UC Berkeley

The realization of on-demand organ and tissue availability could yield a benefit to global health comparable to the curing of cancer, unleashing the full potential of modern transplantation science without its current geographic, socioeconomic, and technical limitations. However, a seemingly simple scientific problem has thus far restricted this reality: our inability to preserve organs outside the body for extended periods of time.

Generally, in order to arrest the metabolism of an ex vivo biologic and prevent it from expiring, one must reduce its temperature to well below the freezing point of water (its principal material component). However, the formation of ice within complex vascularized systems proves catastrophic, and thus a wonderfully fundamental puzzle emerges: How can we prevent water from freezing, when equilibrium thermodynamics demand that it should freeze? Over the past 70 years, efforts to address this question have taken an overwhelmingly chemical approach. However, techniques driven by the use of cytotoxic non-physiological cryoprotectants to modulate freezing point has not yielded clinically-relevant advancements at the whole-organ scale to date, and thus a fundamentally different approach is needed.

This thesis presents theoretical and experimental explorations of an alternative, non-chemical approach to cryopreservation based on the rich and unexplored thermodynamic and kinetic behaviors of water and ice confined under isochoric (constant-volume) conditions. We herein develop fundamental analytical tools with which to probe isochoric phase equilibria and nucleation kinetics across volumes; reveal critical consequences of water confinement at the micro- and macroscales; experimentally demonstrate previously unrealized phase behaviors in isochoric systems in stable equilibrium, metastable equilibrium, and non-equilibrium scenarios; provide initial biological validation of isochoric cryopreservation with whole mammalian organs and tissues; and evince the additional value of isochoric freezing processes in the food industry. We develop several distinct isochoric techniques for varied preservation applications and generate baseline thermodynamic data for each, which, alongside the biological results reported herein, we hope will serve as a foundation from which the community may further advance the unique domain of isochoric freezing.