Cryopreservation is a process used in science and medicine to preserve biological materials by cooling them to extremely low temperatures. This controlled freeze-thaw cycle is a powerful technique for the long-term storage of cells, tissues, and even organs, which can maintain their biological function and viability for decades. While this technology is foundational for modern biotechnology, the physical act of freezing and thawing is inherently damaging to delicate cellular structures. Successfully navigating the transition to cryogenic temperatures, typically near -196°C in liquid nitrogen, requires precise control to mitigate the devastating biophysical changes that occur.
How Freezing Damages Biological Cells
Cellular damage during the freezing phase occurs through two primary mechanisms: mechanical injury from ice crystals and osmotic stress. The cooling rate significantly dictates which type of damage predominates.
When the cooling rate is too rapid, the cell’s internal water does not have sufficient time to escape. This leads to the formation of lethal ice crystals inside the cell, known as intracellular ice formation. These crystals rupture the cell membrane and destroy internal components, causing immediate cell death.
Conversely, a cooling rate that is too slow primarily causes injury outside the cell through solute concentration. As extracellular water turns to ice, the remaining liquid becomes highly concentrated with salts and other solutes, significantly increasing its osmolarity. This draws water out of the cell via osmosis, causing the cell to dehydrate and shrink. The resulting high solute concentration can denature proteins and disrupt the integrity of the cell membrane, leading to “solution effects” injury.
Thawing Rate
The subsequent thawing process introduces unique threats to cell viability. A slow thawing rate allows for a phenomenon known as recrystallization. During this period, the small ice crystals formed during freezing merge and grow into larger, more structurally damaging crystals. These larger crystals exert greater mechanical force on surrounding biological structures, often resulting in cell rupture.
The rehydration of the shrunken, dehydrated cells presents a second challenge during thawing. As the extracellular ice melts, the highly concentrated surrounding solution is diluted, becoming hypotonic relative to the cell’s interior. Water rushes back into the cells to restore osmotic equilibrium, causing rapid swelling. If the cell membrane cannot re-equilibrate quickly enough, the cell can burst, a form of osmotic lysis. To minimize damage, cells are typically thawed rapidly, often by immersion in a warm water bath to achieve a warming rate around 37°C per minute.
Strategies for Protecting Cells from Damage
The primary strategy for mitigating freeze-thaw damage involves the introduction of cryoprotective agents (CPAs), specialized molecules that shield cells from biophysical stresses. Permeating CPAs, such as Dimethyl Sulfoxide (DMSO) and glycerol, penetrate the cell membrane and replace some of the intracellular water.
By increasing the total concentration of solutes, CPAs effectively lower the freezing point of the solution and reduce the amount of water that turns into ice. This action lessens the degree of cell dehydration during cooling, thereby reducing solution effects and osmotic stress. CPAs also help minimize the formation of large ice crystals by encouraging the formation of an amorphous, glass-like solid state known as vitrification.
While CPAs are necessary for cell survival during freezing, many, particularly DMSO, become toxic to cells at ambient temperatures. Consequently, the thawing protocol requires the prompt and careful dilution and removal of the CPA to prevent chemical toxicity.
Essential Applications in Science and Industry
The successful control of the freeze-thaw cycle is foundational to several scientific and medical disciplines, allowing for the long-term storage of biological materials.
In reproductive medicine, cryopreservation is routinely used to bank gametes and embryos for fertility preservation and assisted reproductive technologies. Stored cells can remain viable for many years, offering individuals the option to delay childbearing or protect fertility before undergoing medical treatments like chemotherapy.
Cryopreservation is also the backbone of tissue banking and regenerative medicine, enabling the creation of accessible inventories of stem cells, blood components, and tissue grafts for transplantation and transfusion. In research and pharmaceutical development, the ability to freeze and store cell lines maintains a consistent supply of biological material for experiments, ensuring reproducible results and the development of new therapeutics.