Cryopreservation is the specialized process of cooling and storing biological material, such as cells, tissues, or organs, at ultra-low temperatures to maintain their viability over an extended period. This technique suspends all biological activity, including metabolism and enzymatic reactions, effectively stopping the degradation and aging of the sample. By placing materials into a state of suspended animation, scientists can preserve them for decades, enabling a vast array of medical and research applications. The challenge is achieving this deep-freeze state without the destructive consequences of simple freezing.
Understanding Cell Damage During Freezing
The process of freezing water inside and outside a cell poses two distinct threats to the cellular structure. The most direct injury comes from the mechanical disruption caused by ice crystal formation. If the cooling rate is too rapid, water inside the cell does not have enough time to exit, leading to the formation of sharp, internal ice crystals that puncture and destroy the cell’s membrane and organelles.
A second threat is known as “solution effects” or osmotic shock. As the temperature drops, water outside the cell freezes, concentrating the solutes (like salts and proteins) in the remaining liquid. This exclusion process creates a hypertonic, or highly concentrated, extracellular environment. This imbalance draws water out of the cell to equalize the solute concentration, causing the cell to shrink, dehydrate, and suffer toxic effects from the high salt concentration.
Cryoprotective Agents and How They Work
To counteract the destructive forces of freezing, scientists rely on specialized chemicals known as Cryoprotective Agents (CPAs), which are added to the cell suspension before cooling. These agents work primarily by lowering the freezing point of the solution, delaying the onset of ice formation. By replacing water molecules, CPAs also prevent them from bonding to form crystalline structures.
CPAs are categorized based on their ability to permeate the cell membrane. Penetrating CPAs, such as Dimethyl Sulfoxide (DMSO) and glycerol, are small molecules that pass directly into the cell’s interior. Once inside, they replace some intracellular water, mitigating cell shrinkage and preventing the formation of lethal ice crystals inside the cytoplasm. Glycerol is commonly used for preserving red blood cells, while DMSO is the preferred choice for most other mammalian cells and stem cells.
Non-penetrating CPAs, such as sucrose or high-molecular-weight polymers, remain outside the cell. These agents draw water out of the cell before cooling begins, reducing the amount of water available to form ice crystals inside the cell. Non-penetrating agents also help stabilize the cell membrane and are frequently used with penetrating CPAs. This combination increases the overall cryoprotective effect while managing the toxicity caused by high concentrations of penetrating agents.
Methods of Preservation and Storage
Successful cryopreservation requires carefully controlling the rate at which the temperature is lowered, utilizing one of two main procedural methods. The traditional approach is slow freezing, also called controlled-rate cooling, which balances the risks of mechanical and osmotic damage. Cells are cooled gradually, often at a rate of approximately 1°C per minute, using a specialized programmable freezer.
This slow rate allows time for water to osmotically exit the cell and freeze externally, minimizing the formation of intracellular ice. The process involves an initial hold just below freezing to induce ice nucleation, followed by a controlled decrease in temperature down to about -80°C. Samples are then plunged into liquid nitrogen for long-term storage.
The second, more advanced technique is vitrification, which completely avoids ice crystal formation by turning the entire solution into a glass-like solid. Vitrification requires higher concentrations of CPAs and an ultra-rapid cooling rate to prevent water molecules from organizing into a crystal lattice. Samples are often plunged directly into liquid nitrogen at -196°C. Because it bypasses ice crystal formation entirely, vitrification is preferred for sensitive biological materials, such as oocytes and embryos, resulting in higher survival rates.
The final step for long-term preservation requires storage in the vapor or liquid phase of nitrogen, maintained at a stable temperature of -196°C. At this deeply cryogenic temperature, all chemical and biological processes are effectively halted, allowing the material to remain stable for decades.
Major Applications in Medicine and Research
Cryopreservation is an indispensable technology across modern medicine and biological research, with applications spanning fertility, regenerative therapy, and biobanking. In reproductive medicine, it is routinely used for fertility preservation, allowing individuals to safeguard their reproductive potential. This includes the cryopreservation of sperm, mature eggs (oocytes), and embryos for use in future in vitro fertilization (IVF) treatments.
The technology is also a foundation for cell-based therapies, particularly in stem cell banking. Hematopoietic stem cells, often collected from bone marrow or umbilical cord blood, are routinely cryopreserved for use in treating blood disorders and certain cancers. This ensures a readily available, viable source of therapeutic cells for future transplantation or research.
Beyond individual cells, cryopreservation is successfully applied to preserve various tissues for reconstructive and transplant surgery. Examples include the banking of skin grafts, heart valves, and bone or ligament tissues. While preserving whole, complex organs for long-term banking remains a significant challenge due to the difficulty of uniformly delivering CPAs and preventing ice damage throughout the organ, progress is being made.