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Oocyte vitrification has become a widely adopted method in clinical practice. However, the solidification behavior and its impact on oocytes during the ultrarapid cooling process remain poorly understood. In this study, we established a system and methodology to observe crystallization behavior in oocytes during quench cooling and warming. Subsequently, the threshold concentration of cryoprotective agents (CPAs) required for oocyte vitrification was determined through a visualization method. The results demonstrated that the ice front could not be observed in the image sequence when using 16.5% DMSO +16.5% EG during high-speed quench cooling (2821.58°C/min). Finally, oocytes were encapsulated with an antifreezing hydrogel (7.5% EG +7.5% DMSO +0.5% alginate) and subjected to high-speed quench cooling. No ice crystals appeared in the antifreezing hydrogel-encapsulated oocytes at a low concentration of osmotic CPA (2.4 M). This research opens up new possibilities for oocyte vitrification with a reduced concentration of CPA.

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Long-term cryopreservation of human umbilical vein endothelial cells (HUVECs) is important and beneficial for a variety of biomedical research and applications. In this study, we investigated HUVEC's cryobiological characteristics and parameters that are indispensable for predicting and determining an optimal cooling rate to prevent lethal intracellular ice formation (IIF) and severe cell dehydration during the cryopreservation processes. The parameters include cell membrane hydraulic conductivity (i.e., cell membrane water permeability), Lp, cell membrane water permeability activation energy, Elp, and osmotically inactive volume of a cell Vb. Cryomicroscopy was used to study the IIF phenomena and cell volume excursion at various cooling rates, 1, 10, and 20°C/min, respectively, based on which the cryobiological parameters were determined using biophysical and mathematical models. Results from this research work laid an important cryobiological foundation for the optimization of HUVEC's cryopreservation conditions.

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Freezing of natural biomaterials results in the formation of ice crystals and the generation of hypertonicity, both of which are deleterious to biomaterials. Although the cryopreservation of cells, tissues, and even organs has been achieved empirically by vitrification using cryoprotectants, the underlying mechanisms are poorly understood. To better understand the crystallization and vitrification processes of cryoprotected cells, onion epidermal cells immersed in dimethyl sulfoxide (DMSO) solutions with different concentrations were employed as platforms. The crystallization and vitrification dynamic processes of the individual and multiple monolayer cells were recorded using a high-speed microscope camera and the forms of the intracellular and extracellular ices were further confirmed by corresponding Raman spectra. The effects of DMSO concentration and cooling/warming rate on both processes were investigated and the findings were of an important significance to improve the understanding of the mechanisms of intracellular ice formation in individual cells and the ice propagation between adjacent cells. It is expected to provide a theoretical basis for cryopreservation by vitrification and point toward a new pathway for developing cryopreservation protocols.

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To cryopreserve cells, it is essential to avoid intracellular ice formation during cooling and warming. One way to achieve this is to convert the water inside the cells into a non-crystalline glass. It is currently believed that to accomplish this vitrification, the cells must be suspended in a very high concentration (20-40%) of a glass-inducing solute, and subsequently cooled very rapidly. Herein, we report that this belief is erroneous with respect to the vitrification of one-cell rat embryos. In the present study, one-cell rat embryos were vitrified with 5 µL of EFS10 (a mixture of 10% ethylene glycol (EG), 27% Ficoll, and 0.45 M sucrose) in cryotubes at a moderate cooling rate, and warmed at various rates. Survival was assessed according to the ability of the cells to develop into blastocysts and to develop to term. When embryos were vitrified at a 2613 °C/min cooling rate and thawed by adding 1 mL of sucrose solution (0.3 M, 50 °C) at a warming rate of 18 467 °C/min, 58.1 ± 3.5% of the EFS10-vitrified embryos developed into blastocysts, and 50.0 ± 4.7% developed to term. These rates were similar to those of non-treated intact embryos. Using a conventional cryotube, we achieved developmental capabilities in one-cell rat embryos by rapid warming that were comparable to those of intact embryos, even using low concentrations (10%) of cell-permeating cryoprotectant and at low cooling rates.

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Quantitative information about the kinetics and cumulative probability of intracellular ice formation is necessary to develop minimally damaging freezing procedures for the cryopreservation of cells and tissues. Conventional cryomicroscopic assays, which rely on indirect evidence of intracellular freezing (e.g., opacity changes in the cell cytoplasm), can yield significant errors in the estimated kinetics. In contrast, the formation and growth of intracellular ice crystals can be accurately detected using temporally resolved imaging methods (i.e., video recording at sub-millisecond resolution). Here, detailed methods for the setup and operation of a high-speed video cryomicroscope system are described, including protocols for imaging of intracellular ice crystallization events and stochastic analysis of the ice formation kinetics in a cell population. Recommendations are provided for temperature profile design, sample preparation, and configuration of the video acquisition parameters. Throughout this chapter, the protocols incorporate best practices that have been drawn from two decades of experience with high-speed video cryomicroscopy in our laboratory.

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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.

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Immediate post-thaw evaluation of membrane integrity has proven to yield overestimates of cell survival under conditions that preclude intracellular ice formation (IIF). However, prominent theories on the mechanisms of intracellular nucleation suggest a damaged membrane can reseal, prompting us to evaluate whether immediate post-thaw assessments of membrane integrity can in fact underestimate cell survival under conditions that promote IIF. HUVEC and HepG2 monolayers were treated with 1.4 M DMSO and frozen to -25 °C under conditions that formed either 0% or 100% IIF. Membrane integrity was evaluated both immediately and 24 h post-thaw, with metabolic activity assessments performed 24 h post-thaw as a secondary measure of survival. Treatment with 1.4 M DMSO and nucleation of 100% IIF resulted in a drastic increase in the relative percent of membrane intact cells following a 24 h culture period (HUVEC: 90.2% ± 0.7%; HepG2: 70.4% ± 4.0%), which correlated with 24 h post-thaw metabolic activity. These differences between the immediate and 24 h post-thaw membrane integrity assessments were significantly more than those seen in the absence of either IIF or DMSO treatment. Therefore, a high incidence of IIF in DMSO-treated monolayers may lead to erroneous underestimates of cell survival when conducting immediate post-thaw assessments of membrane integrity.

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To promote the recovery of cells that undergo intracellular ice formation (IIF), it is imperative that the recrystallization of intracellular ice is minimized. Hepatocytes are more prone to IIF than most mammalian cells, and thus we assessed the ability of novel small molecule carbohydrate-based ice recrystallization inhibitors (IRIs) to permeate and function within hepatocytes. HepG2 monolayers were treated with N-(4-chlorophenyl)-d-gluconamide (IRI 1), N-(2-fluorophenyl)-d-gluconamide (IRI 2), or para-methoxyphenyl-ß-D-glycoside (IRI 3) and fluorescent cryomicroscopy was used for real time visualization of intracellular ice recrystallization. Both IRI 2 and IRI 3 reduced rates of intracellular recrystallization, whereas IRI 1 did not. IRI 2 and IRI 3, however, demonstrated a marked reduction in efficiency in the presence of the most frequently used permeating cryoprotectants (CPAs): glycerol, propylene glycol (PG), dimethyl sulfoxide (DMSO), and ethylene glycol (EG). Nevertheless, IRI 3 reduced rates of intracellular recrystallization relative to CPA-only controls in the presence of glycerol, PG, and DMSO. Interestingly, IRI preparation in trehalose, a commonly used non-permeating CPA, did not impact the activity of IRI 3. However, trehalose did increase the activity of IRI 1 while decreasing that of IRI 2. While this study suggests that each of these compounds could prove relevant in hepatocyte cryopreservation protocols where IIF would be prominent, CPA-mediated modulation of intracellular IRI activity is apparent and warrants further investigation.

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The key to optimizing the cryopreservation strategy of human adipose-derived stem cells (hADSCs) is to identify the biophysical characteristics during freezing. Systematic freezing experiments were conducted under a cryo-microscope system to investigate the cryoinjury mechanism for hADSCs at different cooling rates. By simultaneously fitting morphological change data to the water-transport equation at 5, 10 and 20 °C/min, the plasma membrane hydraulic conductivity, Lpg, and activation energy, ELp, were determined. Moreover, the optimal cooling rate was also predicted by using mathematical model methods. Additionally, the surface-catalyzed nucleation (SCN) parameters were calculated by fitting in numerical models, Ω0SCN and k0SCN were determined at cooling rates of 30, 45 and 60 °C/min. These results may provide potential application value for cryopreservation of hADSCs.

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This paper discusses the role of ice crystal formation in causing or contributing to the difficulties that have been encountered in attempts to develop effective methods for the cryopreservation of some tissues and all organs. It is shown that extracellular ice can be severely damaging but also that cells in situ in tissues can behave quite differently from similar cells in a suspension with respect to intracellular freezing. It is concluded that techniques that avoid the formation of ice altogether are most likely to yield effective methods for the cryopreservation of recalcitrant tissues and vascularised organs.

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Cryopreservation encompasses several interconnect disciplines including physiology and cryophysics. This chapter reviews the current techniques for cryopreservation of plant genetic resources (PGRs). Vitrification is an effective ice crystal avoidance mechanism for hydrated cells and tissues. With any cryopreservation method, whole or partial parts of specimens which are sufficiently dehydrated can be vitrified by rapid cooling in liquid nitrogen (LN). Techniques discussed are the vitrification protocol, encapsulation-vitrification protocol, droplet vitrification protocol (DV), vitrification protocol using cryo-plates (V cryo-plate), and air dehydration protocol using cryo-plates (D cryo-plate). In these DV, V, and D cryo-plate protocols, specimens to be cryopreserved are immersed directly into LN on aluminum foil strips or cryo-plates; removal from LN to rewarming solution results in a high level of plant regrowth with ultrarapid cooling and warming. The protocols were applied to a wide array of plant species including wild and multi-ploid species, although fine tuning of the protocols was required for successful application to specific plant species and lines. These three protocols efficiently complement each other and appear highly promising to facilitate large-scale cryobanking of PGRs in genebanks. Cryo-scanning electron microscopy makes it possible to examine the cellular and water behavior in plant tissues when immersed in LN. It has been verified that tissues cryopreserved by the process of vitrification and the cryo-plate protocols are cryopreservation methods for reliable long-term preservation of PGRs.

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Intracellular ice formation (IIF) is the major cause of death in cells subjected to freezing. The occurrence of intracellular ice prevents the penetration of light into the camera and makes the image dark. Therefore, the gray-level variation can reflect the IIF. However, cell deformation is accompanied with IIF, especially for larger cells. It is necessary to account this entire phenomenon together in a single method. In this paper, the normalized parameter C defined by the gray-level variation depending on the displacement was defined to reflect the gray-level change of each pixel point in the region of interest of the image. The process of IIF of onion epidermal cells and 293T cells was analyzed by this method.

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Preventing intracellular ice formation is essential to cryopreserve cells. Prevention can be achieved by converting cell water into a non-crystalline glass, that is, to vitrify. The prevailing belief is that to achieve vitrification, cells must be suspended in a solution containing a high concentration of glass-inducing solutes and cooled rapidly. In this study, we vitrified 1-cell mouse embryos and examined the effect of the cooling rate, the warming rate, and the concentration of cryoprotectant on cell survival. Embryos were vitrified in cryotubes. The vitrification solutions used were EFS20, EFS30, and EFS40, which contained ethylene glycol (20, 30 and 40% v/v, respectively), Ficoll (24%, 21%, and 18% w/v, respectively) and sucrose (0.4 0.35, and 0.3 M, respectively). A 5-µl EFS solution suspended with 1-cell embryos was placed in a cryotube. After 2 min in an EFS solution at 23 °C, embryos were vitrified by direct immersion into liquid nitrogen. The sample was warmed at 34 °C/min, 4,600 °C/min and 6,600 °C/min. With EFS40, the survival was low regardless of the warming rate. With EFS30 and EFS20, survival was also low when the warming rate was low, but increased with higher warming rates, likely due to prevention of intracellular ice formation. When 1-cell embryos were vitrified with EFS20 and warmed rapidly, almost all of the embryos developed to blastocysts in vitro. Moreover, when vitrified 1-cell embryos were transferred to recipients at the 2-cell stage, 43% of them developed to term. In conclusion, we developed a vitrification method for 1-cell mouse embryos by rapid warming using cryotubes.

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Cryosurgery is a minimally invasive treatment that utilize extreme low temperatures to destroy abnormal tissues. The clinical monitoring methods for cryosurgery are almost based on the visualization of the iceball. However, for a normal cryosurgery process, the effective killing region is always smaller than the iceball. As a result, the end of the cryosurgery process can only be judged by the surgeons according to their experience. The subjective judgement is one of the main reasons for poor estimation of tumor ablation, and it sparks high probability of recurrence and metastasis associate with cryosurgery. Being different from the previous optimization studies, we develop a novel approach with the aid of nanoparticles to enlarge the effective killing region of entire iceball, and thus it greatly decrease the difficulty of precise judgement of the cryosurgery only by applying the common clinical imaging methods. To verify this approach, both the experiments on a tissue-scale phantom with embedded living HepG2 cells in agarose and on a cell-scale cryo-microscopic freeze-thaw stage are performed. The results indicate that the introduction of the self-synthesized Fe3O4 nanoparticles significantly improved cell killing in the cryosurgery and the range of killing is extended to the entire iceball. The potential mechanism is further revealed by the cryo-microscopic experiments, which verifies the presence of Fe3O4 nanoparticles can significantly enhance the probability of intracellular ice formation and the cell dehydration during freezing hence it promote precise killing of the cells. These findings may further promote the widespread clinical application of modern cryosurgery.

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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).

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Stem cells are important for regenerative medicine mainly due to their multilineage differentiation capacity. However, the cells rapidly loose this capability during culturing. Cryopreservation preserves the differentiation potential of the cells, until they are needed. In this study, specific cell properties of multipotent stromal cells (MSCs), from the common marmoset monkey Callithrix jacchus MSCs derived from amnion (Am) and bone marrow (Bm) were studied in order to predict optimal cooling rates for cryopreservation. Cell volume behaviour in anisotonic media, hydraulic membrane permeability at supra as well as subzero temperatures, and time point of intracellular ice formation (IIF) were investigated by Coulter Counter and cryomicroscopy. Cryopreservation outcome was studied using the predicted and experimentally determined cooling rate followed by 24 h re-cultivation. Little differences in osmotically inactive volume were found between amnion (0.27 × Vo) and bone marrow (0.28 × Vo) derived MSCs. The activation energy for water transport at suprazero temperature was found to be similar for both cell types; 4.4 ± 0.2 and 5.0 ± 0.15 kcal mol-1 for amnion and bone marrow derived MSCs, respectively. At subzero temperatures in the absence of dimethyl sulfoxide (Me2SO), the activation energy for water transport increased to 24.8 ± 3 kcal mol-1 and 27.4 ± 0.9 kcal mol-1 for Am and BmMSCs respectively. In the presence of Me2SO, activation energies were found to be 11.6 ± 0.3 kcal mol-1 and 19.5 ± 0.5 kcal mol-1 respectively. Furthermore, Me2SO was found to decrease the incidence of intracellular ice formation. The predicted optimal cooling rates of 11.6 ± 0.9 °C/min (AmMSCs) and 16.3 ± 0.5 °C/min (BmMSCs) resulted in similar post-thaw viability values compared to the experimentally determined optimal cooling profiles of 7.5 °C/min to -30 °C, followed by 3 °C/min to -80 °C.

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Advancement in biomedical simulation and imaging modality have catalysed the development of in silico predictive models for cryoablation. However, one of the main challenges in ensuring the accuracy of the model prediction is the use of proper thermal and biophysical properties of the patient. These properties are difficult to measure clinically and thus, represent significant uncertainty that can affect the model prediction. Motivated by this, a sensitivity analysis is carried out to identify the model parameters that have the most significant impact on the lesion size during cryoablation. The study is initially carried out using the Morris method to screen for the most dominant parameters. Once determined, analysis of variance (ANOVA) is performed to quantitatively rank the order of importance of each parameter and their interactions. Results from the sensitivity analysis revealed that blood perfusion, water transport and ice nucleation parameters are critical in predicting the lesion size, suggesting that the acquisition of these parameters should be prioritised to ensure the accuracy of the model prediction.

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Quantitative evaluation of the inherent correlation between cell cryoinjuries and intracellular ice formation (IIF) together with recrystallization (IIR) is of primary importance for both optimization of biopreservation and cryotherapy. The objective of this study is to thoroughly explore the roles of IIF on cell viability by using pig iliac endothelium cells (PIECs) as model cells during freezing and thawing. The experimental results indicated that both the probabilities of IIF (PIF) and IIR (PIR) increased along with the increase of cooling rates (p < 0.05) during the freeze-thaw cycles at cooling rates of 40, 60, 80, 100, and 150°C/min and the same warming rates of 100°C/min in phosphate-buffered saline-based solutions with or without 1 M DMSO. Viability evaluation with Hoechst 33342/propidium iodide double staining showed that most of the cells were killed (viability <20%) by the abovementioned freeze-thaw cycles, which indicated that the cooling rates investigated were all too rapid since large amounts of IIF and IIR were introduced. Another interesting phenomenon is that the presence of a low concentration of DMSO (1 M) tends to improve cell viability while increasing the PIF and PIR during freezing/thawing, contrary to the common belief that larger PIF corresponds to greater cryoinjury. This may be attributed to the intrinsic protection effect of DMSO by reduction of solution injury or other potential injuries. These findings may be of potential application value for both cryopreservation and cryosurgery by providing helpful additions to the existing studies on investigation of cryoinjuries of PIECs.

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In this study, mature female mice of the ICR strain were induced to superovultate, mated, and collected at either zygote or early morula stages. Embryos suspended in 1 M ethylene glycol in PBS containing 10 mg/L Snomax for 15 min, then transferred in sample holder to Linkam cryostage, cooled to and seeded at 7 °C, and then observed and photographed while being cooled to -70 °C at 0.5-20 °C/min. Intracellular ice formation (IIF) was observed as abrupt ''flashing''. Two types of flashing or IIF were observed in this study. Extracellular freezing occurred at a mean of -7.7 °C. In morulae, about 25% turned dark within ±1 °C of extracellular ice formation (EIF). These we refer to as "high temperature'' flashers. In zygotes, there were no high temperature flashers. All the zygotes flashed at temperatures well below the temperature for EIF. Presumably high temperature flashers were a consequence of membrane damage prior to EIF or damage from EIF. We shall not discuss them further. In the majority of cases, IIF occurred well below -7.7 °C; these we call ''low temperature'' flashers. None flashed with cooling rate (CR) of 0.5 °C/min in either zygotes or morulae. Nearly all flashed with CR of 4 °C/min or higher, but the distribution of temperatures is much broader with morulae than with zygotes. Also, the mean flashing temperature is much higher with morulae (-20.9 °C) than with zygotes (-40.3 °C). We computed the kinetics of water loss with respect to CR and temperature in both mouse zygotes and in morulae based on published estimates of Lp and it is Ea. The resulting dehydration curves combined with knowledge of the embryo nucleation temperature permits an estimate of the likelihood of IIF as a function of CR and subzero temperature. The agreement between these computed probabilities and the observed values are good.

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A few species of nematodes can survive extensive intracellular freezing throughout all their tissues, an event that is usually thought to be fatal to cells. How are they able to survive in this remarkable way? The pattern and distribution of ice formed, after freezing at -10°C, can be observed using freeze substitution and transmission electron microscopy, which preserves the former position of ice as white spaces. We compared the pattern and distribution of ice formed in a nematode that survives intracellular freezing well (Panagrolaimus sp. DAW1), one that survives poorly (Panagrellus redivivus) and one with intermediate levels of survival (Plectus murrayi). We also examined Panagrolaimus sp. in which the survival of freezing had been compromised by starvation. Levels of survival were as expected and the use of vital dyes indicated cellular damage in those that survived poorly (starved Panagrolaimus sp. and P. murrayi). In fed Panagrolaimus sp. the intracellular ice spaces were small and uniform, whereas in P. redivivus and starved Panagrolaimus sp. there were some large spaces that may be causing cellular damage. The pattern and distribution of ice formed was different in P. murrayi, with a greater number of individuals having no ice or only small intracellular ice spaces. Control of the size of the ice formed is thus important for the survival of intracellular freezing in nematodes.

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