Complex cell preservation methods, such as attached monolayers, have failed to achieve a level of success that would provide insights and pathways for potential whole organ preservation. Ice crystal growth during freezing can cause both mechanical and osmotic damage to cells, and the ability to control this process by using ice recrystallisation inhibitors has been shown to result in enhanced cryopreservation outcomes. A variety of antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) have been identified in organisms, of which all are icebinding proteins that are crucial for the species survival. Three different macromolecular cryoprotectants are evaluated: poly(vinyl alcohol) (PVA) due to its high ice recrystallisation inhibition activity, polyproline as a possible AF(G)P mimic, and a polyampholyte due to its scalable synthesis and precise 1:1 ratio of cationic/anionic groups. We also evaluated three potential osmoprotectants: alanine due to the heavy alanine rich regions of AF(G)Ps, betaine for its osmoprotecting properties, and proline due to its previous use as a cryoprotectant and its implications as an osmoprotectant. The macromolecular cryoprotectants and small molecule osmolytes were examined for their physical interactions with ice (Chapter 2), toxicity and proliferation impacts (Chapter 3), the ability to successfully cryopreserve mammalian cells along with post-freeze viability (Chapter 4), and finally, the membrane permeability at various steps throughout the freezing processes was evaluated (Chapter 5). Only PVA was found to have strong ice activity, minimal toxicity was found and proline was shown to down-regulate growth, osmolytes plus PVA or polyproline, and polyampholyte alone, were found to cryopreserve cell monolayers, and polyampholyte showed improved membrane permeability post-freeze. The application of these approaches could provide next generation cryopreservation strategies for many different cell types
