Cryopreservation is a key step in delivering safe and effective treatments to patients in clinic. Cells and biological materials are sensitive not only to temperature, but also how fast they are cooled and the point at which the freezing process is stopped. Here, we summarize best practices in cryopreservation, including results from our study on the post-thaw viability of immortalized T cells after controlled cooling.
Controlled-rate freezing based on science
Every cell system cools differently depending on membrane permeability, dehydration capacity, and tolerance for cryoprotectant. Because differences abound, even within cell types, it is critical to determine an optimal cooling rate before cryostorage. Freezing must proceed in a controlled, linear way to maximize post-thaw viability.
Effective cooling balances drops in temperature with associated intracellular dehydration. In biological systems, ice forms only from pure water molecules. Fats, salts, sugars, cryoprotectant, solutes, and cells all condense and fall into channels between ice crystals. Cells primed with cryoprotectant dehydrate to remain at an osmotic equilibrium with their surrounding media. And the rate of that dehydration is critical to cell viability.
Cells that are cooled too quickly do not dehydrate adequately, and cells that carry too much water form ice that damages their membranes and hastens their demise. On the other hand, freezing too slowly can also diminish cell recovery. Doing so will give cells ample time to dehydrate, but their internal solute concentrations will increase to cytotoxic levels.
An ideal protocol starts with chilling samples to 4°C ‒ just above a suspension’s freezing point ‒ and adding cryoprotectant. Then, controlled freezing can begin. Optimal cooling averages 1°C/min for most mammalian somatic cell suspensions. Although nonsomatic cells (e.g., sperm and red blood cells) cool effectively at 10°C/min, this rate will usually be too fast for somatic cells. A rate of 0.1°C/min ‒ which is ideal for preserving organoids and tissue-engineered constructs ‒ often works but takes considerable time.
Some researchers use an ice-nucleation plunge step to start ice formation, but Cytiva teams do not recommend such steps because they are not proven to be effective. However, setting a hold between -30°C and -35°C (60 to 80 minutes into a process) can promote bulk ice formation in large-volume (e.g., > 20 mL) containers.
Researchers also question when it is safe to stop controlled cooling. Some suggest -80°C or even -120°C, the point of extracellular glass transition, when molecular mobility stops. The -80°C protocol exists perhaps because it matches the temperature of dry ice, which was essential to mechanical freezers. Yet little evidence supports such deep-freeze protocols.
To determine a safe point, Cytiva teams examined the viability of immortalized T cells after controlled cooling to eight end points between 4°C and -100°C. On reaching a set temperature, cells were transferred to storage, thawed, cultured, and then counted and assessed for relative fluorescence at 24, 48, and 72 hours post-thaw. Samples transferred before they were cooled to -50°C exhibited sharp drops in viability. But transferring samples at temperatures cooler than -50°C did not improve cell viability substantially.
Cytiva's findings suggest that it is safe to stop controlled cooling after cells undergo their intracellular glass transition (-47°C). At that point, cells will be maximally dehydrated, and metabolism will cease. Because containers can experience unanticipated transient heating during controlled cooling, researchers are advised to cool drug product to -60°C.