Vitrification is enabling successful cryopreservation of progressively larger organs. Heating the whole volume of an organ simultaneously instead of only the surface during recovery from vitrification has been vital to this success. Volumetric warming enables faster and more uniform warming. Faster warming reduces ice crystal growth, ice recrystallization, and toxic effects of cryoprotectants by reducing ice growth time and cryoprotectant exposure time during warming. Nanowarming of intravascular magnetic nanoparticles by alternating magnetic fields, and direct dielectric warming by alternating electric fields, have both been used successfully for volumetric warming of vitrified organs. Dielectric warming predates vitrification, having been studied intermittently in cryobiology since the 1950s. Most early research was empirical, using microwaves at 915 MHz or 2.45 GHz because of wide availability of magnetron generators and microwave ovens. With greater theoretical understanding, interest later grew in frequencies near 400 MHz and lower. Larger energy absorption and smaller wavelengths inside tissue warming from vitrification instead of thawing from freezing made optimum frequencies for vitrification even lower, below 100 MHz for organs more than 10 cm in diameter. Recent dielectric warming research has used 27, 40, and 55 MHz, achieving heating rates of 200 - 700 °C/min. The advantages of dielectric warming not being dependent upon exogenous particles or vascular volume, high energy efficiency, and ability to monitor temperature by impedance behavior during warming, are counterbalanced by the need for organ immersion before vitrification and pre-warming to a uniform starting temperature above the glass transition temperature. With measurements of two electrical properties of a vitrification solution, permittivity and loss factor as function of temperature, detailed theoretical analysis and modeling of dielectric warming is possible. Comparatively little research has been done on dielectric warming at frequencies optimal for vitrification. This review covers the history, theory, equipment types, practical aspects, and future directions of dielectric warming of cryopreserved tissue.

Organs cryopreserved by vitrification benefit from fast warming to avoid growth and recrystallization of ice that may nucleate during cooling and during warming. Rapid warming is especially important for tissue that doesn't absorb the full concentration of perfused cryoprotectants. Nanowarming uses an oscillating magnetic field to heat magnetic nanoparticles introduced into blood vessels during cryoprotectant perfusion. Dielectric warming uses an oscillating electric field to directly heat water and cryoprotectant molecules everywhere inside an organ. The efficiency of dielectric warming peaks at a particular solution viscosity and temperature that depends on the field oscillation frequency. Below that temperature, field uniformity is very important for uniform warming. An 800 W 55 MHz dielectric warming system was constructed that reached peak warming efficiency at -60 °C instead of -70 °C previously observed at 27 MHz when using the M22 vitrification solution. The shape of capacitor plates that formed the electric field for organ warming was optimized by computer simulation. Computer simulation also provided insights into the effects of organ container shape on internal field uniformity, confirming the theoretical prediction that ellipsoidal shapes are optimum. Dielectric materials surrounding the organ container during warming were used with beneficial effect. In physical experiments with constant field warming at 55 MHz, warming rates peaked near 200 °C/min for ∼15 g rabbit kidneys in 45 mL total volume, and near 700 °C/min for ∼5 g porcine ovaries in 15 mL total volume. Of three rabbit kidneys vitrified, dielectrically warmed under slightly varying conditions, and then transplanted, one survived long-term with return to normal clinical function (serum creatinine <2 mg/dL) in the recipient animal still living 17 months later. The mass of the kidney was 13.9 g, by an order of magnitude the largest vitrified vital organ successfully returned to clinically normal function to date.

Organs cryopreserved by vitrification are exposed to the lowest possible concentration of cryoprotectants for the least time necessary to successfully avoid ice formation. Faster cooling and warming rates enable lower concentrations and perfusion times, reducing toxicity. Since warming rates necessary to avoid ice formation during recovery from vitrification are typically faster than cooling rates necessary for vitrification, warming speed is a major determining factor for successful vitrification. Dielectric warming uses an oscillating electric field to directly heat water and cryoprotectant molecules inside organs to achieve warming that's faster and more uniform than can be achieved by heat conduction from the organ surface. This work studied 27 MHz dielectric warming of rabbit kidneys perfused with M22 vitrification solution. The 27 MHz frequency was chosen because its long wavelength and penetration depth are suitable for human organs, because it had an anticipated favorable temperature of maximum dielectric absorption in M22, and because it's an allocated frequency for industrial and amateur use with inexpensive amplifiers available. Previously vitrified kidneys were warmed from -100 °C by placement in a 27 MHz electric field formed between parallel capacitor plates in a resonant circuit. Power was varied during warming to maintain constant electric field amplitude between the plates. Maximum power absorption occurred near -70 °C, with a peak warming rate near 150 °C/min in 50 mL total volume with approximately 500 W power. After some optimization, it was possible to warm ∼13 g vitrified kidneys with unprecedentedly little injury from medullary ice formation and a favorable serum creatinine trend after transplant. Distinct behaviors of power absorption and system tuning observed as a function of temperature during warming are promising for non-invasive thermometry and future automated control of the warming process at even faster rates with user-defined temperature dependence.

Information Theoretic Death (loss of brain structures encoding memory and personality to an extent that it’s physically impossible for any technology to infer them) is NOT Cardiopulmonary Death (legal death determined by irreversible cessation of breathing and heartbeat, taking into account what resuscitation measures are available or intended, if any). If the cryonics patient is legally dead, then there is an established cryonics protocol to treat the patient as a person rather than as a corpse ̶ for example, cryobiological vitrification (ice-free cryopreservation). Long term cryonics care means no further deterioration. Future developments may allow inference to healthy brain structure, and recovery of the original person to full health.

Human biostasis, the preservation of a human when all other contemporary options for extension of quality life are exhausted, offers the speculative potential for survival via continuation of life in the future. While provably reversible preservation, also known as suspended animation, is not yet possible for humans, the primary justification for contemporary biostasis is the preservation of the brain, which is broadly considered the seat of memories, personality, and identity. By preserving the information contained within the brain’s structures, it may be possible to resuscitate a healthy whole individual using advanced future technologies. There are numerous challenges in biostasis, including inadequacies in current preservation techniques, methods to evaluate the quality of preservation, and potential future revival technologies. In this report, we describe a roadmap that attempts to delineate research directions that could improve the field of biostasis, focusing on optimizing preservation protocols and establishing metrics for querying preservation quality, as well as pre- and post-cardiac arrest factors, stabilization strategies, and methods for long-term preservation. We acknowledge the highly theoretical nature of future revival technologies and the importance of achieving high-fidelity brain preservation to maximize the potential of future repair technologies. We plan to update the research roadmap biennially. Our goal is to encourage multidisciplinary communication and collaboration in this field.

To assess safety and effectiveness of percutaneous image-guided cryoablation of hepatic tumors adjacent to the gallbladder.Twenty-one cryoablation procedures were performed to treat 19 hepatic tumors (mean size, 2.7 cm; range, 1.0–5.0 cm) adjacent to the gallbladder in 17 patients (11 male; mean age, 59.2 y; range, 40–82 y) under computed tomography (n = 15) or magnetic resonance imaging (n = 6) guidance in a retrospective study. All tumors (mean size, 2.67 cm; range, 1.0–5.0 cm) were within 1 cm (mean, 0.4 cm) of the gallbladder; seven (33%) were contiguous with the gallbladder. Primary outcomes included complication rate and severity and postprocedure gallbladder imaging findings. Secondary outcomes included technical success and technique effectiveness at 6 months.Complications occurred in six of 21 procedures (29%); one (5%) was severe. Ice balls extended into the gallbladder lumen in 20 of 21 procedures (95%); no gallbladder-related complications occurred. The most common gallbladder imaging finding was mild, asymptomatic focal wall thickening after nine of 21 procedures (42%), which resolved on follow-up. Technical success was achieved in 19 of 21 sessions (90%). Six-month follow-up was available for 16 tumors; of these, all but two (87%) had no imaging evidence of local tumor progression.Percutaneous cryoablation of hepatic tumors adjacent to the gallbladder can be performed safely and successfully. Although postprocedural gallbladder changes are common, they are self-limited and clinically inconsequential, even when the ice ball extends into the gallbladder lumen.

Abstract Vitrification can dramatically increase the storage of viable biomaterials in the cryogenic state for years. Unfortunately, vitrified systems ≥3 mL like large tissues and organs, cannot currently be rewarmed sufficiently rapidly or uniformly by convective approaches to avoid ice crystallization or cracking failures. A new volumetric rewarming technology entitled “nanowarming” addresses this problem by using radiofrequency excited iron oxide nanoparticles to rewarm vitrified systems rapidly and uniformly. Here, for the first time, successful recovery of a rat kidney from the vitrified state using nanowarming, is shown. First, kidneys are perfused via the renal artery with a cryoprotective cocktail (CPA) and silica‐coated iron oxide nanoparticles (sIONPs). After cooling at −40 °C min−1 in a controlled rate freezer, microcomputed tomography (µCT) imaging is used to verify the distribution of the sIONPs and the vitrified state of the kidneys. By applying a radiofrequency field to excite the distributed sIONPs, the vitrified kidneys are nanowarmed at a mean rate of 63.7 °C min−1. Experiments and modeling show the avoidance of both ice crystallization and cracking during these processes. Histology and confocal imaging show that nanowarmed kidneys are dramatically better than convective rewarming controls. This work suggests that kidney nanowarming holds tremendous promise for transplantation.

Vitrification is an alternative to cryopreservation by freezing that enables hydrated living cells to be cooled to cryogenic temperatures in the absence of ice. Vitrification simplifies and frequently improves cryopreservation because it eliminates mechanical injury from ice, eliminates the need to find optimal cooling and warming rates, eliminates the importance of differing optimal cooling and warming rates for cells in mixed cell type populations, eliminates the need to find a frequently imperfect compromise between solution effects injury and intracellular ice formation, and can enable chilling injury to be "outrun" by using rapid cooling without a risk of intracellular ice formation. On the other hand, vitrification requires much higher concentrations of cryoprotectants than cryopreservation by freezing, which introduces greater risks of both osmotic damage and cryoprotectant toxicity. Fortunately, a large number of remedies for the latter problem have been discovered over the past 35 years, and osmotic damage can in most cases be eliminated or adequately controlled by paying careful attention to cryoprotectant introduction and washout techniques. Vitrification therefore has the potential to enable the superior and convenient cryopreservation of a wide range of biological systems (including molecules, cells, tissues, organs, and even some whole organisms), and it is also increasingly recognized as a successful strategy for surviving harsh environmental conditions in nature. But the potential of vitrification is sometimes limited by an insufficient understanding of the complex physical and biological principles involved, and therefore a better understanding may not only help to improve present outcomes but may also point the way to new strategies that may be yet more successful in the future. This chapter accordingly describes the basic principles of vitrification and indicates the broad potential biological relevance of this alternative method of cryopreservation.

PurposeHerein, we evaluate the use of MRI as a tool for assessing iron oxide nanoparticle (IONP) distribution within IONP perfused organs and vascularized composite allografts (VCAs) (i.e., hindlimbs) prepared for cryopreservation.MethodsMagnetic resonance imaging was performed on room‐temperature organs and VCAs perfused with IONPs and were assessed at 9.4 T. Quantitative T1 mapping and ‐weighted images were acquired using sweep imaging with Fourier transformation and gradient‐echo sequences, respectively. Verification of IONP localization was performed through histological assessment and microcomputer tomography.ResultsQuantitative imaging was achieved for organs and VCAs perfused with up to 642 mMFe (36 mgFe/mL), which is above previous demonstrations of upper limit detection in agarose (35.7mMFe [2 mgFe/mL]). The stability of IONPs in the perfusate had an effect on the quality of distribution and imaging within organs or VCA. Finally, MRI provided more accurate IONP localization than Prussian blue histological staining in this system, wherein IONPs remain primarily in the vasculature.ConclusionUsing MRI, we were able to assess the distribution of IONPs throughout organs and VCAs varying in complexity. Additional studies are necessary to better understand this system and validate the calibration between T1 measurements and IONP concentration.

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.

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.

Vitrification is an alternative approach to cryopreservation that enables hydrated living cells to be cooled to cryogenic temperatures in the absence of ice. Vitrification simplifies and frequently improves cryopreservation because it eliminates mechanical injury from ice, eliminates the need to find optimal cooling and warming rates, eliminates the importance of differing optimal cooling and warming rates for cells in mixed cell type populations, eliminates the need to find a frequently imperfect compromise between solution effects injury and intracellular ice formation, and enables cooling to be rapid enough to "outrun" chilling injury, but it complicates the osmotic effects of adding and removing cryoprotective agents and introduces a greater risk of cryoprotectant toxicity during the addition and removal of cryoprotectants. Fortunately, a large number of remedies for the latter problem have been discovered over the past 30+ years, and the former problem can in most cases be eliminated or adequately controlled by careful attention to technique. Vitrification is therefore beginning to realize its potential for enabling the superior and convenient cryopreservation of most types of biological systems (including molecules, cells, tissues, organs, and even some whole organisms), and vitrification is even beginning to be recognized as a successful strategy of nature for surviving harsh environmental conditions. However, many investigators who employ vitrification or what they incorrectly imagine to be vitrification have only a rudimentary understanding of the basic principles of this relatively new and emerging approach to cryopreservation, and this often limits the practical results that can be achieved. A better understanding may therefore help to improve present results while pointing the way to new strategies that may be yet more successful in the future. To assist this understanding, this chapter describes the basic principles of vitrification and indicates the broad potential biological relevance of vitrification.

During vitrification, nucleation of ice crystals occurs most rapidly at low temperatures during later stages of cooling, while actual growth of ice crystals is most rapid at warmer temperatures near the solution melting point. Warming rates required to avoid significant ice growth during recovery from vitrification are therefore larger than the cooling rates required during vitrification, when the sample is relatively un-nucleated. Large samples vitrified by external conduction cooling therefore require more rapid warming by internal means if damaging ice growth and cryoprotectant toxicity are to be avoided. Oscillating electric fields coupling to polar molecules (dielectric heating) and mobile ions (ohmic heating) can achieve this. For uniform warming, the oscillation frequency must be low enough to deposit energy inefficiently into the sample (skin depth >> sample size). However the inefficiency cannot be so low that the electric field strength necessary for a desired warming rate causes dielectric breakdown of the sample or surrounding air (arcing). The wavelength inside and outside the sample must also be much larger than the sample to avoid field nodes and antinodes. For vitrified samples of tens or hundreds of grams (human organs), the skin depth and wavelength constraints are met by frequencies of tens of megahertz. Dielectric absorption as a function of frequency is maximal at the Debye relaxation frequency of dipoles. This frequency increases as viscosity decreases, and is therefore temperature-dependent. It's desirable to choose a frequency with maximal absorption at the temperature at which the ice growth rate is maximal so that the warming rate is fastest when ice growth is fastest. This also promotes temperature uniformity by slowing warming in sample regions that warm past the temperature of maximal energy absorption. In the vitrification solution M22, the target temperature for maximum warming is approximately −60°. The corresponding frequency appears to be on the order of 30 MHz, which fortuitously also meets the aforementioned skin depth and wavelength constraints. Unlike dielectric heating that has a characteristic temperature of maximum absorption for a given frequency, ohmic heating only increases as temperatures increases, leading to "thermal runaway." It's therefore desirable to use a cryoprotectant carrier solution of low ionic strength, such as lactose/mannitol-based LM5. Boundary conditions of electric fields at dielectric interfaces also make sample geometry important for achieving a uniform internal field. Previous work in our laboratory with a 27 MHz 200-watt RF source demonstrated peak warming rates of 160 °C during warming a vitrified 20-mL cylindrical volume of M22 in LM5, and half that rate in a vitrified rabbit kidney in the same volume, with 3 °C and 15 °C maximum internal temperature differences respectively. Greater temperature non-uniformity within the organ vs. plain solution reflects decreased dielectric absorption within the non-polar lipid-rich renal pelvis. The maximum warming rate achievable within organs will likely be determined by such differential energy absorption, and the maximum tolerable temperature non-uniformities that result. Future study requires higher power, larger samples, variable frequency, and detailed measurements of cryoprotected tissue electrical properties as a function of frequency to permit accurate modeling.

Cryopreservation requires transformation of a biological material into a solid state for long-term stability. As a sample solidifies during cooling, mechanical stress accumulates due to changing temperature gradients and differential thermal contraction of heterogeneous sample components. If stress becomes larger than the material strength of the sample, fracturing will occur. The extraordinary low critical cooling rate of M22 vitrification solution (0.1 °C/min) has allowed our laboratory to study fracture avoidance during vitrification of multi-liter volumes of solution. A few general observations are notable. Rapid cooling through high sub-zero temperatures ( > 80 °C) is permissible and desirable to reduce risk of ice growth and cryoprotectant toxicity, but cooling should slow as the glass transition temperature (−123 °C) is approached to allow more time for stress relaxation. Glass containers facilitate fracturing by differential thermal contraction stress because vitrification solutions adhere to hydrophilic glass surfaces. Containers made from polyethylene or similar hydrophobic materials can eliminate this source of stress by allowing samples to contract away from container surfaces as they cool. Tissue permeated with cryoprotectant is generally more resistant to fracturing than the same volume of pure vitrification solution, making fracture-free vitrification of a pure solution a conservative test for suitability of a cooling protocol for fracture avoidance. Following these principles, our laboratory has routinely cooled M22-perfused rabbit kidneys to −135 °C without fracturing. Although not believed necessary for long-term storage, a vitrified kidney was also successfully recovered from liquid nitrogen immersion storage without fracturing using the following protocol: Standard perfusion and cooling to −135 °C, 75 min @ −130 °C, 60 min @ −160 °C, 120 min @ −188 °C, 13 days @ −196 °C (under liquid nitrogen), 90 min @ −185 °C, 90 min @ −155 °C, 18 h @ 135 °C, followed by standard warming, cryoprotectant removal, and transplantation with no hemorrhaging or other indication of fractures. For further exploration, a 200 mL bottle (greater than the mass of a human kidney) of 105% M22 was successfully cooled from ∼ 0 °C to liquid nitrogen temperature without fracturing using a simple convection cooling protocol: Placement in 110 °C vapor for 90 min, 170 °C vapor for 4 h, −186 °C vapor for 19 h, then immersion in liquid nitrogen (−196 °C) for 90 min prior to removal and observation that the solution had no fractures. Liquid nitrogen is far colder than necessary for long-term storage; −135 °C to −140 °C is likely satisfactory to arrest ice nucleation over time periods of clinical interest. These experiences suggest that fracture avoidance may be one of the easier problems of human tissue and organ banking. Rather than gross fractures, thermomechanical stress is a suspected cause of more subtle, but still-significant lesions that have sometimes been observed after transplantation of vitrified rabbit kidneys. Developing protocols for cooling as quickly as possible to minimize ice nucleation, while avoiding damaging thermal strain within tissue, is vital to the future success of organ banking. Finite element analysis computer models will be invaluable for this purpose.

The components of cryopreservation solutions can be classified into three general classes: (1) carrier solutes, which are the non-penetrating osmolytes, pH buffers, and nutritive ingredients that support viability of cells at hypothermic temperatures; (2) bulk cryoprotectants, which are ingredients (either membrane-penetrating or non-penetrating) typically added at multi-percent concentrations that reduce availability of bulk liquid water for ice formation by hydrogen bonding and dilution; (3) solutes that specifically interact with ice nucleating particles or ice crystals to inhibit or modify the growth of ice, and which are typically effective at very low concentrations. Class 3 solutes may exhibit ice nucleation inhibition (INI), ice growth inhibition (IGI), and/or ice recrystallization inhibition (IRI). Such solutes are valuable additives in vitrification solutions because they can replace much larger concentrations of more toxic bulk cryoprotectants while achieving similar suppression of ice formation. Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) are the prototypical examples of class 3 solutes. Fahy proposed in 1995 that synthetic analogs of AFPs would be useful additives for vitrification solutions, especially if lower molecular weights conveyed higher mobility in the high viscosity of vitrification solutions at low temperature. Subsequently, low molecular weight versions of the polymers polyvinyl alcohol (PVA) and polyglycerol (PGL) were found to show efficacy for numerous applications as “ice blockers” in vitrification solutions. PGL is apparently a specific INI against ice-nucleating protein contaminants, while PVA exhibits general INI, IGI, and IRI activity. Low molecular weight PVA and PGL are backbones of the advanced M22 and VM3 vitrification solutions. There are other INIs in the literature, such as flavonol glycosides, and new families of synthetic IRI inhibitors that may also be useful for cryopreservation. Even in circumstances in which ice nucleation cannot be avoided during vitrification, injury might be prevented if IRIs can keep ice crystals sufficiently small during warming. There is, however, a deficiency of small synthetic molecules with the same broad anti-ice activity as PVA. Small molecules are preferred because of greater mobility and decreased contribution to solution viscosity (low viscosity being important for perfusion cryoprotection of organs). Even though syndiotactic PVA oligomers have stable conformations with excellent alignment of hydroxyls for bonding to the basal plane of ice, a custom synthesized four “mer” PVA oligomer (1,3,5,7-heptanetetrol) was found to be inactive as an ice blocker in our laboratory. Subsequent structure-activity studies by Gibson’s group showed that PVA IRI activity ceases somewhere between 10 and 19 mers (400–800 MW). Evidently if a molecule is small, molecular models showing structural matching to an ice crystal surface, such as is seen with cyclohexanediols and triols, are not sufficient to establish identity as an ice blocker. Nor are empirical results at concentrations high enough (∼>1 mg/g) to enable bulk cryoprotective effects. Until good models and understanding of how known synthetic ice blockers or IRIs work are developed, prospects for finding or making new ones are limited. Protein-based compounds are understood better, but have obstacles of cost, stability in cryoprotectant solutions, and possible antigenicity.