Ice-nucleation by mineral dust applied to cell cryopreservation and atmospheric science

Heterogeneous ice-nucleation is a poorly understood physical process. This poses great challenges to understanding how ice formation in mixed-phase clouds affects Earth’s current and future climate. Also, cryopreservation of biological material is hampered by the physical hazards of uncontrolled ice-nucleation. The aim of this thesis is to apply understanding about materials known to trigger heterogeneous ice-nucleation, and in particular, mineral particles to improving cryopreservation processes. The learnings from this are fed back into atmospheric sciences. Cell monolayers frozen in multiwell plate format could be used for in-vitro high-throughput toxicology screening which could streamline the development of new lifesaving drugs and therapies. In this thesis it is shown how controlling ice-nucleation is essential for good recovery when cells are frozen in this way. First it is described how aqueous liquid aliquots will supercool to degree that is related to volume and in 96-well plates this can be by over 20 °C before freezing occurs. Using plated cultures of both primary and immortalized animal cells it was then shown unambiguously that when ice-nucleation is controlled post-thaw recovery is significantly improved. Moreover, a causal link was established by correlating ice-nucleation temperature with cell survival rates. Following on from this a strategy for controlling ice-nucleation in 96-well plates in a scaled up and biocompatible manner was tested. A highly effective mineral ice-nucleator previously discovered by the atmospheric science community, LDH1, was incorporated into IceStart arrays to control ice-nucleation in 96-well plates. The remarkable ice-nucleating properties of LDH1 were characterized and it was found that supercooling could almost be eliminated in 100 µL water and cryoprotectant solution droplets with as little as 0.05 mg. When trialed with plated immortalised hepatocyte cell cultures, post-thaw survival was significantly improved. Finally, the stability of mineral ice-nucleators was investigated, particularly in response to heat. This has been overlooked to date and has a crucial role in the detection of biological ice-nucleating particles the atmosphere. While some minerals such as K-feldspar are largely stable, quartz particles lose ice-nucleating activity when heated in water. This is a surprising result which drives refinement of the established heat test for biological INP and gives some clues about nature of ice-nucleating sites on minerals.