Cryopreservation is a process that involves cooling and storing biological materials at extremely low temperatures to preserve them for extended periods. This technique halts biological activity and prevents degradation, allowing cells, tissues, or organs to maintain their viability and structural integrity for future use. This is commonly achieved using liquid nitrogen, which maintains temperatures around -196°C.
The Science of Cryopreservation
Cryopreservation relies on the principle that extremely low temperatures slow down or halt metabolic processes within cells. This reduction in metabolic activity minimizes the depletion of energy stores and the accumulation of harmful waste products, preserving cells. However, the freezing of water, a major component of biological material, presents a significant challenge. As water freezes, it can form ice crystals, which can damage cell membranes and cellular structures.
Ice crystal formation can occur both inside and outside cells. Ice crystals can puncture cell membranes and damage organelles, while extracellular ice formation leads to osmotic stress and cellular dehydration. To counteract these damaging effects, cryoprotective agents (CPAs) are introduced. These molecules, such as dimethyl sulfoxide (DMSO) and glycerol, penetrate cell membranes and depress the freezing point of water, reducing ice formation.
CPAs also increase the viscosity of the solution, which can promote vitrification, a process where the solution solidifies into an amorphous, glass-like state without forming damaging ice crystals. Some CPAs also interact with water molecules, helping biological materials retain their structure. This allows the cells to survive the freezing and thawing processes with minimal damage.
Biological Materials Preserved
Cryopreservation is widely applied for long-term storage of diverse biological materials. In reproductive medicine, for instance, it is commonly used to preserve reproductive cells like sperm, eggs (oocytes), and embryos. This allows individuals and couples to preserve their fertility for future use, especially when medical treatments might compromise it or for family planning. Embryo cryopreservation has revolutionized in vitro fertilization (IVF) by allowing unused embryos to be stored and used in subsequent treatments, increasing efficiency and reducing patient discomfort.
Stem cells are another category of biological materials preserved. These cells, including hematopoietic stem cells, are preserved for use in regenerative medicine and disease treatment. Cryopreserved stem cells are also used in basic research to study cell biology, development, and disease models, providing a stable supply of cells for experimentation.
Various tissues are also routinely cryopreserved for medical and research purposes. This includes storage of blood components, such as red blood cells and platelets, for transfusions, and tissues like blood vessels, heart valves, and skin grafts for transplantation. Cryopreservation extends the usability of these tissues, making them available when needed for surgical procedures and expanding the pool of transplantable materials. Beyond human applications, cryopreservation is also employed in biodiversity conservation, preserving non-human samples like animal cells, plant germplasm (seeds and pollen), and microorganisms (bacteria and fungi) for future research and efforts to preserve endangered species.
The Cryopreservation Process and Storage
The cryopreservation process begins with the careful preparation and selection of the biological material to ensure its viability and suitability for preservation. Once selected, cryoprotective agents (CPAs) are introduced to the sample. These agents, such as glycerol or DMSO, are added to the solution surrounding the cells to reduce the freezing point and minimize the formation of damaging ice crystals during cooling. The specific type and concentration of CPA used can vary depending on the biological material.
Following the addition of CPAs, the biological material undergoes a controlled cooling phase. This controlled rate of cooling is important to prevent rapid cellular dehydration or the formation of large, destructive intracellular ice crystals. Specialized controlled-rate freezers are often used, which can precisely manage the temperature decrease, typically cooling cells from room temperature to around -90°C to -130°C. While a uniform cooling rate of about 1°C per minute is often effective, optimal rates can differ based on cell type and water permeability.
For long-term preservation, cryopreserved samples are typically transferred to and stored in liquid nitrogen. This ultra-low temperature, around -196°C (-321°F), ensures the sustained viability of the material for extended periods, potentially indefinitely. Samples are usually stored in specialized cryogenic vials within liquid nitrogen dewars, which are vacuum-jacketed tanks designed to maintain these extremely cold conditions. Vapor phase storage within these dewars is preferred to minimize the risk of contamination and potential vial rupture associated with direct immersion in liquid nitrogen.
The final stage is the thawing process, which is equally important for successful revival of the preserved material. Thawing is performed rapidly, by immersing the cryovial in a 37°C water bath. Rapid thawing helps to minimize the formation of harmful ice crystals that can occur during rewarming, especially if the process is too slow. Once thawed, the cryoprotective agents are carefully removed, and the cells are then gently transferred to a suitable culture medium to resume their normal functions.