David P. Eisenberg

Thermal expansion of the cryoprotectant cocktail DP6 combined with synthetic ice modulators in presence and absence of biological tissues

This study explores physical effects associated with the application of cryopreservation via vitrification using a class of compounds which are defined here as synthetic ice modulators (SIMs). 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. A sub-category of SIMs, referred to in the literature as synthetic ice blockers (SIBs), are compounds that interact directly with ice nuclei or crystals to modify their structure and/or rate of growth. The current study is part of an ongoing effort to characterize thermo-mechanical effects during vitrification, with emphasis on measuring the physical property of thermal expansion-the driving mechanism to thermo-mechanical stress. Materials under investigation are the cryoprotective agent (CPA) cocktail DP6 in combination with one of the following SIMs: 12% polyethylene glycol 400, 6% 1,3 cyclohexanediol, and 6% 2,3 butanediol. Results are presented for the CPA-SIM cocktail in the absence and presence of bovine muscle and goat artery specimens. This study focuses on the upper part of the cryogenic temperature range, where the CPA behaves as a fluid for all practical applications. Results of this study indicate that the addition of SIMs to DP6 allows lower cooling rates to ensure vitrification and extends the range of measurements. It is demonstrated that the combination of SIM with DP6 increases the thermal expansion of the cocktail, with implications for the likelihood of fracture formation-the most dramatic outcome of thermo-mechanical stress.

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.

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 specimens 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.

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.

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.

Stress–Strain Measurements in Vitrified Arteries Permeated With Synthetic Ice Modulators

This study measures the Youngs 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. Youngs modulus was measured to be 0.92-3.01 GPa, with no clear indication of SIM dependency on the Youngs modulus. Instead, the range of values is attributed to variations between specimens of the same species.

The Effect of Synthetic Ice Blockers on Thermal Expansion of the Cryoprotective Cocktail DP6

Cryopreservation is the only alternative for long-term preservation of high-quality biomaterials, where the availability of reliable techniques for preservation of multicellular structures and organs represents an unmet medical need. Developing cryopreservation techniques revolves around controlling the formation of ice crystals, which is known to be lethal to living cells. Cryopreservation is typically achieved in the presence of cryoprotective agents (CPAs), which exhibit a dramatic increase in viscosity with decreasing temperature. Subject to high cooling rates, the rapidly elevating viscosity of the CPA suppresses ice crystallization and promotes vitrification (vitreous means glassy in Latin). Unfortunately, available CPAs are known to be toxic at the relevant concentrations which permit vitrification. One potential method of reducing CPA concentration, and thereby achieving conditions more favorable to the tissue, is with the introduction of the so-called synthetic ice blockers (SIBs)—the subject matter of the current study.© 2011 ASME

Thermal expansion of cryoprotective agents and synthetic ice blockers

It has been recently reported that adding various synthetic ice blockers (SIBs) to the cryoprotective agent (CPA) cocktail DP6 decreased the onset of crystallization, which is a favorable condition for cryopreservation - the preservation of tissues at very low temperatures. DP6 is a CPA cocktail which has drawn significant attention in the cryobiology community in recent years. The current study focuses on thermal expansion measurements of tissue specimens permeated with the same SIB-CPA mixtures. Results of this study indicate lower thermal expansion in an SIB-DP6-tissue system when compared with the pure SIB-DP6 mixture, which may increase thermo-mechanical stress under some conditions. It is concluded that the inclusion of SIBs have the potential of improving the outcome of large-scale cryopreservation due to decreased tendency to crystallization.