Title

Advancements in Cryomacroscopy with Applications to Cryopreservation by Vitrification

Date of Award

Fall 9-2014

Embargo Period

1-27-2015

Degree Type

Dissertation (CMU Access Only)

Degree Name

Doctor of Philosophy (PhD)

Department

Mechanical Engineering

Advisor(s)

Yoed Rabin

Abstract

The role of cryopreservation for tissue banking and transplant medicine is undisputable, being the only practical method for long-term storage of high quality biomaterials. Ideally, cells, tissues, and organs could be preserved indefinitely at cryogenic temperatures, where metabolic activity and the natural degradation mechanisms of the biomaterial are arrested. Techniques for successful cryopreservation have been developed over the past five decades for several tissue types. However, successful techniques are generally related to small specimens, in the scale-range of cell clusters to small-organized tissues (μm to mm range), with stem cells and corneas as examples. Cryopreservation of large specimens (cm and above) has been accomplished only in cases where the mechanical functionality has a higher priority than the recovery of biological functionality, with heart valves as an example. Yet, the science and technology of cryopreservation have dramatically advanced in recent years, and cryopreservation of large tissues and organs is closer to becoming a practical reality than ever before. In classical cryopreservation, the low-concentration cryoprotective agents (CPAs) are used to control ice formation- the cornerstone of cryoinjury. However, classical cryopreservation has proven ineffective in preventing damage to complex mammalian tissues, where vitrification is investigated as an alternative cryopreservation application (vitreous means glassy in Latin). Vitrification is achieved by rapidly cooling high-concentration CPAs, resulting in a sample trapped in glassy domain. The high concentrations and rapid cooling rates applied have created additional obstacles, such as toxicity and thermal stresses, with potentially detrimental outcome. In this context cryopreservation research is focused on finding a balance between these three competing effects. The subject matter of this study is the development of new prototype devices to provide real-time visualization and documentation of cryopreservation of large samples in situ; these devices belong to a unique classification termed cryomacroscopy. In the absence of cryomacroscopy techniques, common practices for macro-scale visualization are based on end-state observations, at room and storage temperatures. Due to the path dependent nature of vitrification, this practice may miss many physical phenomena associated with cryopreservation that could be captured with cryomacroscopy such as: (i) heterogeneous ice nucleation frequently associated with low-concentration CPAs and slow cooling rates; (ii) homogeneous ice nucleation frequently associated with high-concentration CPAs and rapid cooling; (iii) deformation of CPAs due to thermal contraction; (iv) undesired solute precipitation effects; and (v) fracture formation due to thermo-mechanical stress. The implementation of polarized light is introduced in this study, serving as a contrast enhancement technique for the improved visualization of contaminants and ice crystals in the CPA domain. Furthermore, polarized light is used for the first time to create photoelasticity effects in the study of thermo-mechanical stresses during vitrification. Photoelasticity techniques are presented in this study to investigate the conditions leading to fracturing, as well as the formation of stress concentrations. Photoelasticity is also demonstrated in the study of annealing-where stress relaxation is facilitated by an intermediate temperature-hold above glass transition. A thermal model is presented in this study to correlate observed phenomena with the thermal history within the specimen. Related investigations include: (i) crystallization within the CPA; (ii) removal of dissolved gases by pre-freezing in an effort to eliminate potential ice nucleation sites; (iii) incorporation of the enthalpy method to track the development of a freezing front; (iv) identifying conditions of solute precipitation from the vehicle solutions used to mitigate osmotic effects during cell permeation; (v) cooling rate effects on the developing stresses during vitrification; (vi) identifying the optimal temperatures to enable stress relaxation; and (vii) evaluation of thermal gradients in the domain during annealing. Lastly, cryomacroscopy observations are demonstrated on a special class of chemicals compound, recently termed synthetic ice modulators (SIMs), which represent the cutting edge in the field of cryobiology. SIMs are may be added to the CPA cocktail to inhibit ice growth. SIMs may permit vitrification in the presence of lower concentration CPAs, which may reduce toxicity effects. In conclusion, the cryomacroscope prototypes presented in this study may be developed as tools: (1) to facilitate the development of new materials and protocols; (2) to aid in the quality control of mass production of cryopreservation products; and (3) to facilitate advanced cryopreservation research.

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