When a living cell is frozen, it is threatened with destruction by ice crystals and osmotic stress. The physical forces of the phase transition from liquid to solid are enormous - and without protection, no cell can survive this process unscathed. This is where cryoprotective agents (CPAs) come into play: chemical substances that specifically protect cells from the damaging effects of freezing.
The choice of the right cryoprotectant, its concentration and the protocol for its addition and removal are just as crucial to the success of cryopreservation as the quality of the freezer used. In this article, we explore the scientific basis of the most important CPAs and their practical application in combination with Consarctic® cryotechnology.
CPAs develop their protective effect via two main mechanisms:
Due to their presence in the solution, CPAs lower the freezing point and increase the viscosity. This slows down the formation of ice crystals and gives the cells more time to adapt to the changed osmotic conditions.
Certain CPAs attach directly to the cell membrane and stabilize the lipid bilayer. They thus prevent the mechanical rupture of the membrane by ice crystals and the loss of cell integrity.
DMSO is the most commonly used cryoprotectant in biomedical research and clinical applications. It is a penetrating (permeating) CPA that can pass through the cell membrane and thus reduce both intracellular and extracellular ice crystal formation.
Glycerol is another classic permeating CPA with a long history in cryopreservation - it was the first cryoprotectant discovered in 1949 by Christopher Polge for the preservation of sperm.
Trehalose is a non-permeating disaccharide that occurs naturally in cold-resistant organisms such as tardigrades. It works by forming a glass-like matrix around the cell and stabilizing the membrane from the outside.
The choice of CPA and freezing protocol are inextricably linked. An automatic freezer such as the BIOFREEZE® must match the cooling rate precisely to the CPA protocol used.
For DMSO-based protocols, a controlled cooling rate of typically -1°C/min is optimal. The TC-Active function of the BIOFREEZE® detects the released crystallization heat and automatically compensates for it - a decisive advantage over passive freezing methods.
Extremely fast cooling rates are required for vitrification with trehalose-containing media. Here, the Controlled Rate Freezer is less important than direct immersion in liquid nitrogen. However, the subsequent safe long-term storage in a Consarctic® nitrogen tank remains equally critical for both methods.
The scientific community is investing significant resources in the development of new, less toxic CPAs. Approaches include:
Regardless of which CPA protocol a facility uses, Consarctic® cryoproducts offer the flexibility and precision needed to achieve optimal results. From programmable freezing curves to safe long-term storage in the gas phase, we provide the technical foundation for your successful cryopreservation.
Would you like to support your CPA protocols with optimal technology? Talk to our application specialists about the ideal device configuration for your specific freezing protocol.