Is isochoric vitrification feasible?

This study investigates the feasibility of ice-free isochoric vitrification for cryopreservation applications using mathematical modeling, computation tools, and the underlying principles of thermo-mechanics. This study is triggered by an increasing interest in the possibility of isochoric vitrification, following promising experimental results of isochoric cryopreservation. In general, isochoric cryopreservation is the preservation of biological materials in subzero temperatures in a rigid-sealed container, where some ice crystallization creates favorable pressure elevation due to the anomaly of water expansion upon ice Ih formation. Vitrification on the other hand is the transformation of liquid into an amorphous solid in the absence of any crystals, which is typically achieved by rapid cooling of a highly viscous solution. The current study presents a mathematical model for vitrification under variable pressure conditions, building upon a recently published thermo-mechanics modeling approach for isochoric cryopreservation. Using the physical properties of dimethyl sulfoxide (DMSO) as a representative cryoprotective agent (CPA), this study suggests that vitrification under isochoric conditions is not feasible, essentially since the CPA solution contracts more than the isochoric chamber by an order of magnitude. This differential contraction can lead to absolute zero pressure in the isochoric chamber, counteracting the premise of the isochoric cryopreservation process. It is concluded that the only alternative to prevent ice formation while benefiting from the potential advantages of higher pressures is to create the required pressures by external means, and not merely by passively enclosing the specimen in an isochoric chamber.

Thermal Analyses of Nanowarming-Assisted Recovery of the Heart From Cryopreservation by Vitrification.

This study explores thermal design aspects of nanowarming-assisted recovery of the heart from indefinite cryogenic storage, where nanowarming is the volumetric heating effect of ferromagnetic nanoparticles excited by a radio frequency electromagnet field. This study uses computational means while focusing on the human heart and the rat heart models. The underlying nanoparticle loading characteristics are adapted from a recent, proof-of-concept experimental study. While uniformly distributed nanoparticles can lead to uniform rewarming, and thereby minimize adverse effects associated with ice crystallization and thermomechanical stress, the combined effects of heart anatomy and nanoparticle loading limitations present practical challenges which this study comes to address. Results of this study demonstrate that under such combined effects, nonuniform nanoparticles warming may lead to a subcritical rewarming rate in some parts of the domain, excessive heating in others, and increased exposure potential to cryoprotective agents (CPAs) toxicity. Nonetheless, the results of this study also demonstrate that computerized planning of the cryopreservation protocol and container design can help mitigate the associated adverse effects, with examples relating to adjusting the CPA and/or nanoparticle concentration, and selecting heart container geometry, and size. In conclusion, nanowarming may provide superior conditions for organ recovery from cryogenic storage under carefully selected conditions, which comes with an elevated complexity of protocol planning and optimization.

Mathematical modeling of surface deformation during vitrification.

Mathematical modeling of surface deformation during cryopreservation by vitrification is presented in this study. The specific problem under consideration is of a cryoprotective agent (CPA) solution vitrifying in a vial, following previously obtained cryomacroscopy observations. A multiphysics solution is proposed in this study, combining coupled effects associated with heat transfer, fluid mechanics, and solid mechanics. Consistent with previous investigations, this study demonstrates that surface deformation is the result of material flow, which is the combined outcome of temperature gradients developed during the inward cooling process, the tendency of the material to change its volume with temperature, and the exponential increase in material viscosity with the decreasing temperature. During this process, the behavior of the CPA changes from liquid to a solid-like amorphous material, where the arrested flow in the vitrified state results in mechanical stresses. Results of this study show a good qualitative agreement of surface deformation with previously obtained experimental data, and support prior investigations to explain fracture tendencies propagating from the deformed surface. Results of this study also highlight the effect of heat convection in the CPA at the early stage of cooling.

Analysis of crystallization during rewarming in suboptimal vitrification conditions: a semi-empirical approach.

Circumventing ice formation is critical to successful cryopreservation by vitrification of large organs. While ice formation during the cooling part of the cryogenic protocol is dictated by the evolving thermal conditions, ice formation during the rewarming part of the cryogenic protocol is also dependent on the history of cooling and storage conditions. Furthermore, while the exothermic effect of ice crystallization during cooling tends to adversely slow down the desired high cooling rates to ensure ice-free preservation, the same effect under some conditions tends to assist acceleration of rewarming during recovery of the specimen from cryogenic storage when limited crystallization does occur. The current study proposes a computational framework to study the thermal effects of crystallization during recovery from cryogenic storage, using a semi-empirical approach to account for the relationship between latent heat effects and the rewarming rate. This study adds another layer of computational capabilities to a recent study investigating similar effects during cooling. Results of this study demonstrate that the thermal effects of crystallization on the local cooling and rewarming rates cannot be neglected. It further explains how crystallization during rewarming helps in increasing the rewarming rate and, thereby, affects rewarming-phase crystallization. Counterintuitively, this study suggests that the fastest possible rewarming rate at the outer surface of the domain in an inwards rewarming problem is not always advantageous, while the proposed computational tool is essential to find an intermediate optimal rate.

Thermomechanical stress analysis of rabbit kidney and human kidney during cryopreservation by vitrification with the application of radiofrequency heating.

This study presents a computational framework for thermomechanical stress analysis in a specimen undergoing cryopreservation, with emphasis on radiofrequency (RF) heating for recovering from cryogenic storage. In particular, this study addresses cryopreservation by vitrification, where the specimen is stored in the amorphous phase (vitreous means glassy). In broad terms, the relatively high cooling and rewarming rates necessary for vitrification result in differential thermal expansion in the specimen, which is the driving force for thermomechanical stress. Thermomechanical stress can lead to structural damage, such as fractures or plastic deformation, rendering the specimen useless. Not without technical difficulties, those hazardous effects during the rewarming phase of the protocol can be mitigated by applying volumetric heating, with RF heating as an attractive means. The proposed computational framework in this study addresses the coupled electromagnetic, thermal and solid mechanics fields, using commercially available solvers. This study advances from a spherical-case benchmark to realistic models of the rabbit kidney and the human kidney. Results of this study suggest that structural damage to the brittle material can be prevented when stress relaxation is facilitated around the glass transition temperature. Furthermore, this study suggests that volumetric heating is necessary to surpass the critical rewarming rate, while benefiting from lowering the overall thermomechanical stress during recovery from cryogenic storage. More broadly, the computational framework presented here can be used for the optimization of the RF heating parameters, chamber specifics, specimen container shape, and the thermal protocol in order to preserve structural integrity in the specimen.

Scaling Effects on the Residual Thermomechanical Stress During Ice-Free Cooling to Storage Temperature.

Cryopreservation via vitrification (glass formation) is a promising approach for long-term preservation of large-size tissues and organs. Unfortunately, thermomechanical stress, which is driven by the tendency of materials to change size with temperature, may lead to structural failure. This study focuses on analysis of thermomechanical stress in a realistic, pillow-like shape cryobag as it is cooled to cryogenic storage, subject to sufficiently high cooling rates to facilitate vitrification. Contrary to common perception, it is demonstrated in this study that the maximum stress in the specimen does not necessarily increase with increasing size of the specimen. In fact, the maximum stress is affected by the combination of two competing effects, associated with the extent of the temperature gradients within the specimen and its overall volume. On one hand, the increase in specimen size gives rise to more prominent temperature gradients, which can intensify the thermomechanical stress. On the other hand, the temperature distribution at the core of larger specimens is more uniform, which leads to a larger portion of the specimen transitioning from fluid to a glassy material almost instantaneously, which carries a moderating effect on the overall mechanical stress at the glassy state (i.e., lower residual stress). In conclusion, this study demonstrates the role of container shape optimization in reducing the thermomechanical stress during cooling.

Thermal analysis of marginal conditions to facilitate cryopreservation by vitrification using a semi-empirical approach.

This study aims at the thermal analysis of marginal conditions leading to cryopreservation by vitrification, which appears to be the only alternative for indefinite preservation of large-size tissues and organs. The term "marginal conditions" here refers to cooling rates in close range with the so-called critical cooling rate, above which crystallization is avoided. The analysis of thermal effects associated with partial crystallization during vitrification is associated with the coupled phenomena of heat transfer and kinetics of crystallization. This study takes a practical, semi-empirical approach, where heat transfer is analyzed based on its underlying theoretical principles, while the thermal effects associated with partial crystallization are taken into account by means of empirical correlations. This study presents a computation framework to solve the coupled problem, while presenting a proof-of-concept for DP6 as a representative cryoprotective agent. The thermal effects associated with crystallization at various relevant cooling rates are measured in this study by means of differential scanning calorimetry. Results of this study demonstrate that, due to the thermal effects associated with partial crystallization, the cooling rate at the center of a large organ may lag behind the cooling rate in its surroundings under some scenarios, but may also exceed the surroundings cooling rate in other scenarios, leading to counter-intuitive effects associated with partial crystallization.

Vitrification tendency and stability of DP6-based vitrification solutions for complex tissue cryopreservation.

Vitrification tendency and stability of the amorphous state were analyzed by means of differential scanning calorimetry (DSC) for the vitrification solution DP6, with and without additional solutes to enhance ice suppression. This study is a part of an ongoing research effort to characterize the thermophysical and mechanical properties of DP6 and its derivatives, and their qualities as cryoprotective solutions. DP6 was determined to have a critical cooling rate necessary to ensure vitrification of 2.7 °C/min. The following additional solutions were tested: DP6 + 6% (2R, 3R) 2,3-butanediol, DP6 + 6% 1,3-cyclohexanediol, DP6 + 6% (0.175M) sucrose, DP6 + 12% PEG 400, and DP6 + 17.1% (0.5 M) sucrose. The additives decreased the critical cooling rate of the DP6 solution to rates below 1 °C/min that were not quantifiable by the DSC techniques used. The following critical warming rates necessary to avoid devitrification were identified for DP6 and the modified solutions, respectively: 189 °C/min, 5 °C/min, ≈ 1 °C/min, 15 °C/min, <1 °C/min, and <1 °C/min. Glass transition temperatures and melting temperatures were also measured. Sucrose was the least effective additive on a per mass basis, with 1,3-cyclohexanediol appearing to be the most effective additive for suppressing ice formation in DP6.

Thermal Analyses of a Human Kidney and a Rabbit Kidney During Cryopreservation by Vitrification.

This study focuses on thermal analysis of the problem of scaling up from the vitrification of rabbit kidneys to the vitrification of human kidneys, where vitrification is the preservation of biological material in the glassy state. The basis for this study is a successful cryopreservation protocol for a rabbit kidney model, based on using a proprietary vitrification solution known as M22. Using the finite element analysis (FEA) commercial code ANSYS, heat transfer simulations suggest that indeed the rabbit kidney unquestionably cools rapidly enough to be vitrified based on known intrarenal concentrations of M22. Scaling up 21-fold, computer simulations suggest less favorable conditions for human kidney vitrification. In this case, cooling rates below -100 °C are sometimes slower than 1 °C/min, a rate that provides a clear-cut margin of safety at all temperatures based on the stability of rabbit kidneys in past studies. Nevertheless, it is concluded in this study that vitrifying human kidneys is possible without significant ice damage, assuming that human kidneys can be perfused with M22 as effectively as rabbit kidneys. The thermal analysis suggests that cooling rates can be further increased by a careful design of the cryogenic protocol and by tailoring the container to the shape of the kidney, in contrast to the present cylindrical container. This study demonstrates the critical need for the thermal analysis of experimental cryopreservation and highlights the unmet need for measuring the thermophysical properties of cryoprotective solutions under conditions relevant to realistic thermal histories.

Thermo-mechanical stress analysis of cryopreservation in cryobags and the potential benefit of nanowarming.

Cryopreservation by vitrification is the only promising solution for long-term organ preservation which can save tens of thousands of lives across the world every year. One of the challenges in cryopreservation of large-size tissues and organs is to prevent fracture formation due to the tendency of the material to contract with temperature. The current study focuses on a pillow-like shape of a cryobag, while exploring various strategies to reduce thermo-mechanical stress during the rewarming phase of the cryopreservation protocol, where maximum stresses are typically found. It is demonstrated in this study that while the level of stress may generally increase with the increasing amount of CPA filled in the cryobag, the ratio between width and length of the cryobag play a significant role. Counterintuitively, the overall maximum stress is not found when the bag is filled to its maximum capacity (when the filled cryobag resembles a sphere). Parametric investigation suggests that reducing the initial rewarming rate between the storage temperature and the glass transition temperature may dramatically decrease the thermo-mechanical stress. Adding a temperature hold during rewarming at the glass transition temperature may reduce the thermo-mechanical stress in some cases, but may have an adverse effect in other cases. Finally, it is demonstrated that careful incorporation of volumetric heating by means on nanoparticles in an alternating magnetic field, or nanowarming, can dramatically reduce the resulting thermo-mechanical stress. These observations display the potential benefit of a thermo-mechanical design of the cryopreservation protocols in order to prevent structural damage.

A new method for temperature-field reconstruction during ultrasound-monitored cryosurgery using potential-field analogy.

The current study aims at developing computational tools in order to gain information about the thermal history in areas invisible to ultrasound imaging during cryosurgery. This invisibility results from the high absorption rate of the ultrasound energy by the frozen region, which leads to an apparent opacity in the cryotreated area and a shadow behind it. A proof-of-concept for freezing-front estimation is demonstrated in the current study, using the new potential-field analogy method (PFAM). This method is further integrated with a recently developed temperature-field reconstruction method (TFRM) to estimate the temperature distribution within the frozen region. This study uses prostate cryosurgery as a developmental model and trans-rectal ultrasound imaging as a choice of practice. Results of this study indicate that the proposed PFAM is a viable and computationally inexpensive solution to estimate the extent of freezing in the acoustic shadow region. Comparison of PFAM estimations and experimental data shows an average mismatch of less than 2 mm in freezing-front location, which is comparable to the uncertainty in ultrasound imaging. Comparison of the integrated PFAM + TFRM scheme with a full-scale finite-elements analysis (FEA) indicates an average mismatch of 0.9 mm for the freezing front location and 0.1 mm for the lethal temperature isotherm of -45 °C. Comparison of the integrated PFAM + TFRM scheme with experimental temperature measurements show a difference in the range of 2 °C and 6 °C for selected points of measurement. Results of this study demonstrate the integrated PFAM + TFRM scheme as a viable and computationally inexpensive means to gain information about the thermal history in the frozen region during ultrasound-monitored cryosurgery.

Polarized light scanning cryomacroscopy, part I: Experimental apparatus and observations of vitrification, crystallization, and photoelasticity effects.

Cryomacroscopy is an effective means to observe physical events affecting cryopreservation success in large-size specimens. The current study aims at integrating polarized-light in the study of large-size cryopreservation, using the scanning cryomacroscope as a development platform. Results of this study demonstrate polarized light as a visualization enhancement means, including the following effects: contaminants in the CPA solution, crystallization, fracture formation, thermal contraction, and solute precipitation. In addition, photoelasticity effects are used to demonstrate the development of residual stresses and the potential for stress relaxation above the glass transition temperature. Furthermore, this study suggests that the ability to periodically switch between non-polarized light and polarized light is an essential feature of investigation. When using polarized light for example, a dark region may represent a free-of-stress and free-of-crystals material, or fully crystallized material, which may potentially experience mechanical stress; switching to a non-polarized light would help to distinguish between the different cases. The analysis of thermo-mechanical stress in cryopreservation is essentially based on four key elements: identification of physical events, knowledge of physical properties, thermal analysis of the specimen, and description of the mechanical behavior of the cryopreserved material (also known as the constitutive law). With the above knowledge, one can investigate the conditions to preserve structural integrity. While the current study aims at identification of physical events, critical knowledge on physical properties and mechanical behavior has already been developed in previous studies. The companion manuscript (Part II) aims at providing means for thermal analysis in the specimen, which will serve as the basis for a multi-scale analysis of thermo-mechanical stress in large-size specimens.

Thermal conductivity of the cryoprotective cocktail DP6 in cryogenic temperatures, in the presence and absence of synthetic ice modulators.

The thermal conductivity of the cryoprotective agent (CPA) cocktail DP6 in combination with synthetic ice modulators (SIMs) is measured in this study, using a transient hot-wire method. DP6 is a mixture of 3 M dimethyl sulfoxide (DMSO) and 3 M propylene glycol, which received significant attention in the cryobiology community in recent years. Tested SIMs include 6% 1,3Cyclohexanediol, 6% 2,3Butanediol, and 12% PEG400 (percentage by volume). This study integrates the scanning cryomacroscope for visual verification of crystallization and vitrification events. It is demonstrated that the thermal conductivity of the vitrifying CPA cocktail decreases monotonically with the decreasing temperature down to -180 °C. By contrast, the thermal conductivity of the crystalline material increases with decreasing temperature in the same temperature range. Results of this study demonstrate that the thermal conductivity may vary by three fold between the amorphous and crystalline phases of DP6 below the glass transition temperature of DP6 (Tg = -119 °C). The selected SIMs demonstrate the ability to inhibit crystallization in DP6, even at subcritical cooling rates. An additional ice suppression capability is observed by the Euro-Collins as a vehicle solution, disproportionate to its volume ratio in the cocktail. The implication of the observed thermal conductivity differences between the amorphous and crystalline phases of the same cocktail on cryopreservation simulations is significant in some cases and must be taken into account in thermal analyses of cryopreservation protocols.

Polarized light scanning cryomacroscopy, part II: Thermal modeling and analysis of experimental observations.

This study aims at developing thermal analysis tools and explaining experimental observations made by means of polarized-light cryomacroscopy (Part I). Thermal modeling is based on finite elements analysis (FEA), where two model parameters are extracted from thermal measurements: (i) the overall heat transfer coefficient between the cuvette and the cooling chamber, and (ii) the effective thermal conductivity within the cryoprotective agent (CPA) at the upper part of the cryogenic temperature range. The effective thermal conductivity takes into account enhanced heat transfer due to convection currents within the CPA, creating the so-called Bénard cells. Comparison of experimental results with simulation data indicates that the uncertainty in simulations due to the propagation of uncertainty in measured physical properties exceeds the uncertainty in experimental measurements, which validates the modeling approach. It is shown in this study that while a cavity may form in the upper-center portion of the vitrified CPA, it has very little effect on estimating the temperature distribution within the domain. This cavity is driven by thermal contraction of the CPA, with the upper-center of the domain transitioning to glass last. Finally, it is demonstrated in this study that additional stresses may develop within the glass transition temperature range due to nonlinear behavior of the thermal expansion coefficient. This effect is reported here for the first time in the context of cryobiology, using the capabilities of polarized-light cryomacroscopy.

The Grand Challenges of Organ Banking: Proceedings from the first global summit on complex tissue cryopreservation.

The first Organ Banking Summit was convened from Feb. 27 - March 1, 2015 in Palo Alto, CA, with events at Stanford University, NASA Research Park, and Lawrence Berkeley National Labs. Experts at the summit outlined the potential public health impact of organ banking, discussed the major remaining scientific challenges that need to be overcome in order to bank organs, and identified key opportunities to accelerate progress toward this goal. Many areas of public health could be revolutionized by the banking of organs and other complex tissues, including transplantation, oncofertility, tissue engineering, trauma medicine and emergency preparedness, basic biomedical research and drug discovery - and even space travel. Key remaining scientific sub-challenges were discussed including ice nucleation and growth, cryoprotectant and osmotic toxicities, chilling injury, thermo-mechanical stress, the need for rapid and uniform rewarming, and ischemia/reperfusion injury. A variety of opportunities to overcome these challenge areas were discussed, i.e. preconditioning for enhanced stress tolerance, nanoparticle rewarming, cyroprotectant screening strategies, and the use of cryoprotectant cocktails including ice binding agents.

Thermomechanical Stress in Cryopreservation Via Vitrification With Nanoparticle Heating as a Stress-Moderating Effect.

This study focuses on thermomechanical effects in cryopreservation associated with a novel approach of volumetric heating by means on nanoparticles in an alternating electromagnetic field. This approach is studied for the application of cryopreservation by vitrification, where the crystalline phase is completely avoided-the cornerstone of cryoinjury. Vitrification can be achieved by quickly cooling the material to cryogenic storage, where ice cannot form. Vitrification can be maintained at the end of the cryogenic protocol by quickly rewarming the material back to room temperature. The magnitude of the rewarming rates necessary to maintain vitrification is much higher than the magnitude of the cooling rates that are required to achieve it in the first place. The most common approach to achieve the required cooling and rewarming rates is by exposing the specimen's surface to a temperature-controlled environment. Due to the underlying principles of heat transfer, there is a size limit in the case of surface heating beyond which crystallization cannot be prevented at the center of the specimen. Furthermore, due to the underlying principles of solid mechanics, there is a size limit beyond which thermal expansion in the specimen can lead to structural damage and fractures. Volumetric heating during the rewarming phase of the cryogenic protocol can alleviate these size limitations. This study suggests that volumetric heating can reduce thermomechanical stress, when combined with an appropriate design of the thermal protocol. Without such design, this study suggests that the level of stress may still lead to structural damage even when volumetric heating is applied. This study proposes strategies to harness nanoparticles heating in order to reduce thermomechanical stress in cryopreservation by vitrification.

Stress-Strain Measurements in Vitrified Arteries Permeated With Synthetic Ice Modulators.

This study measures the Young's modulus in vitrified blood vessels below the glass transition temperature in conditions relevant to cryogenic storage and focuses on the cryoprotective agents (CPAs) cocktail DP6 mixed with synthetic ice modulators (SIMs). Small steplike strain changes were observed during the loading without affecting the bulk behavior, suggesting microfracture occurrences resembling previous observation on microfracture formation under compression in crystallized blood vessels. Young's modulus was measured to be 0.92-3.01 GPa, with no clear indication of SIM dependency on the Young's modulus. Instead, the range of values is attributed to variations between specimens of the same species.

Thermal expansion of vitrified blood vessels permeated with DP6 and synthetic ice modulators.

This study provides thermal expansion data for blood vessels permeated with the cryoprotective cocktail DP6, when combined with selected synthetic ice modulators (SIMs): 12% polyethylene glycol 400, 6% 1,3-cyclohexanediol, and 6% 2,3-butanediol. The general classification of SIMs includes molecules that modulate ice nucleation and growth, or possess properties of stabilizing the amorphous state, by virtue of their chemical structure and at concentrations that are not explained on a purely colligative basis. The current study is part of an ongoing effort to characterize thermo-mechanical effects on structural integrity of cryopreserved materials, where thermal expansion is the driving mechanism to thermo-mechanical stress. This study focuses on the lower part of the cryogenic temperature range, where the cryoprotective agent (CPA) behaves as a solid for all practical applications. By combining results obtained in the current study with literature data on the thermal expansion in the upper part of the cryogenic temperature range, unified thermal expansion curves are presented.

The Scanning Cryomacroscope - A Device Prototype for the Study of Cryopreservation.

A new cryomacroscope prototype-a visualization device for the in situ analysis of cryopreserved biological samples-is presented in the current study. In order to visualize samples larger than the field of view of the optical setup, a scanning mechanism is integrated into the system, which represents a key improvement over previous cryomacroscope prototypes. Another key feature of the new design is in its compatibility with available top-loading controlled-rate cooling chambers, which eliminates the need for a dedicated cooling mechanism. The objective for the current development is to create means to generate a single digital movie of an experimental investigation, with all relevant data overlaid. The visualization capabilities of the scanning cryomacroscope are demonstrated in the current study on the cryoprotective agent dimethyl sulfoxide and the cryoprotective cocktail DP6. Demonstrated effects include glass formation, various regimes of crystallization, thermal contraction, and fracture formation.

On the Effects of Thermal History on the Development and Relaxation of Thermo-Mechanical Stress in Cryopreservation.

This study investigates the effects of the thermal protocol on the development and relaxation of thermo-mechanical stress in cryopreservation by means of glass formation, also known as vitrification. The cryopreserved medium is modeled as a homogeneous viscoelastic domain, constrained within either a stiff cylindrical container or a highly compliant bag. Annealing effects during the cooling phase of the cryopreservation protocol are analyzed. Results demonstrate that an intermediate temperature-hold period can significantly reduce the maximum tensile stress, thereby decreasing the potential for structural damage. It is also demonstrated that annealing at temperatures close to glass transition significantly weakens the dependency of thermo-mechanical stress on the cooling rate. Furthermore, a slower initial rewarming rate after cryogenic storage may drastically reduce the maximum tensile stress in the material, which supports previous experimental observations on the likelihood of fracture at this stage. This study discusses the dependency of the various stress components on the storage temperature. Finally, it is demonstrated that the stiffness of the container wall can affect the location of maximum stress, with implications on the development of cryopreservation protocols.