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Cryopreservation represents one if not the only long-term option for tissue and perhaps future organ banking. In one particular approach, cryopreservation is achieved by completely avoiding ice formation (or crystallization) through a process called vitrification. This "ice-free" approach to tissue banking requires a combination of high-concentration cryoprotective additives such as M22 (9.4 M), VS55 (8.4 M), or DP6 (6 M) and sufficiently fast rates of cooling and warming to avoid crystallization. In this article, we report the temperature-dependent specific heat capacity of the above-mentioned cryoprotective additives in small volumes (10 mg sample pans) at rates of 5°C/min and 10°C/min using a commercially available differential scanning calorimetry (TA Instruments Q1000), in the temperature range of -150°C to 30°C. This data can be utilized in heat-transfer models to predict thermal histories in a cryopreservation protocol. More specifically, the effects of temperature dependence of specific heat due to the presence of three different phases (liquid, ice, and vitreous phase) can dramatically impact the thermal history and therefore the outcome of the cryopreservation procedure. The crystallization potential of these cryoprotectants was also investigated by studying cases of maximal and minimal crystallization in VS55 and DP6, where M22 did not crystallize under any rates tested. To further reduce crystallization in VS55 and DP6, a stabilizing sugar (sucrose) was added in varying concentrations (0.15 M and 0.6 M) and was shown to further reduce crystallization, particularly in VS55, at modest rates of cooling (1°C/min, 5°C/min, and 10°C/min).

The use of thermal based therapies for treatment of atrial fibrillation is increasing. While numerous reports are available in the literature regarding the efficacy of cryotherapy on pulmonary vein survival, there are no reports specifically at the cellular level that establish thermal thresholds and mechanisms of cellular destruction. The current article reports on the response of HL-1 cardiomyocytes to cooling rates and end temperatures during cryothermal exposure. The focus is on establishment of in vitro thresholds while also establishing mechanisms of action due to biophysical events (i.e. intracellular ice formation and water transport).

The current study presents a new and novel analysis of heat release signatures measured by a differential scanning calorimeter (DSC) associated with water transport (WT), intracellular ice formation (IIF) and extracellular ice formation (EIF). Correlative cryomicroscopy experiments were also performed to validate the DSC data. The DSC and cryomicroscopy experiments were performed on human dermal fibroblast cells (HDFs) at various cytocrit values (0-0.8) at various cooling rates (0.5-250 °C/min). A comparison of the cryomicroscopy experiments with the DSC analysis show reasonable agreement in the water transport (cellular dehydration) and IIF characteristics between both the techniques with the caveat that IIF measured by DSC lagged that measured by cryomicroscopy. This was ascribed to differences in the techniques (i.e. cell vs. bulk measurement) and the possibility that not all IIF is associated with visual darkening. High and low rates of 0.5 °C/min and 250 °C/min were chosen as HDFs did not exhibit significant IIF or WT at each of these extremes respectively. Analysis of post-thaw viability data suggested that 10 °C/min was the presumptive optimal cooling rate for HDFs and was independent of the cytocrit value. The ratio of measured heat values associated with IIF (q(IIF)) to the total heat released from both IIF and water transport or from the total cell water content in the sample (q(CW)) was also found to increase as the cooling rate was increased from 10 to 250 °C/min and was independent of the sample cytocrit value. Taken together, these observations suggest that the proposed analysis is capable of deconvolving water transport and IIF data from the measured DSC latent heat thermograms in cell suspensions during freezing.

While studies on the freezing of cells in suspension have been carried out extensively, corresponding studies with cells in the attached state and in tissue or tissue-equivalents are less developed. As attachment is a hallmark of the tissue state it is important to understand its impact on biophysics and viability to better apply freezing towards tissue preservation. The current study reports on observed biophysical response changes observed during freezing human dermal fibroblasts in suspension, attached cell, and fibrin tissue-equivalent models. Specifically, intracellular ice formation is shown to increase and dehydration is inferred to increase from suspension to attached systems. Biophysical model parameters fit to these experimental observations reflect the higher kinetics in the attached state. Post-thaw viability values from fast cooling rates were higher for suspension systems, and correlated well with the amount of IIF observed. On the other hand, viability values from slow cooling rates were higher for attached systems, although the degree of dehydration was predicted to be comparable to suspension cells. This disconnect between biophysics and viability predictions at slow rates clearly requires further investigation as it runs counter to our current understanding of dehydration injury in cells. This may suggest a possible protective effect of the attachment state on cell systems.

It is well accepted in cryobiology that the temperature history and cooling rates experienced in biomaterials during freezing procedures correlate strongly with biological outcome. Therefore, heat transfer measurement and prediction in the cryogenic regime is central to the field. Although direct measurement of temperature history (i.e. heat transfer) can be performed, accuracy is usually achieved only for local measurements within a given system and cannot be readily generalized to another system without the aid of predictive models. The accuracy of these models rely upon thermal properties which are known to be highly dependent on temperature, and in the case of significant cryoprotectant loading, also on crystallized fraction. In this work, we review the available thermal properties of biomaterials in the cryogenic regime. The review shows a lack of properties for many biomaterials in the subzero temperature domain, and especially for systems with cryoprotective agents. Unfortunately, use of values from the limited data available (usually only down to -40 degrees C) lead to an underestimation of thermal property change (i.e. conductivity rise and specific heat drop due to ice crystallization) with lower temperatures. Conversely, use of surrogate values based solely on ice thermal properties lead to an overestimation of thermal property change for most biomaterials. Additionally, recent work extending the range of available thermal properties to -150 degrees C has shown that the thermal conductivity will drop in both PBS and tissue (liver) due to amorphous/glassy phases (versus crystalline) of biomaterials with the addition of cryoprotective additives such as glycerol. Thus, we investigated the implications of using approximated or constant property values versus measured temperature-dependent values for predicting temperature history during freezing in PBS (phosphate-buffered saline) and porcine liver with and without cryoprotectants (glycerol). Using measured property values (thermal conductivity, specific heat, and latent heat of phase change) of porcine liver, a standard was created which showed that values based on surrogate ice properties under-predicted cooling times, while constant properties (i.e. based on limited data reported near the freezing point) over-predicted cooling times. Additionally, a new iterative numerical method that accommodates non-equilibrium cooling effects as a function of time and position (i.e. crystallization versus amorphous phase) was used to predict temperature history during freezing in glycerol loaded systems. Results indicate that in addition to the increase in cooling times due to the lowering of thermal diffusivity with more glycerol, non-equilibrium effects such as the prevention of maximal crystallization (i.e. amorphous phases) will further increase required cooling times. It was also found that the amplified effect of non-equilibrium cooling and crystallization with system size prevents the thermal history to be described with non-dimensional lengths, such as was possible under equilibrium cooling. These results affirm the need to use accurate thermal properties that incorporate temperature dependence and crystallized fraction. Further studies are needed to extract thermal properties of other important biomaterials in the subzero temperature domain and to develop accurate numerical methods which take into account non-equilibrium cooling events encountered in cryobiology when partial or total vitrification occurs.

Though cryopreservation of mouse sperm yields good survival and motility after thawing, cryopreservation of rat sperm remains a challenge. This study was designed to evaluate the biophysics (membrane permeability) of rat in comparison to mouse to better understand the cooling rate response that contributes to cryopreservation success or failure in these two sperm types. In order to extract subzero membrane hydraulic permeability in the presence of ice, a differential scanning calorimeter (DSC) method was used. By analyzing rat and mouse sperm frozen at 5 degrees C/min and 20 degrees C/min, heat release signatures characteristic of each sperm type were obtained and correlated to cellular dehydration. The dehydration response was then fit to a model of cellular water transport (dehydration) by adjusting cell-specific biophysical (membrane hydraulic permeability) parameters L(pg) and E(Lp). A "combined fit" (to 5 degrees C/min and 20 degrees C/min data) for rat sperm in Biggers-Whitten-Whittingham media yielded L(pg) = 0.007 microm min(-1) atm(-1) and E(Lp) = 17.8 kcal/mol, and in egg yolk cryopreservation media yielded L(pg) = 0.005 microm min(-1) atm(-1) and E(Lp) = 14.3 kcal/mol. These parameters, especially the activation energy, were found to be lower than previously published parameters for mouse sperm. In addition, the biophysical responses in mouse and rat sperm were shown to depend on the constituents of the cryopreservation media, in particular egg yolk and glycerol. Using these parameters, optimal cooling rates for cryopreservation were predicted for each sperm based on a criteria of 5%-15% normalized cell water at -30 degrees C during freezing in cryopreservation media. These predicted rates range from 53 degrees C/min to 70 degrees C/min and from 28 degrees C/min to 36 degrees C/min in rat and mouse, respectively. These predictions were validated by comparison to experimentally determined cryopreservation outcomes, in this case based on motility. Maximum motility was obtained with freezing rates between 50 degrees C/min and 80 degrees C/min for rat and at 20 degrees C/min with a sharp drop at 50 degrees C/min for mouse. In summary, DSC experiments on mouse and rat sperm yielded a difference in membrane permeability parameters in the two sperm types that, when implemented in a biophysical model of water transport, reasonably predict different optimal cooling rate outcomes for each sperm after cryopreservation.

The latent heat during phase change of water-NaCl binary mixture was measured using a differential scanning calorimeter, and the magnitude for two distinct phase change events, water/ice and eutectic phase change, were analyzed considering the phase change characteristics of a binary mixture. During the analysis, the latent heat associated with each event was calculated by normalizing the amount of each endothermic peak with only the amount of sample participating in each event estimated from the lever rule for the phase diagram. The resulting latent heat of each phase change measured is 303.7 +/- 2.5 J/g for water/ice phase change, and 233.0 +/- 1.6 J/g for eutectic phase change, respectively regardless of the initial concentration of mixture. Although the latent heats of water/ice phase change in water-NaCl mixtures are closely correlated, further study is warranted to investigate the reason for smaller latent heat of water/ice phase change than that in pure water (335 J/g). The analysis using the lever rule was extended to estimate the latent heat of dihydrate as 115 J/g with the measured eutectic and water/ice latent heat values. This new analysis based on the lever rule will be useful to estimate the latent heat of water-NaCl mixtures at various concentrations, and may become a framework for more general analysis of latent heat of various biological solutions.

Atrial fibrillation (AF) is the irregular beating of the heart affecting an estimated 2.7 million Americans and responsible for 26 billion dollars of the nation’s annual healthcare cost. Cryoablation is a clinically approved technique for the treatment of paroxysmal AF in which an ablation catheter rapidly cools the cardiac tissue. Through freezing, discrete lesions are created in the region where the pulmonary veins join with the atria in order to terminate the conduction of abnormal electrical signals responsible for AF. While the efficacy of AF cryoablation is well known, the thermal thresholds and mechanisms of cryoinjury that determine efficacy are not known thereby hindering development of the next generation of AF cryoablation systems. This study begins to address this by improving understanding of the mechanisms and thermal thresholds of cryodestruction in a cardiac muscle cell line (HL-1). Specifically, we determine biophysical parameters governing known mechanisms of injury (membrane permeability and likelihood of intracellular ice formation, IIF). Additionally we correlate these biophysics and thermal history (cooling rate and end = temperature) to post-thaw viability in vitro in HL-1s guide optimal lesion formation and thus cryoablation device design. HL-1 cells were cultured using previously published methods. Cells were harvested using trypsinization and kept as a suspension on ice until needed. A temperature stage was used in conjunction with a light microscope to observe freezing of the samples. The controlled thermal history included 3 variables including: cooling rate (0.5 to 130 °C/min), end temperature (−5 to −60 °C), and hold time (0–5 min). These were varied in an experimental matrix to determine the importance of each. Before cooling, samples were pre-nucleated by applying a chilled needle on the outer edge of the sample. Change in water content within cells (cellular dehydration) was measured based on observed changes to projected cell area during freezing. IIF events were measured based on observing a sudden change in opacity (darkening) of the cell interior. Measured data were applied to water transport and nucleation models to determine membrane permeability and IIF nucleation parameters. Additionally, post-thaw viability of samples were determined after a rapid thaw and 15 min incubation period using a fluorescence membrane dye exclusion assay. For cases when the end temperature was set as −20 °C the highest viability of around 15% was observed at a cooling rate of 5 °C/min, while slower or faster cooling rates resulted in reduced viability. Subsequent measurements with cooling rate set as 5 °C/min and varied end temperatures resulted in viability values around 94%, 72%, 15%, 6% and less than 5% at −5 °C, −10 °C, −20 °C, −40 °C, and −60 °C, respectively. Our results suggest a large difference in post-thaw cell viability dependent on cooling rate and end temperature, while difference in hold times (0, 1, and 5 min) appeared less important. The application of this work to pulmonary vein freezing numerical models, and to future work in more translational systems such as attached (monolayer, artificial constructs) cell systems and tissues will be discussed.

While cryopreservation through vitrification holds great promise, practical application has been limited to smaller systems (cells and thin tissues) due to diffusive transfer and phase-change limitations, which are typically manifested as devitrification and cracking failures during thaw. Here we describe a new approach for rapidly and uniformly heating cryopreserved biospecimens with radiofrequency (RF) excited magnetic nanoparticles (mNPs). Importantly, heating rates can be increased several fold over conventional boundary heating and are independent of sample size and dielectric properties, overcoming the inhomogeneity associated with microwave thawing. Proof-of-principle experiments in aqueous and cryoprotectant solutions are presented and then modeling is used to further illustrate the potential of this innovative approach. Three embodiments of mNP suspensions are studied – aqueous, 6 M glycerol in phosphate buffered saline, and 8.4 M VS55 cryoprotectant solution (Mehl, Cryobiology 30 (1993) 509–518). The iron oxide mNPs used are commercially available (Ferrotec, Inc.) and have been previously shown to produce significant heating in aqueous suspensions (Etheridge and Bischof, ABME 41 (2013) 78–88). Nanoparticle concentrations up to 10 mg Fe/ml are shown to have negligible impact on the freezing behavior of these solutions, including the glass transition, critical cooling/warming rates, and specific heat, as measured by differential scanning calorimetry. One milliliter mNP suspensions at 5 and 10 mg Fe/ml in water and cryoprotectant solution were frozen in liquid nitrogen (at sufficient rates to achieve vitrification in the cryoprotectant samples) and then reheated in an insulated, 370 kHz and 20 kA/m RF coil. Sample temperatures were monitored with 40-gauge thermocouples embedded prior to cooling. RF fields can produce interference in metallic thermocouples, but this was characterized and found to be negligible for the fine gauge thermocouples used. Heating rates at the glass transition of approximately 200 ° C/min were achieved in the 10 mg Fe/ml samples, avoiding devitrification in the cryoprotectant solutions. Since RF fields at frequencies Source of funding: This work was supported by the Minnesota Futures Grant Program, NSF/CBET #1066343, and the University of Minnesota Institute for Engineering Seed Grant Program. Conflict of interest: None declared. bischof@umn.edu

There is a lack of information on the effect of cryoprotective agents (CPAs) on the thermal properties of biomaterials at cryobiologically relevant temperatures (i.e.

Background Cardiac balloon and catheter cryoablations for the treatment of atrial fibrillation have been gaining attention as approaches for treating arrhythmias, in particular for isolation of the pulmonary veins (PV). Despite widespread and growing clinical use of cardiac cryoablation, there are still questions regarding dosing and treatment time, which may affect both efficacy and collateral injury. To date injury thresholds for therapy of cardiac and/or PV tissues are largely unreported in the literature. Methods Slices of ventricular myocardium ( n = 11) and left atrial tissue ( n = 6) from female Yorkshire Cross swine hearts were dissected including the native endocardial surface. Samples were placed in an infrared imaging apparatus consisting of a plastic petri dish with central ablation probe (Galil Medical) and 2 mm of Sylgard polymer formed to the bottom. The tissue slices were impaled on the central cryoprobe and infrared thermography was captured using a Flir A20 looking from the top down. Subsequently, samples were cultured for 24 h in a cell culture incubator (myocardium) at 37 °C for optimal staining with 1% TTC in Trizma buffer for 1 h at 37 °C. Photographs of the resultant TTC staining of each sample were captured and a custom Matlab program was used to normalize staining intensity and correlate the average staining intensity with the average thermal profile at any radial distance from the cryoprobe. The relationship between the staining intensity and end temperature was fit with a sigmoidal curve. Samples with a fit of greater than r 2 > 0.99 were analyzed. The transition from dead to injured tissue was defined as the point on the curve fit where the staining ratio was 10% of the range between the lower and upper asymptotes of the sigmoid (i.e., 10% of the grayscale value change at the lesion margin closest to the lesion). The maximum cooling rate correlated to this ratio and end temperature was then calculated from the thermal profiles. Eight thermal profiles were averaged to obtain a characteristic thermal profile for each sample. This approach has led to the creation of a database of thermal profiles that results in either completely or partially necrotic tissues. Results Initial results suggest that cooling rates of −103 ± 49 and −72 ± 9 °C/min and end temperatures of approximately −21 ± 6 and −17.2 ± 3.2 °C are necessary for complete necrosis of ventricular and left atrial tissue, respectively. Using a two-tailed T -test assuming equal variance the difference in end temperature and cooling rate between ventricular and left atrial tissue were non-significant ( P = .21, P = .20 respectively). Conclusions Experiments are ongoing to determine cryothermal injury thresholds of tissues associated with cardiac ablation, such as: lung and esophagus. The TTC staining protocol is in the process of being validated by comparison to H& E stain read by a blinded pathologist. Cellular viability assays are being performed with dye exclusion assays and results are being compared with tissue assays. Source of funding: GAANN Fellowship, Institute for Engineering in Medicine at the University of Minnesota, Medtronic, Inc. Source of funding: Medtrain, Inc. Conflict of interest: Ryan Goff has an Internship with Medtronic, Inc. goffx073@umn.edu

Surgery, radiation and chemotherapy remain the mainstay of current cancer therapy. However, treatment failure persists due to the inability to achieve complete local control of the tumor and curtail metastatic spread. Vascular disrupting agents (VDAs) are a class of promising systemic agents that are known to synergistically enhance radiation, chemotherapy or thermal treatments of solid tumors. Unfortunately, there is still an unmet need for VDAs with more favorable safety profiles and fewer side effects. Recent work has demonstrated that conjugating VDAs to other molecules (PEG, NGR) or nanoparticles (liposomes, gold) can greatly reduce toxicity of one prominent VDA (tumor necrosis factor alpha, TNF- α ). In this report, we show the potential of a gold conjugated TNF- α nanoparticle (NP-TNF) to improve multimodal cancer therapies with VDAs. In a dorsal skin fold and hindlimb murine xenograft model of prostate cancer, we found that NP-TNF disrupts endothelial barrier function and induces a significant increase in vascular permeability within the first 1–2 h followed by a dramatic 80% drop in perfusion 2–6 h after systemic administration. We also demonstrate that the tumor response to the nanoparticle can be verified using dynamic contrast-enhanced magnetic resonance imaging (MRI), a technique in clinical use. Additionally, multimodal treatment with thermal therapies at the perfusion nadir in the sub- and supra- physiological temperature regimes increases tumor volumetric destruction by over 60% and leads to significant tumor growth delays compared to thermal therapy alone. Lastly, NP-TNF was found to enhance thermal therapy in the absence of neutrophil recruitment, suggesting that immune/inflammatory regulation is not central to its power as part of a multimodal approach. Our data demonstrates the potential of nanoparticle-conjugated VDAs to significantly improve cancer therapy by preconditioning tumor vasculature to a secondary insult in a targeted manner with limited systemic toxicity or inflammatory response. We anticipate our work to direct investigations into more potent tumor vasculature specific combinations of VDAs and nanoparticles with the goal of transitioning optimal regimens into clinical trials.

Cryoablation for atrial fibrillation is a clinically approved technique used to control irregular heart rhythms due to abnormal electrical signals conducted from the pulmonary veins to the atria. The ablation catheter rapidly cools the cardiac tissue to freezing temperatures creating a discrete lesion which terminates these abnormal electrical pulses. Benefits of this approach include decreased risk of thrombus formation, well demarcated lesions, and preservation of extracellular matrix and endothelial integrity. However, possible complications include phrenic nerve injury and pulmonary vein stenosis (although these are not unique to the cryoablative approach). Therefore, further understanding of the mechanisms and thresholds of cryoinjury are required. This study begins to address these issues by improving predictive model accuracy through demonstrating the importance of accurate, temperature-dependent tissue properties. The direct relationship between thermal history and treatment outcome, combined with the difficulties of realtime, clinical thermometry, make heat transfer modeling and numerical predictions important aspects of cryoablation studies. The accuracy of the models depends on numerous factors including the kinetics and energy release during phase change phenomena and knowledge of thermal properties as a function of temperature. However, insufficient data for tissue thermal properties in the subzero domain results in a reliance on property estimations, generally based on water–ice data or weight averaged values from known materials. This study focused on expanding the thermal properties database for biological tissue in the subzero regime for use in cryoablation modeling. Results for porcine myocardium as well as ultrasound gel (a substitute phantom used for cryoprobe characterization) are reported. A differential scanning calorimeter was used to measure the specific heat and to observe latent heat effects. The thermal conductivity was measured using self-heating thermistors, employing both transient pulse decay and quasi-steady constant power methods. Specific heats in porcine myocardium were slightly less than those for ice, ranging from 0.93 J/gK at −150 °C to 2.35 J/gK at −21 °C. Specific heat behavior for ultrasound gel could be bracketed into two ranges, in which the values measured were less than those for ice at temperatures below the glass transition temperature (around −110 °C), and progressively higher than those for ice with temperatures above the glass transition. Thermal conductivities of porcine myocardium rose gradually at lower temperatures, ranging from 1.52 W/mK at −10 °C to 2.31 at −148 °C, and were about 50% less than those values for ice. Thermal conductivities of ultrasound gel were similar to those of porcine myocardium, but with an inflection point at the glass transition temperature after which the rise in thermal conductivity was attenuated. The importance of accurate, temperature-dependent thermal properties is demonstrated by comparing the predicted thermal history versus those estimated from constant property values.