Cryopreservation is the practice of preserving biological materials by cooling them to very low temperatures, typically -196°C, the boiling point of liquid nitrogen. At these temperatures, nearly all biological activity ceases, effectively pausing the processes that lead to cellular decay. This allows for the long-term storage of cells, tissues, and other structures for future use.
Within cryopreservation, vitrification is an advanced method that involves cooling biological samples so rapidly that the water inside them becomes a glass-like solid. This process avoids the formation of damaging ice crystals, improving outcomes for delicate biological materials sensitive to traditional freezing methods.
The Vitrification Process
The core objective of vitrification is to solidify a sample while preventing the formation of ice crystals. With their sharp, crystalline structure, ice crystals can pierce and shred delicate cellular components like membranes and organelles, leading to cell death. Vitrification bypasses this danger by transforming water into an amorphous, non-crystalline solid state, much like glass. This is achieved through a combination of cryoprotectant agents and an extremely high rate of cooling.
To prepare a sample, it is first exposed to high concentrations of cryoprotectants, such as ethylene glycol and dimethyl sulfoxide (DMSO), which penetrate the cell. Their function is to dramatically increase the viscosity, or thickness, of the fluid inside. This high viscosity physically obstructs water molecules from organizing into the rigid structure of an ice crystal as the temperature drops.
Once the cells are saturated with cryoprotectants, they undergo ultra-rapid cooling. The sample is plunged directly into liquid nitrogen, causing its temperature to plummet at rates that can exceed 20,000°C per minute. This speed does not allow enough time for ice nucleation—the initial step of crystal formation—to occur, locking water molecules into a stable, glass-like state.
Vitrification vs. Slow-Freezing
The traditional method, known as slow-freezing, operates on a principle of controlled dehydration. In this process, samples are cooled at a much slower, controlled rate, often around 1°C per minute. This gradual temperature drop encourages ice to form in the medium outside the cells first. As external ice crystals grow, they draw water out of the cells through osmosis, causing the cells to dehydrate. This cellular dehydration increases the concentration of solutes inside the cells, which lowers their freezing point and reduces the amount of water available to form ice internally. The process is delicate: if cooling is too slow, cells can suffer from excessive dehydration, but if it is too fast, lethal intracellular ice can form.
In contrast, vitrification avoids ice formation altogether by using high concentrations of cryoprotectants and flash-freezing rates. This eliminates the risk of mechanical damage from ice. The trade-off is that the high concentrations of cryoprotectants can be toxic to cells, so exposure time must be precisely managed.
Applications of Vitrification
Vitrification has several important applications in medicine and research. One of its most prominent uses is in human reproductive medicine, where it is a standard procedure in many in vitro fertilization (IVF) clinics for the cryopreservation of human oocytes (eggs) and embryos. Because oocytes are large, single cells with high water content, they are particularly vulnerable to damage from ice crystal formation, and vitrification has dramatically improved their post-thaw survival rates compared to slow-freezing.
Beyond reproduction, vitrification is used in the banking of stem cells for future therapeutic use. Stem cells, which have the potential to develop into many different cell types, are used for regenerative medicine and research. Vitrification allows these cells to be stored long-term without significant loss of viability or function.
The technology is also expanding into the preservation of more complex biological structures. Researchers are using vitrification to store tissues and organoids—miniature, lab-grown organs used for drug testing and disease modeling. Preserving these intricate structures without ice damage supports advancements in tissue engineering and provides a platform for studying human development and disease.
The Warming Process and Viability
The warming process is as important as cooling for ensuring a sample remains viable. The primary danger during warming is a phenomenon known as devitrification, where ice crystals can form as the glassy solid transitions back into a liquid. This occurs because the vitrified state is metastable, meaning it can crystallize if given enough time in a specific temperature range.
To prevent devitrification, the warming process must be just as rapid as the initial cooling. Warming rates must be high enough to move the sample through the critical temperature zone before ice has a chance to nucleate and grow. If warming is too slow, small ice crystals can form and then merge into larger, more damaging crystals in a process called recrystallization.
Viability refers to the ability of the preserved cells to function normally after being warmed. For a vitrified embryo, viability means it can continue to develop and has the potential for implantation. For an oocyte, it means it can be successfully fertilized. Thanks to refined protocols, survival rates for vitrified samples are high, with studies reporting that over 90% of vitrified human embryos survive the warming process with their structure intact.