Cryo-storage is an important procedure in biological research and therapies to preserve the functions of cells, tissues, and organs. It is widely used to freeze down cell lines and primary cells including stem and immune cells, which are increasingly popular for cell-based therapy. This step is also essential for temporary preservation of tissues and organs before further study and transplantation. In this article, the principles of cryo-preservation will be highlighted, along with an explanation of how it is commonly performed. There will also be a discussion on emerging materials to enhance the efficacy of cryo-storage and thawing.
Principles of cryo-preservation
During cryo-storing, biological constructs are subject to low temperature, typically -80 °C, using a programmable freezer or -196 °C with liquid nitrogen. This slows down most biochemical processes in the cell that can cause damage and death. However, during freezing, cells will inevitably suffer from dehydration and solute toxicity as water is drawn out of their intracellular spaces, thus increasing intracellular solute concentrations to toxic levels. The formation of intracellular and extracellular ice crystals can also mechanically damage cells. Therefore, a programmable freezer is commonly used to regulate the rate of freezing to minimize ice crystal formation.
In the mid-1980s, the concept of vitrification was introduced. Instead of water crystallizing into ice, water undergoes vitrification to become a glassy ‘solid liquid’ in the presence of a class of chemicals known as cryo-protectants. Despite many years of research, there is no consensus on the mechanism of cyro-protection. Cell membrane-permeating cryoprotectants such as dimethyl-sulfoxide (DMSO) are thought to function by reducing intracellular ice crystal formation. On the other hand, non-cell membrane permeating cryoprotectants such as hydroxyethyl starch are found to increase extracellular viscosity, thus limiting the rate of dehydration and osmotic stress. They also possibly decrease the formation of extracellular ice crystals to minimize mechanical damage.
How is cryo-storing done?
Cryo-preservation is a standard protocol in most labs. Cryoprotectants are typically added to a mixture containing cell media and serum with a volume ratio of 1:4:5. It is useful to have a high concentration of serum to minimize cell dehydration and solute toxicity. Cells usually in the range of 2-5 million per mL are then mixed thoroughly in the liquid mixture before being frozen down in a programmable freezer or liquid nitrogen tank. During thawing, the cell mixture is warmed up in a 37°C water bath for one to two minutes. Next, the cells are washed with culture media to remove cryoprotectants to restore them back to their physiological states. Note that the washing step should be done immediately as chemical cryoprotectants such as DMSO can adversely affect cell viability and biological functions even with short exposure. It has been reported that poor adherence to cryopreservation and thawing protocol can cause irreversible damage to organelles and ultra-structures such as mitochondria, cytoskeleton, and cell membrane. High intracellular concentrations of cryoprotectants like DMSO have also been shown to induce oxidative stress and metabolic dysregulation.
Innovations in cryo-preservation
Cryo-preservation is a main factor for the clinical success of red blood cell storage and transfusion, and cryo-storing of embryos for reproductive medicine. With rising interest in cell therapy, including the use of stem cells for regenerative medicine and immune cells for cancer immunotherapy, there is a growing need for better cryo-preservation. Below are some recent innovations using natural biomolecules and magnetic particles for cryo-storing and thawing.
The formidable ‘water-bears’ can survive freezing by replacing most of their internal water content with the natural sugar, trehalose, to avoid toxic ice crystallization. As trehalose is unable to permeate the cell membrane, Rao et al. ingeniously conjugated trehalose to nanoparticles which were then delivered intracellularly to provide cryopreservation. Tamás and colleagues also demonstrated that extracellular, but not intracellular, localization of naturally-occurring antifreeze proteins could promote the viability of cell monolayer by three-fold. Recently, Akiyama et al. developed a method known as superflash freezing where they made use of inkjet printing to rapidly freeze cells encapsulated in droplets without any cryoprotectants.
In addition to better materials for cell freezing, scientists have also created new methods to regulate cell thawing. Wang and colleagues found that by providing a rapidly increasing and spatially homogenous warming rate during thawing, ice crystals that are formed during cryo-preservation could be melted faster and uniformly, thus minimizing damage to cells. The team synthesized magnetic nanoparticles and used magnetic induction heating to thaw stem cells. They showed that cells thawed using their technique displayed better viability, proliferative capacity, and had minimal damage to their organelles compared to their counterparts thawed using water baths. Importantly, the stem cells also better retained their ability to differentiate to different lineages. A similar strategy called nano-warming was employed by Manuchehrabadi et al. to thaw porcine heart tissues. The team demonstrated that the use of magnetic nanoparticle heating was superior to conventional convective heating as it could minimize thermal mechanical stress and improve cryo-preservation outcomes.
With rising use of patient-derived cells, organoids and xenografts, coupled with increasing popularity in cell-based therapy, the demand for better cryo-preservation protocols and reagents will only become stronger. For instance, cryo-preservation of chimeric antigen receptor T cells for cancer immunotherapy is known to kill 10 to 30 percent of cells and affect critical biological functions like cell proliferation. The lack of better protocols and cryo-protectants meant that T cells have to be grown to larger numbers, which can delay and increase the cost of treatment. Unfortunately, based on the few numbers of new literature in this area of research each year, it can be deduced that the scientific community has not yet responded to this unmet clinical and industrial need.