Cryopreservation is a scientific method for preserving biological materials, such as cells, tissues, or organs, by cooling them to extremely low temperatures, typically -196°C. At these frigid temperatures, all biological activity effectively comes to a halt. This suspension of activity prevents degradation, allowing for the long-term storage of biological specimens. The ability to preserve living cells for extended periods holds importance, particularly in stem cell research and therapy, where maintaining cell viability and function is crucial for their future use.
Understanding Cryopreservation
Extremely low temperatures are necessary to arrest cellular activity completely. At temperatures above freezing, even slightly, cellular processes continue, albeit slowly, leading to degradation and eventual cell death. As water within cells freezes, it forms ice crystals, which can cause significant mechanical damage to cell membranes and internal structures. Furthermore, as water turns to ice, the remaining unfrozen liquid becomes highly concentrated with solutes, leading to osmotic stress that can fatally dehydrate cells.
To counteract these damaging effects, cryoprotective agents (CPAs) are introduced. These substances, such as dimethyl sulfoxide (DMSO) and glycerol, work by increasing the solute concentration within and around cells, which lowers the freezing point of water and reduces the amount of ice formed. CPAs also interact with water molecules, forming hydrogen bonds that displace water and help maintain the structural integrity of biological molecules like proteins and DNA. While CPAs are beneficial, their concentration must be carefully managed, as high concentrations can become toxic to cells.
The Process of Stem Cell Cryopreservation
The cryopreservation of stem cells begins with their collection and preparation. This involves isolating stem cells from their source, such as bone marrow, umbilical cord blood, or adipose tissue, and then performing quality control assessments. Once the cells are deemed suitable, they are suspended in a specialized cryopreservation medium.
A precise concentration of cryoprotective agents (CPAs), commonly 10% DMSO, is then slowly added to the cell suspension. DMSO facilitates the movement of water across the cell membrane and helps prevent intracellular ice formation by increasing the intracellular solute concentration. Following CPA addition, the cells undergo a controlled freezing process, typically using either slow freezing or vitrification methods. Slow freezing involves gradually cooling the cells at a controlled rate, often around 1°C per minute, allowing water to slowly leave the cells and minimize ice crystal formation. Vitrification, on the other hand, is a rapid cooling technique that uses higher CPA concentrations to transform the cell suspension into a glass-like solid without any ice crystal formation.
After the freezing process, the cryopreserved stem cells are transferred to long-term storage, commonly in liquid nitrogen tanks at -196°C. When the stem cells are needed for use, they undergo a rapid thawing procedure, typically by immersing the cryovials in a warm water bath at 37°C. Following thawing, the CPAs are carefully removed from the cells to prevent toxicity, and the cells are then prepared for their intended application.
Applications of Cryopreserved Stem Cells
Cryopreserved stem cells have applications across scientific and medical fields. In regenerative medicine, these cells are used in cell-based therapies to repair or replace damaged tissues and treat diseases. For instance, hematopoietic stem cells, often sourced from bone marrow or umbilical cord blood, are routinely cryopreserved for use in transplants to treat blood cancers and other blood disorders.
Beyond clinical treatments, cryopreserved stem cells are important tools in research and drug discovery. They provide biological models for studying disease mechanisms, testing the efficacy and toxicity of new drugs, and understanding cell differentiation processes. Biobanking initiatives rely on cryopreservation to store collections of stem cells, cancer cells, and genetic material, which facilitates ongoing research and the development of targeted therapies.
Cord blood banking allows parents to store their newborn’s umbilical cord blood for potential future use in the child or a compatible family member. This provides a readily available source of stem cells for transplantation. Banking and retrieving stem cells also supports personalized medicine, enabling the use of a patient’s own cells for tailored treatments, reducing the risk of immune rejection.
Factors Affecting Viability and Future Use
The success of stem cell cryopreservation is influenced by several factors. The specific stem cell type plays a role, as different cell types may respond uniquely to freezing and thawing protocols. For example, embryonic stem cells and pancreatic islets may benefit from rapid cooling, while hematopoietic and mesenchymal stem cells often fare better with slower cooling rates. The initial quality of the cell sample, including its viability and purity before freezing, also impacts post-thaw recovery.
The effectiveness and concentration of cryoprotective agents are important, as they must protect the cells from ice formation and osmotic stress without causing toxicity. Precise control over both the freezing and thawing protocols is also important. Deviations from optimal cooling or warming rates can lead to increased intracellular ice formation or osmotic damage, compromising cell integrity. After thawing, post-thaw cell viability assessment is performed using methods such as cell counting and functional assays to confirm cell survival and biological activity. Ongoing research aims to improve CPA formulations and refine freezing and thawing techniques to enhance post-thaw cell viability and expand the applications of cryopreserved stem cells.