The last century has seen remarkable advancement in theoretical and practical therapeutics. One such development is cell cryo-preservation: the process of freezing cells and preserving them for future transfusions. Current methods involve the introduction of cryo-protectants, such as DMSO and glycerol, and freezing to levels below which biochemical reactions cannot occur.
Cryo-preserved cells are vulnerable to Ice Recrystallisation (IR). This is the formation of large ice crystals at the expense of smaller crystals and occurs during repeated freezing and thawing events. These crystals can rupture cell membranes and thus post-thaw cell numbers are often much lower than the number frozen. IR is a major factor in causing primary graft failure in transplantation patients.
Red blood cell transfusions are life changing procedures to those who have suffered from significant blood loss or for those suffering from illnesses such as leukaemia. In order to freeze samples of red blood cells (RBCs), the cells undergo an extensive glycerisation process – 40% V/V. However prior to transfusion, glycerol must be removed to less than 1% v/v to prevent haemolysis, but de-glycerisation is a lengthy process which prevents this method of being used in emergency situations.
Current cryo-protectants do not control or inhibit IR, thus it is necessary to find new cryo-protectants that can control IR to improve the success of the cryo-preservation process.
July’s Paper of the Month by Briard et al., discovered a novel class of carbohydrate derivatives with ice recrystallisation inhibiting properties that retain potency at lower glycerol percentages. The new ice recrystallisation inhibitors (IRIs) are of low molecular weight making them ideal additives to cellular systems. Briard et al., tested the ability of these molecules to reduce ice recrystallisation and ultimately improve the survival rate of cryo-preserved cells.
The group used human red blood cells (RBCs) to test the properties of the IRIs as experimental assay procedures have long since been established for RBCs. One aspect of the experiments treated the RBCs to 15% glycerol, compared to the 40% clinical standard, in an attempt to reduce the post thaw processing time.
They recreated typical cryo-cell conditions to get a real estimate of the effect of IR inhibitors on frozen cell samples. They used the Linkam FDCS196 to freeze the RBC samples and image the effect of the IRIs on ice crystal sizes. The inhibitor’s presence was found to reduce mean ice crystal size, resulting in a higher rate of cell integrity.
The transportation of cells between storage and medical facilities results in frozen samples being exposed to ambient temperatures repeatedly. Such phenomena are known as transient warming events (TWEs) and are understood to debilitate cell viability. The cyclic exposure to ambient temperatures followed by freezing temperatures can increase the size of ice crystals explaining the damage to the cells.
The group tested the effect of IRIs on crystal size during TWEs. In the presence of the inhibitors, mean ice crystal size was not found to increase during TWEs, however in the absence of the IRIs the crystal size increased. The increasing size of ice crystals play a major role in the reduction of post thaw viability.
Professor Ben, of the research group, discusses the motivation behind his work and the role of the FDCS196:
“The Linkam cryostage is an essential piece of equipment to perform the studies we described. Our motivation to design these compounds is twofold. Firstly we have always been interested in understanding the nature of the cryo-injury that cells/tissues sustain upon freezing and secondly we use this information to design new and greatly improved cryo-protectants that will enable the many novel cellular and regenerative therapies that are currently being developed. The current problem is that there is no shelf life to these cellular products as they cannot be cryo-preserved efficiently.”
These studies found aryl-glycosides to be the most potent of their IRIs. Their experiments confirmed the introduction of IRIs to the 15% glycerol samples significantly increased post-thaw viability to approximately 90% for aryl-glycosides. Cryo-microscopy confirmed the ability of the inhibitors to reduce mean ice crystal size and IR during TWEs, which in turn increased post thaw viability. Their work provides promising results by addressing issues with current cryo-protectants and with further study, may pave a way for improved cellular cryo-preservation in the future.
By Tabassum Mujtaba
Briard, J. G. et al. (2016). Small molecule ice recrystallization inhibitors mitigate red blood cell lysis during freezing, transient warming and thawing. Sci. Rep. 6, 23619