How are Cryoprotective Agents (CPAs) used in cryopreservation?
Cryopreservation is a key enabling technology used in many industries and having constant, reliable and effective biological materials at the end of the thawing process is critical. Cryoprotective agents (CPAs), or also known as cryoprotectants, are chemical compounds which protect the inside and outside of the cells from the physical and chemical stresses that occur during the cryopreservation process. They allow for the cryopreserved biomaterial to remain viable after being thawed and are necessary for the cryopreservation process. The origins of cryopreservation date back to the 1800s, and it is closer to the 1900s that CPAs were being adopted for use during cryopreservation. Some commonly used CPAs include glycerol, DMSO or hydroxyethyl starch (HES). Freezing solutions typically contain relatively low CPA concentrations near 10%, and these are added in a single step.
CPAs are commonly used to protect against ice formation which causes freezing damage to the biological tissue when being cooled. CPAs reduce ice formation at any temperature by increasing the total concentration of all the solutes present in the system. They lower the melting point of water and hence protect the cells. The cells first shrink by osmosis in response to the high solute concentration outside the cell and then swell as the penetrating CPA enter the cell. Within several minutes, the CPA concentration inside and outside the cell will equalize. The cells will return to a volume defined by the tonicity of the carrier solution. The cells or tissues are then ready for freezing. CPAs are divided into two categories depending on their cell membrane permeability. Penetrating CPAs (CPAs that cross cell membranes and enter cells) are often used with non-penetrating CPAs (ones that do not enter cells) because ice more readily forms extracellularly than intracellularly.
While CPAs are essential to the cryopreservation process, they do not fully prevent the deadly effects of ice recrystallization. Ice recrystallization is a process whereby ice crystals formed during the freezing process grow larger at the expense of smaller ice crystals, and this leads to lower cell recovery and decreased cell function. CPAs also do not protect against the cell damage that ice crystals cause when samples are inappropriately stored or experience transient warming during handling, storage, and shipment.
Effective cryopreservation would be possible if there was no restriction to the quantity of CPAs that can be employed. However, commonly used CPAs are toxic by nature and can cause damage to the cells that they are meant to preserve.
Research done on some of the CPAs available has found that it is possible to reduce the toxicity of the CPAs by combining them. For example, it’s been shown that combining DMSO and Ethylene glycol (EG) resulted in reduced toxicity for buffalo oocytes. Understanding the permutations concerning the combination of various CPAs could be of the ways of understanding the mechanisms of CPA toxicity as well as coming up with better combinations.
At PanTHERA CryoSolutions, our scientists have been working hard at bettering the cryopreservation process and hence increasing the quality of thawed cells. PanTHERA’s first-of-its-kind ice recrystallization inhibitors (IRIs) are novel small carbohydrate-based compounds. In comparison to the traditional CPAs that do not control ice growth, PanTHERA’s products can control ice crystal growth during the cryopreservation processing of biological samples.
PanTHERA’s IRIs allow for lower required concentrations of CPAs needed to achieve stable storage, and hence, reduce toxicity-associated damage caused by the CPAs. In addition, the IRIs guard against the harmful impact of transient warming and allow for higher temperate storage conditions. More information on the IRIs can be found here.
About PanTHERA CryoSolutions
PanTHERA CryoSolutions is a Canadian corporation that designs and manufactures cryopreservation solutions for cells, tissues and organs for research and clinical markets. Our patented ice recrystallization inhibitor (IRI) technology exceeds other products by providing superior cryopreservation and increasing post-thaw cell recovery and function for our customers. The technology enables the use of significantly less costly storage and transportation systems limiting the need for liquid nitrogen use for some cell therapy applications.