Cryopreservation provides critical protection for cell therapies by minimizing genetic changes. But cooling too slowly or quickly risks diminishing cell viability upon thaw. On 11 March 2020, Peter Kilbride (senior research scientist) and Julie Meneghel (cryobiologist), both of Cytiva (formerly GE Healthcare Life Sciences), discussed the importance of controlled-rate cryopreservation. Illustrating how mammalian cells change when frozen, Kilbride and Meneghel offered concrete cryopreservation strategies and identified temperatures at which it is safe to stop controlled cooling and transfer drug product to cryogenic storage.

The Presentation
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 postthaw 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 also can diminish cell recovery. Doing that will give cells ample time to dehydrate, but their internal solute concentrations will increase to cytotoxic levels.

An ideal protocol begins with chilling of samples to 4 °C — just above a suspension’s freezing point — and addition of 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, that usually will 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 (60–80 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 postthaw. Samples transferred at warmer than –50 °C exhibited sharp drops in viability. But drug-product transfers at lower temperatures 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 products to –60 °C.

Questions and Answers
How long can samples be stored safely? Once cooled below –120 °C, cells can be stored indefinitely because no biological or physical events will occur. Researchers should beware of transient warming when containers are closely packed. But studies confirm that drug product remains stable below –120 °C even after 20–25 years.

How do bags of different film types affect cell-therapy storage? Bag types do not influence cryostorage outcomes. But large volumes result in significant releases of latent heat. A temperature hold can account for that. Large volumes also reduce supercooling before ice nucleation, which can support viability.

Will changing cryoprotectant viscosity bolster cryopreservation? Changing the viscosity changes the protocol. Glycerol is more viscous than dimethyl sulfoxide (DMSO), needing slower cooling to maintain cell viability. Cooling too quickly with highly viscous protectants prevents ice from forming fully. Low viscosity tends to be best, although some cells tolerate glycerol or sugars better than they do DMSO.

More Online
The full presentation of this webcast can be found on the BioProcess International website at the link below.

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