The impact of tetrabutylammonium lactate (TBAL) semiclathrate former on the viability of Saccharomyces cerevisiae

Cell suspensions are stored long term via cryopreservation in mechanical ultra freezers or in liquid nitrogen with the addition of cryoprotective agents (CPAs). Cell viability is often compromised by ice formation, which causes osmotic stress from concentrated solutes and mechanical damage from ice recrystallization. There is a need for novel CPA formulations and alternative approaches. Some ionic polyalkyl-onium salts, miscible in water, form ice-like semiclathrate crystals. Semiclathrates can dissociate at temperatures greater than 0 °C and, unlike ice, incorporate semiclathrate forming solutes into the crystalline phase, which can reduce the residual environment's tonicity. How these properties impact cell survival has yet to be explored. In this study, Saccharomyces cerevisiae viability was assessed after exposure to -70 °C with different mixtures of the semiclathrate former tetrabutylammonium lactate (TBAL). The results show that while TBAL is impermeable to the cells, toxicity, perhaps due to cation interactions with the cell membrane caused a loss of culturability above 0.6 M TBAL, which limited analysis to only partial semiclathrate forming conditions. However, at a low concentration (60 mM TBAL), where a small amount of semiclathrate formation is predicted to follow ice formation, the viability significantly increased compared to the saline only control and was similar to conventional CPAs at this condition. While further work is needed to decouple the impact of semiclathrate formation from other effects, and the toxicity of TBAL is currently limiting - the work presents the basis of a novel approach that may have applicability in some cryopreservation protocols.