loss from cell death during thawing. Xiaoyu (Sherwin) Zhu, Scientific Support Specialist at Corning Life Sciences, recommends using cell cultures at 90% confluency, using a minimum cell density of 10⁶ cells/mL, and changing the medium 24 hours before freezing. “In addition, you should carefully check for any contamination in the culture before cryopreservation,” he says. “For example, cells can be cultured in antibiotic-free medium for several passages, which allows time for any hidden, resistant contaminants to be easily detected.”

Freezing media

One of the key ingredients in freezing media are cryoprotective agents (CPAs), which act to protect the cells from ice crystals when the temperature drops. For research applications, dimethylsulfoxide (DMSO) is a common CPA used at 5–20% along with a protein such as human serum albumin. “The choice of CPA(s) and their concentrations must be carefully considered, as each CPA will have different levels of cytotoxicity depending on cell type,” says Julie Meneghel, Cryobiologist at Cytiva. “The exposure time of CPAs to biological material in the unfrozen state should be limited, both pre-freeze and especially post-thaw.”

“Fetal bovine serum is also used in cryopreservation—95% serum and 5% DMSO is a good recipe for some sensitive cell lines, especially hybridomas,” says Zhu. “Some cell types are sensitive to DMSO so we use 5–20% (v/v) glycerol as replacement.”

CPAs for therapeutic cells are more limited because they will be infused into humans. “For therapeutic use, serums are normally avoided, and the amount of DMSO is limited,” says Meneghel. “This is achieved either through cryopreserving at a higher cell density, or by reducing the concentration of DMSO.”

The freezing process

“Extracellular ice is inevitable, but intracellular ice can be very damaging,” says Meneghel. Avoiding damage from intracellular ice formation means cooling cells slowly and steadily. This allows ice to form in the medium, which initiates an important shuffle of water and solutes: increasingly osmolality outside the cells causes water to leave cells.

This partial dehydration is crucial to successful cryopreservation, because it means that intracellular conditions do not favor the formation of ice crystals. “However, if cooling is too fast, cells won't have time to dehydrate and ice crystallization inside cells then becomes thermodynamically favorable,” says Meneghel. “Cells experiencing this on cooling will not be viable post-thaw; if in doubt, it is generally better to cool more slowly than more quickly for this reason.” Steubing recommends the standard, commonly used cooling rate of about 1°C/min. “Usually, we add freeze medium and place the cells at -20°C for 40 minutes, store the cells at -80°C overnight, and then transfer the cells into liquid nitrogen,” he says.

Although the cryopreservation of cells destined for research or therapeutic uses is similar, there are some important distinctions to keep in mind when handling therapeutic cells, which are often primary or stem cells or lymphocytes. “These cells are more sensitive to the temperature change and tend to lose viability during the cryopreservation,” says Kyung-A (Katie) Song, Scientific Support Manager at Corning Life Sciences. “To protect viable cells, we often use CPA recipes that include human or bovine serum albumin and other factors, and freeze at a higher density of cells for research applications.”

The thawing process

“Please remember, ‘thaw quickly, freeze slowly,'" says Song. “Fast cooling will reduce the cell viability, or cause cell death.” Thawing cells quickly, and removing or diluting CPAs, are paramount. After removing a cryovial from vapor phase liquid nitrogen, place it immediately into a 37°C water bath/device. “Rapid thawing provides the best recovery for most cell cultures because it reduces or prevents the formation of damaging ice crystals within cell during rehydration,” says Song.

Steubing recommends preparing sterile materials and media beforehand, keeping the cell culture medium at room temperature. “Work fast once the cells are thawed,” he says. “Do leave a small ice clump in the cryovial before starting to work under the sterile hood.” Because CPAs become toxic upon thawing, don’t allow the sample to warm to the temperature of the bath/device.

The next important step is removing or diluting the CPAs quickly, before they harm your cells. “This is especially true in cell therapies where samples are often warmed containing the CPA before infusion,” says Meneghel. “This warming is not done for a cryobiological reason—we’d prefer that the sample remains cold—but rather that cold-infusion into a patient can be painful.”

Because the freezing media typically contains a high concentration of serum and cryoprotective agents, thawing means transitioning your cells to media containing less of both. “It is important to transfer the cells very slowly into a 15 mL conical tube containing pre-warmed media because the cells will be going from high to low concentration media,” notes Song.

Following these cryopreservation guidelines will help to keep important cell lines or therapeutic cell samples safe until needed—and healthy when thawed for future use.