Although cryopreservation is viewed as an “old school” discipline, modern cryopreservation is going through a growth face. Today’s cryopreservation involves processes and products that are at the forefront of research in various fields such as:
• Personalized medicine
• Diagnostic development
• Stem cell research
• Discovery science
As the need for cryopreserved cells increases, the demands placed on biobanks are evolving and rising at a rapid rate. Researchers now require samples with high cell recovery and products that are biochemically and physiologically identical to its original state at the structural, functional, proteomic, genomic, and reproductive levels. This has caused biobanks to rise to the challenge to adapt to new protocols and strategies to meet the new demands. There have also been studies concluding that the direction and control of the cell’s molecular response to cryopreservation have a great impact on the final outcome.
What is Cryopreservation?
Cryopreservation is a process where cells, organelles, extracellular matrix, tissues, organs, and various biological constructs are preserved by cooling these biospecimens to extremely low temperatures. This can be done using solid carbon dioxide to reach -80°C or liquid nitrogen to reach 196°C. When the temperatures are low enough, any chemical or enzymatic activity that can cause damage or degeneration of the material can be stopped. The methods involved aim to reach the low temperatures without damaging the specimens caused by ice crystal formation when freezing. Traditionally, cryopreservation involves coating the material with cryoprotectants. However, due to the toxicity of many cryoprotectants, new techniques are constantly being developed. By default, cryopreservation should be considered to alter the structure and function of tissues.
Physical Effects of Cryopreservation
Cryopreservation usually involves reducing the temperatures of samples from normal temperatures to a final temperature for storage. Immediately before the cooling phase, samples are placed into a medium with a cryoprotective agent and maintained at about 4°C for equilibration. For optimal preservation, sample cooling is done at a rate of -1°C per minute. This process also helps minimize volume excursions and enables water efflux from cell to extracellular environment. This can cause cellular dehydration. This continues until the temperature is lower than the glass transition temperature. Although beneficial to reduce the likelihood of intracellular ice formation, continued dehydration can cause structural damage to the organelles, membranes, nucleic acids, and proteins. The damage can be detrimental as it can cause activation of degradation pathways (DNA repair, necrosis, apoptosis, etcetera) and delayed molecular based repair. Severe damage can result in loss of the sample.
Molecular Effects of Cryopreservation
Besides the physical effects of cryopreservation, there are also molecular effects due to factors such as:
• Uncoupling of biochemical pathways
• The decrease in metabolic activity and energy
• Activation of stress response pathways
During storage, temperature reduction can cause the metabolism and biochemical pathways to slow down because of low levels of kinetic energy. It is important to note that low temperature only slows and does not stop cellular biochemical reactions. Biochemical reactions will continue regardless of temperature and cause damage to subcellular components, cells, protein, DNA, RNA, viruses, and bacteria. These reactions can lead to the formation and accumulation of toxic compounds like anaerobic metabolism byproducts, free radicals, and waste products. This can lead to the activation of molecular stress responses or direct damage after thawing. When a sample experience stressors, it can lead to biochemical degradation or molecular death once thawed. Some of these factors include:
• Free radical production
• Metabolic uncoupling
• Dysregulation of cellular ion balance
• Cell membrane fluidity and structure changes
• Cryoprotective agent exposure
• Osmotic fluxes
• Calcium release from intracellular stores
When there are production and accumulation of free radicals, it can damage mitochondrial integrity, DNA, and protein in samples or cells. It can also activate apoptotic pathways and unfolded protein response that can cause further degradation.
Effects of Sample Storage Conditions
During storage, the damage continues at both the physical and molecular level. Samples that go through freezing-induced dehydration can lead to continual damage. Biochemically, the reactions continue despite the low temperature that slows it down. Although highly reduced, during storage, energy and oxygen consumption in the sample continues. This also means sample degradation also continues with amplified stress events when the storage temperature increases. Although the specific responses of various processes and reactions are not well understood, it is widely accepted that the temperature during storage greatly affects the time samples before a significant compromise is apparent. Based on estimations, it would take thousands of years before an effect on cryopreserved cultures become noticeable. Storage intervals of 1 to 2 years for DNA, RNA, protein, bacteria, and viruses at -80°C have been reported to be successful. These hold times are for reference only. The repeated process of freeze-thawing will lead to accelerated sample degradation.
Continual physical, molecular, and biochemical responses in the biospecimens have a significant impact on the quality of the biospecimens.
1) Cryopreservation. Wikipedia. Accessed 6/17/2019. https://en.wikipedia.org/wiki/Cryopreservation
2) Baust JM. Biopreservation: The impact of freezing and cold storage on sample quality. Eppendorf. 2016. Accessed 6/17/2019. https://pdfs.semanticscholar.org/faaf/d05959d82a2b43c9e78a6b6124d6f454df20.pdf
3) Baust JM, Corwin WL, VanBuskirk R, Baust JG. Biobanking: The future of cell preservation strategies. Adv Exp Med Biol. 2015; 864: 37-53.