Controlling Ice Growth: From Nucleation to Recrystallization

Abstract

The growing demand for advanced cellular therapies has facilitated a substantial need for efficient cryopreservation methods for a wide range of biological materials. As a result, a wide range of novel cryoprotective agents (CPAs) have been investigated to supplement traditional cryopreservation protocols, with the aim to improve not just post-thaw recovery, but post-thaw viability and functional capacity (e.g. proliferation and differentiation). Controlling ice recrystallization is a strategy to improve the outcome of cryopreservation, and small molecule ice recrystallization inhibitors (IRIs) have proven effective for many complex and clinically relevant cell types. The Ben lab has spent over two decades characterizing a diverse library of IRI active CPAs, however the structural requirements for IRI activity are still not fully understood. The temperature of ice crystal nucleation is also a vital factor impacting cryopreservation outcomes. As an essential step of the cryopreservation process, ice nucleation is the phenomena of an ordered, solid ice embryo forming in supercooled water. Ice nucleation can be considered either homogeneous in pure water, or heterogeneous when a foreign body lowers the energy barrier required for ice nucleation. It has been established in cellular models that it is beneficial to induce controlled, heterogeneous ice nucleation prior to a sample spontaneously nucleating. Through induced nucleation, the degree of supercooling of the sample is limited. This in turn limits thermal shock generated by latent heat release through ice nucleation and can limit the degree of intracellular ice formation observed. The use of induced heterogeneous ice nucleation can also generate a more consistent cryopreservation protocol, as the temperature of spontaneous ice nucleation can be widely variable, even in identically prepared samples. The research described in this thesis leverages the decades of research from the Ben lab to further the understanding of IRI and ice nucleation activity (INA) of small molecules. This work implements an in-house assay for the characterization of ice nucleation activity. With an understanding of small molecule INA activity, the degree of interaction between small molecule INA and IRI activity is examined herein. In parallel, this work reports synthetically accessible scaffolds containing the structural components required for IRI activity in N-functionalized gluconamides. The structure activity relationship investigations elucidate not only a novel set of highly IRI active small molecules, but also an improved understanding of the functional tolerance for further derivatization. Collectively, the work described herein sets the groundwork for the targeted generation of not only specialized small molecule IRIs, but the first attempts to generate dual-action IRI / INA active small molecule CPAs.