Engineering Modeling of Scale-up Cryopreservation by Vitrification: Thermal, Mechanical, and Crystallization Effects

A persistent gap between the supply and demand of organs for transplant medicine and research is well recognized, despite advances in medicine and biotechnology, and despite the elevation of awareness about the benefits of organ donation and transplantation. Organ preservation at low temperatures (cryopreservation) is a promising technique that can potentially increase the availability of organs by improving both the logistics and outcomes of transplantation.

Several approaches for biopreservation have been developed over the century, where cryopreservation is one such approach that enables the preservation of structurally intact living cells and tissues. Single cells and small specimens (μm to mm range) can be readily cryopreserved using classical preservation, limiting the ice formation to the extracellular spaces. Cryoprotective agents (CPAs) are typically added to the biospecimen to control ice formation and growth. However, it becomes increasingly challenging to control and ideally avoid ice formation using low concentration CPAs, where the complexity of cryopreservation increases with the specimen size. The only viable approach to cryopreservation of large organs, such as the heart, kidney, or liver appears to be vitrification (vitreous in Latin means glassy).

During vitrification, the biospecimen is loaded with CPAs, which are characterized by an exponential increase in viscosity with decreasing temperature. When the specimen is cooled fast enough to very low temperatures, such that the mobility of molecules is hampered, crystallization can be completely suppressed, and the material is trapped in an amorphous (vitreous) state. Successful vitrification necessitates rapid cooling and rewarming rate, the magnitude of which is dictated by the concentration and the CPA cocktail used, and the chemical and physical properties of its ingredients. In large specimens, rapid cooling and rewarming rates can result in non-uniform temperature distribution across the specimen, giving rise to thermomechanical stresses, which are driven by differential thermal expansion. Increasing the CPA solution concentration assists in lowering the critical cooling rate (CCR) and critical warming rate (CWR) required to suppress crystallization, but the tradeoff is increased toxicity, which is an inherent CPA property.

The unmet need to advance the science and technology of tissue and organ preservation calls for the development of mathematical modeling of biological systems, in order to investigate the virtually infinite combinations of parameters that affect cryopreservation success, chief of which are toxicity, thermomechanical stresses, and kinetics of crystallization.In the above context, this dissertation focuses on the analyses of heat transfer and thermomechanical stress for the benefits of improved techniques for cryopreservation by vitrification. A computational framework is piecewise presented in this work, with the goal of analyzing the thermal history during vitrification protocols and provide a better understanding of the phenomena associated with phase change in realistic cryopreservation scenarios. The study discusses the marginal conditions leading to cryopreservation by vitrification, where the term marginal conditions refers to cooling rates in close range with the CCR and CWR. Special attention is given in this study to the effects of cooling rate, rewarming rate, storage temperature, and initial temperature and how they affect the extent of crystallization.

Volumetric heating is investigated in this study, as an alternative to convective rewarming during recovery from cryogenic storage. One such example of volumetric heating is the so-called nanowarming effect, which is driven by silica-coated iron oxide nanoparticles (sIONPs), which are excited by a radio-frequency electromagnetic field. A thermal model is presented in this dissertation to investigate this volumetric heat effect on the recovery of rat and human hearts. Computational analysis of nanowarming enables planning and optimization of the protocol, with potential benefits for the design of experiments. This work includes computational model calibration with experimental results. Finally, the study also investigates how the thermal history and non-uniform concentration of nanoparticles affect thermomechanical stresses developed along the cryogenic protocol.