The use of iPTD engineered antifreeze proteins for cryopreservation of cells

Antifreeze proteins from natural sources have been discovered to have cryoprotective function against freezing temperature, and have been tested for the application for cryopreservation of biological materials. However, none has been shown to match the effectiveness of current chemical cryoprotectants, such as dimethyl sulfoxide. One potential limitation with the application of antifreeze proteins is that they may only stay in the extracellular space around cells whereas chemical cryoprotectants can be penetrative. In this thesis project, we have designed, purified and explored the function of antifreeze proteins that were engineered with an intracellular delivery signal peptide, known as iPTD. We showed that iPTD-engineered antifreeze proteins had effective cell surface coverage within 30 minutes of incubation as shown by flow cytometry; however no intracellular protein delivery was observed under multiphoton microscopy. The plasma membrane was protected by iPTD-engineered antifreeze proteins during cryopreservation as seen in Calcein dye release assay, but cell recovery or proliferation was not observed after thawing. Given these properties of iPTD-engineered antifreeze proteins, we used them as red blood cell cryopreservation additives. By adding these modified antifreeze proteins, we were able to reduce the amount of glycerol (used for RBC cryopreservation) necessary to control freeze-induced hemolysis. Furthermore, the quality of thawed red blood cells is higher as protein addition resulted in high retention of intracellular ATP.Medicine, Faculty ofMedicine, Department ofGraduat