Keeping It Cool: Protecting Cryopreserved Samples from Degradation

Cryopreservation requires a slow cooling process to reach temperatures at or near the temperature of liquid nitrogen (−196°C). This method is standard practice in preserving biological samples. In their recent review, Woods et al. (2016) examined cryobiology applications in cryopreservation to identify new solutions to integrate into mainstream cell therapy.¹

Optimal cryostorage begins with the individual sample and its unique composition, which affects the cooling rate. Samples cooled too quickly are at risk of cold shock, while keeping samples at low temperatures for too long can lead to chilling injury.

The authors describe three main constraints that affect the rate of cooling:

1. Cell size or cell surface to volume ratio.
2. Cell membrane permeability (to water and cryoprotectant) and its temperature dependence.
3. Osmotic limits of the cell.

Once a cell reaches cryogenic temperatures, it is essentially suspended in time. Woods et al. explain that a robust cryopreservation and post-thaw processing system will result in an indefinite shelf life, as long as temperature is consistent and samples are protected from background ionization radiation. This threat does indeed degrade samples, but at an extremely slow rate. Estimates suggest that it would take between 200 years and 3,000 years for samples at −196°C to show significant damage from background ionization radiation alone.

Nevertheless, degraded samples are fairly common and certainly problematic for investigators. Other transient events are a common cause of degradation. Any fluctuations in temperature during storage or transfer can take a toll on samples with each freeze–thaw cycle. Along with this, suboptimal warming procedures also affect sample integrity.

Cryoprotective agents such as dimethyl sulfoxide (DMSO) can help protect samples from freeze–thaw; however, DMSO is not compatible with samples stored for clinical use. Also, DMSO can cause cryoinjury if it is not added and removed within the proper time frame.

For further protection against freeze–thaw, the authors describe automated controlled freezing systems such as CryoMed solenoid-driven liquid nitrogen vapor systems (Thermo Scientific) as a potential solution. Since sample transport is also a problem for freeze–thaw, low-tech devices such as the Mr. Frosty freezing container (Thermo Scientific) will store samples at the proper temperature and maintain the temperature during transport.

Woods and colleagues call for a science-driven approach to determine the optimal conditions for sample handling. They suggest eliminating as many degradation risk factors as possible through a thorough evaluation of the biopreservation system.

Reference
1. Woods, E.J., et al. (2016) “Off the shelf cellular therapeutics: Factors to consider during cryopreservation and storage of human cells for clinical use,” Cytotherapy, 18(6) (pp. 697–711), doi: 10.1016/j.jcyt.2016.03.295.