Cryobiology Applications Research Articles

Cryobiology and fertility preservation: A perspective on past, current and future studies

Cryopreservation has been used over many decades for the maintenance of viable biological specimens. Its expansion into the area of fertility preservation has been a natural outcome of the increased risks to human fertility from diseases, such as cancer and its treatment protocols, including radiation and chemo-therapy, and thegeneral lifestyle trend to later marriages. The use of assisted reproductive techniques (ART) in preserving fertility have benefitted significantly from new scientific approaches, such as cryostorage, in which live cells and tissues are stored at low temperatures and revived when necessary. This review focuses on "cryopreservation science monitoring in reproductive biomedicine" to evaluate knowledge, trends, driving forces, impetus, and emerging technologies in order to draw a future roadmap for this field. Our analysis of the field of cryobiology emphasises the significance of strategic planning of cryobiology research to support more its extensive use in therapeutics in the future. The Royan Institute (Tehran, Iran) recognises this need and has developed a plan to engage in multidisciplinary research on the application of cryobiology, including in disease mitigation. We hope that this study can help improve the quality and of public discourse and expert awareness of the role for cryopreservation in fertility within ART.

Advances in Antifreeze Molecules: From Design and Mechanisms to Applications

Freezing can cause serious hazards in various fields, such as cryoinjury in cell and organ cryopreservation, food quality deterioration, and ice accretion on surfaces including energy facilities (wind turbines, overhead transmission lines, and towers), aircraft, etc., causing serious equipment damages and economic losses. To alleviate these problems, antifreeze molecules have attracted significant attention and have been applied extensively in cryopreservation and anti-icing coatings. In this review, the progress on antifreeze molecules is summarized and discussed, including their classifications, mechanisms, and applications. The antifreeze molecules are divided into natural and artificial molecules, and their characteristics are introduced. Moreover, their antifreeze mechanisms are comprehensively outlined involving depression of freezing points, dynamic ice shaping, and ice recrystallization inhibition. Then, the representative applications in cryobiology, anti-icing surfaces, food technology, and agriculture are also summarized and discussed. Finally, the challenges and development prospects of antifreeze molecules are further provided.

An Impedimetric Sensing Probe Based on Printed Circuit Board Technology for Monitoring in Cryobiology Applications

Cryopreservation of living cells is an effective tool for protection, maintenance, and distribution of genetic resources, which involves exposure to cryogenic temperatures and requires precise control over various parameters to avoid potential cell damages. Hundreds of protocols have been reported for cryopreservation of aquatic species, but replicating them is challenging without a reliable monitoring technique during a cryopreservation process. In this work, we aim to use electrical impedance as a monitoring parameter to assist standardization of cryopreservation processes and reporting. Specifically, this paper reports an impedance sensing probe compatible with cryogenic temperatures and conventional containers in cryopreservation of aquatic species based on printed circuit board technology its characterization in cryopreservation conditions including different sperm extenders (buffer) compositions and concentrations, presence of cryoprotectant, and multiple cooling rates. The developed probe based on printed circuit board (PCB) technology shows a capability of measuring conditions during cryopreservation differentiating among samples with different buffer contents and cryoprotectants. The probe also demonstrates the capability to distinguish different cooling regimes and detect phase change phenomena. This PCB-based sensing platform provides quantitative impedance measurement data during the cryopreservation process at sample preparation, cooling, and while frozen. Technology such as this offers opportunities for improving the reproducibility of protocols generated by the aquatic species community and can be made widely available as open hardware.

Laboratory-Scale Isolation of Insect Antifreeze Protein for Cryobiology.

Micromolar concentrations of hyperactive antifreeze proteins (AFPs) from insects can prevent aqueous solutions from freezing down to at least −6 °C. To explore cryopreservation of cells, tissues and organs at these temperatures without ice formation, we have developed a protocol to reliably produce ultrapure Tenebrio molitor AFP from cold-acclimated beetle larvae reared in the laboratory. The AFP was prepared from crude larval homogenates through five cycles of rotary ice-affinity purification, which can be completed in one day. Recovery of the AFP at each step was >90% and no impurities were detected in the final product. The AFP is a mixture of isoforms that are more active in combination than any one single component. Toxicity testing of the purified AFP in cell culture showed no inhibition of cell growth. The production process can easily be scaled up to industrial levels, and the AFP used in cryobiology applications was recovered for reuse in good yield and with full activity.

Three-dimensional printing can provide customizable probes for sensing and monitoring in cryobiology applications

Cryopreservation has been recognized as a powerful tool for long-term preservation of genetic resources. However, the outcomes of cryopreservation by different user groups often vary due to inconsistency in procedures and freezing equipment. Herein, we report on the feasibility of providing customizable sensing probes with three-dimensional (3-D) printing to monitor cryopreservation phenomena. The objectives were to: 1) introduce 3-D printing as a fabrication method for developing customizable probes to be used in cryogenic applications; 2) design and fabricate an example of a 3-D printed sensing probe and multiplexer capable of detecting phase-change phenomena based on quantitative data regarding sample electrical resistance and temperature, and 3) demonstrate the sensing platform in cryopreservation conditions and in combination with a custom-made 3-D printed freezing device. The sensing probe developed was designed to fit within standard 0.5-ml French straws. Phase-transition phenomena were detected by analyzing electrical resistance changes. The quantitative data from this device in conjugation with a 3-D printed freezer rack provided cryopreservation capability with high reproducibility and offered an alternative to expensive programmable freezers. The use of 3-D printing provided flexibility to develop new sensing probes or modify existing designs based on specific needs. After initial prototyping, fabrication, and testing of 3-D printed sensing probes, particularly useful designs can lead to the reduction of variation in performing standardized cryopreservation protocols.

Cold responsive nanoparticles for cryobiological applications

Cold responsive nanoparticles for cryobiological applications

Multivalent Presentation of Ice Recrystallization Inhibiting Polymers on Nanoparticles Retains Activity.

Poly(vinyl alcohol) (PVA) has emerged as the most potent mimic of antifreeze (glyco)proteins ice recrystallization inhibition (IRI) activity, despite its lack of structural similarities and flexible, rather than rigid, backbone. The precise spacing of hydroxyl groups is hypothesized to enable PVA to recognize the prism planes of ice but not the basal plane, due to hydroxyl pattern matching of the ice surface giving rise to the macroscopic activity. Here, well-defined PVA derived from reversible addition–fragmentation chain-transfer (RAFT) polymerization is immobilized onto gold nanoparticles to enable the impact of nanoscale assembly and confinement on the observed IRI activity. Unlike previous reports using star-branched or bottle-brush PVAs, the nanoparticle–PVA retains all IRI activity compared to polymers in solution. Evidence is presented to show that this is due to the low grafting densities on the particle surface meaning the chains are free to explore the ice faces, rather than being constrained as in star-branched polymers. These results demonstrate a route to develop more functional IRI’s and inclusion of metallic particle cores for imaging and associated applications in cryobiology.

Multivalent Display of Antifreeze Proteins by Fusion to Self-Assembling Protein Cages Enhances Ice-Binding Activities.

Antifreeze proteins (AFPs) are small monomeric proteins that adsorb to the surface of ice to inhibit ice crystal growth and impart freeze resistance to the organisms producing them. Previously, monomeric AFPs have been conjugated to the termini of branched polymers to increase their activity through the simultaneous binding of more than one AFP to ice. Here, we describe a superior approach to increasing AFP activity through oligomerization that eliminates the need for conjugation reactions with varying levels of efficiency. A moderately active AFP from a fish and a hyperactive AFP from an Antarctic bacterium were genetically fused to the C-termini of one component of the 24-subunit protein cage T33-21, resulting in protein nanoparticles that multivalently display exactly 12 AFPs. The resulting nanoparticles exhibited freezing point depression >50-fold greater than that seen with the same concentration of monomeric AFP and a similar increase in the level of ice-recrystallization inhibition. These results support the anchored clathrate mechanism of binding of AFP to ice. The enhanced freezing point depression could be due to the difficulty of overgrowing a larger AFP on the ice surface and the improved ice-recrystallization inhibition to the ability of the nanoparticle to simultaneously bind multiple ice grains. Oligomerization of these proteins using self-assembling protein cages will be useful in a variety of biotechnology and cryobiology applications.

Towards large scale expression of antifreeze proteins for cryobiology applications

Towards large scale expression of antifreeze proteins for cryobiology applications

29. Ice-binding proteins and their interactions with ice crystals

Ice-binding proteins (IBPs) depress the freezing point of the body fluids below the melting point, resulting in a thermal hysteresis (TH) that prevents freezing of the organism in supercooled conditions and inhibits ice re-crystallization in frozen tissues. The potential of these proteins in the medical sector, in cryopreservation, in the frozen food industry, and in agriculture is enormous. The mechanisms by which IBPs interact with ice surfaces are still not completely understood and the potential of IBPs as cryoprotecting agents has not yet been realized. At the molecular level it was found that the IBPs coordinate and stabilize water molecules on their binding surface to form an ice-like water film. While this ice-like film is too small to nucleate supper-cooled water it serves as a mechanism of tight binding to ice. Still, the way these IBPs influence ice growth and the activity differences between difference types of IBPs is the subject of current research. We are investigating the interactions of IBPs with ice surfaces. In particular we are interested in the difference between hyperactive antifreeze proteins and moderately active ones, and the dynamic nature of the protein:ice interaction. We have developed novel methods to study these issues, including fluorescence microscopy techniques combined with temperature-controlled microfluidic devices (Celik et al., PNAS 2013, Drori et al., J.R. Soc. Interface 2014, Drori et al., RSC Adv. 2015). These techniques enable the replacement of the IBP solution surrounding an IBP-bound ice crystal by other solutions, without perturbing the system, which enables us to investigate the dynamic nature of the interactions between IBP and ice. The results show that binding of IBP to ice is irreversible, and that the TH-gap is sensitive to the time allowed for the proteins to accumulate on ice surfaces. This sensitivity changes dramatically between different types of IBPs. In a study of ice shaping during growth and melting we have demonstrated a correlation between ice crystal shapes, the shaping process, and the affinity of IBPs for the basal plane (Bar-Dolev et al., J.R. Soc. Interface 2012). Our results point to a connection between the dynamics and level of activity of different types of IBP to their ability to bind to specific ice orientations, in particularly to the basal plane of the ice. These results contribute to an understanding of the mechanisms by which diverse IBPs act that will be critical for the successful use of IBP in cryobiological applications. Supported by ERC, NSF, ISF, and CIHR.

C-13: Application of hydrogel in cryopreservation as a novel cryoprotectant, encapsulation and single-cell-based functional sensing material

Some challenges still remain in cryopreservation and cytotherapy, including (1) cryoinjury due to intracellular ice formation may cause cell loss during cryopreservation. Non-freezing preservation may be achieved by vitrification with ultra-high cooling rate or cryoprotectant concentration; however, this is unfeasible or cytotoxic. (2) Immune rejection response may happen to the transplanted cell products. Microencapsulation of the transplanted cells has been used to immobilize xenograft cells in the last few decades. However, the progress in this field has not met expectations yet. The major problems include biocompatibility, possible function loss during encapsulation, mechanical and chemical stability of the encapsulation membranes, generation reproducibility of the uniform capsules, suitable membrane permeability that protects the implants from immune response of the host and simultaneously permits passage of nutrients and secretions. (3) A fast, simple and reliable quality control assay for the cells along the whole process is desirable. Especially, single-cell-based quality assessment has many advantages. In this work, application of hydrogel (application for rat islet as the example) was studied to address the three challenges listed above. A microfluidic device was applied to generate individual-cell based microencapsulation with uniformity and reproducibility. Meanwhile, biosensing agent was embedded in the hydrogel layer for single-cell-based biosensing, which can act as a simple quality control assay in cryopreservation and cytotherapy. It was found that glass transition of hydrogel may be achieved by tuning the crosslinking network density and water content. This implies that hydrogel can be potentially a novel cryoprotectant for non-freezing preservation purposes. In conclusion, hydrogel can serve as a cryoprotectant, immune barrier, and biosensing agent carrier. Further investigations are needed, especially the properties of hydrogel as cryoprotectant, which may lead to many striking and interesting applications in cryobiology.

064 The dynamic nature of antifreeze protein activity

Ice-binding proteins include antifreeze proteins (AFPs), ice nucleating proteins, ice adhesins, and ice recrystallization inhibition proteins. These proteins have great potential for controlling ice growth in various cryobiological and industrial applications. AFPs have been investigated for several decades. However, due to the unstable nature of ice crystals near their melting temperature, few experimental projects had addressed the kinetics of the AFP – ice interaction. When AFPs adsorb to ice they produce a thermal hysteresis (TH), which is the temperature gap between melting temperature of the ice in the solution of AFP, and the lowered temperature at which the crystal starts to grow. The basis for the relationship between TH and the concentration of AFP in solution is not known. We have used temperature-controlled microfluidic devices and fluorescently labeled AFPs to follow on the surface concentration of AFP on ice crystals. We find that the binding of the proteins to the ice surfaces is irreversible (”Superheating of ice crystals in antifreeze protein solutions” PNAS 2010 and ”Microfluidic experiments reveal that antifreeze proteins bound to ice crystals suffice to prevent their growth” PNAS 2013). Furthermore, we confirm that TH increases with the time that an ice crystal is expose to solutions that contain AFPs. We also find a direct connection between the surface concentrations of AFP and the measured TH. These experimental findings allow for testing different theoretical models regarding the mechanism of AFP’s ability to inhibit ice growth. Understanding the dynamic nature of AFP action will allow for rational design of their use in cryobiology applications. Source of funding: This research was funded by the Europe Research Council (ERC), the Israel Science Foundation (ISF), and the Canadian Institutes of Health Research (CIHR). Conflict of interest: None declared. ido.braslavsky@mail.huji.ac.il

046 Society for Cryobiology: Science, application and relevance of cryobiology today

Cryobiology was established necessarily as an interdisciplinary endeavor bringing together the biological and physical sciences to solve problems seemingly impossible to many lay observers. As Peter Mazur has described, difficulties in cryopreservation stem from a “complex concatenation of conflicting variables”. No single discipline is adequately equipped on its own to resolve the problems encountered in the field. As a result, many advances have been made based on fundamental understanding and application of cryobiology. Through manipulating variables such as the type and concentration of cryoprotectant, addition and removal methods, the use of controlled rate cooling combined with induction of ice nucleation and predictions of intracellular ice formation, these advances have culminated in the widespread use of cryobiology to facilitate research, agriculture and medicine. Indeed, application of the basic science of cryobiology readily lends itself to translation. Any cell or tissue of relevance benefits from the flexibility of shelf life that cryobiology can offer. This requires cryobiologists to interact with practitioners in many technical areas. The recently emerging field of regenerative medicine demonstrates such an example. The large scale manufacturing and banking of cells has been the backbone of this evolving discipline. The current technology for laboratory scale manufacturing and cryopreserved storage of cells has been convincingly successful; yet many important technical and medical issues remain. Many cell types of interest to cellular therapy still exhibit exquisite sensitivity to the thermal stresses they experience during the cryopreservation process, and existing approaches for preserving cells are labor-intensive and operator-dependent; consequently, as allogeneic “off-the-shelf” clinical transplantation or transfusion applications continue to be developed, large-scale manufacturing methods with appropriate quality assurance and quality control has become paramount. Despite its promise, however, the development of efficient and commercially viable processes for industrial scale cell manufacturing and banking is still in its infancy. And while manufacturing or processing of patient-specific cells may not have the same scale-up requirements that large allogeneic production requires, the same quality issues with respect to traceability, documentation, and the ultimate functional viability are just as imperative. The traditional understanding of cryopreservation damage is often focused on immediate post-thaw structural preservation, whereas cryopreservation-induced stress may increase over time, often resulting in the delayed onset of cell death. Since apoptotic cell death takes at least 24 h post-thaw to manifest the “true” or “functional” viability, the development of methods to evaluate this, and subsequently mitigate it are also needed. This is of particular importance where the cells may not succumb until hours or days post-transplant, resulting in seemingly acceptable post thaw results but yielding poor clinical efficacy. Other technical considerations involve the sample container, which must be scalable and should be at least a functionally closed system to meet safety and regulatory requirements. New strategies involving injectable cryoprotectants are also highly desirable for successful application of therapeutic cells. For cell based regenerative medicine to reach its potential in mainstream clinical applications, solutions based in sound scientific understanding of the biology and physical systems will need to continue through translation to ultimately solve these technical issues. Source of funding: None declared. Conflict of interest: None declared. Erik.Woods@CookGBT.com

31. Ice binding proteins and their dynamic interaction with ice

Numerous organisms have evolved specialized ice-binding proteins (IBPs) that act as antifreeze proteins (AFPs) to prevent their body fluids from freezing; as ice recrystallization inhibition (IRI) proteins to inhibit recrystallization of ice in frozen tissues; or as ice nucleating proteins (INPs) to initiate freezing at moderate supercooling temperatures. These proteins have many potential applications in agriculture, food preservation, cryobiology, and biomedical science. The ubiquitous presence of IBPs in such organisms indicates the power of these molecules to enable survival under freezing conditions. Despite this key role in nature, IBPs have been effectively exploited in only one cryopreservation application, namely, ice recrystallization inhibition in ice cream. Several terrestrial organisms, including insects, have developed very active forms of AFPs. These hyperactive AFPs (hypAFPs) have not been used significantly thus far in cryopreservation techniques. The gap between the evident potential of IBPs and their actual applications stems from a lack of understanding of the mechanisms by which IBPs interact with ice surfaces and how these proteins can assist in cryoprotection. We investigate the mechanism by which IBPs inhibit ice crystallization. We study the interactions between IBPs and ice using novel methods that we have developed, including fluorescence microscopy techniques combined with temperature controlled microfluidic devices. Here we report on the investigation of the dynamic nature of these interactions. For this study, the depression in temperature at which crystal growth occurs is compared to the melting point (thermal hysteresis gap measurement) as a function of the time that the proteins are allowed to accumulate on the ice surfaces. We found major differences in the interaction dynamic with ice between different types of AFPs. This finding contributes to the understanding of the mechanisms by which IBPs act. This understanding is a step toward the successful use of IBP in cryobiology applications.

13. Peripheral blood derived hematopoietic progenitor cells (HPC): An overview of a successful application of cryobiology

Hematopoietic progenitor cells (HPC) are primitive pluripotent cells capable of self renewal as well as of differentiation and maturation into all hematopoietic lineages. They can be found in bone marrow, in fetal liver, in umbilical cord blood, and in the mononuclear cell fraction of circulating blood. They are characterized by their colony-forming capacities in different in vitro cell culture assays and by the surface antigen marker CD34. Today, HPC are successfully cryopreserved using methods based on a dimethyl sulphoxide (Me2SO) method proposed by Ashwood-Smith (1961) for bone marrow. Cryopreserved peripheral HPC (in combination with patient high-dose chemotherapy or irradiation) have become a “standard” blood component for the treatment of several malignant diseases in more than 10,000 patients in Europe per year, e.g. lymphoma, myeloma, leukemia, and germ cell tumors. Nowadays HPC are usually obtained by apheresis from peripheral blood after stimulation with a cytokine named G-CSF (granulocyte colony stimulating factor). During cooling, heat is removed either by computer-controlled and liquid nitrogen (LN2) operated machines or in mechanical (−80 °C) refrigerators. Stiff et al. (1983) have demonstrated that the addition of 6% hydroxyethyl starch (HES) reduced the “original” concentration of Me2SO (10%) by half. A serious problem regarding frozen peripheral HPC is the danger of clotting after thawing. Although it is highly advisable to use as little anticoagulant – mostly ACD-A (acid–citrate–dextrose, formula A) or heparin – as possible during the apheresis procedure from the patient’s point of view, this may lead to problems during the following processing and after thawing. So if low anticoagulant concentrations have been used during the apheresis procedure, we recommend adding additional ACD-A to the final apheresis product. There are reports in the literature (e.g., Douay et al., 1982; Goldman et al., 1978) on Me2SO toxicity at room temperature. As a consequence of these and other reports, in clinical protocols diluted and precooled Me2SO solutions are usually added at 0–4 °C. In an investigation with highly pure Me2SO (pharmaceutical grade), however, no impairment (colony assay) within the first hour was found when 5% or 10% of Me2SO were added at either 4 or 37 °C (Rowley and Anderson, 1993). Cool-down kinetics depend on several parameters: geometry (“undefined,” cylindrical, or plate shaped), sample thickness, composition (type and concentration of cryoprotectants), and thermal properties (heat capacity, heat conductivity) of the freezing bag and (if used) the surrounding metal envelope. Therefore, temperature time-histories measured in small reference samples (e.g., tubes) may differ significantly from those measured in the corresponding product bags. With regard to cryoprotectant concentration and cooling rates, we did not find significant differences in cell recovery and clonogenicity (Sputtek et al., 1997), provided at least 5% Me2SO was present and cooling rates did not exceed 5 °C/min. Our most recent data supports the conclusion that a reliable −150 °C freezer is as suitable for long-term storage of HPC as is storage in the vapor phase over LN2. There was no systematic decline in viability measured in terms of six different parameters after more than 16 months storage in a −150 °C freezer (Sputtek et al. 2011). In our hands cell counts dropped significantly within 5 h after thawing compared to a high recovery when post thaw storage was performed at 20 °C and 0 °C, respectively. Therefore, we recommend to continue the common practice of thawing the frozen HPC units at the patient’s bedside.

83. A coupled heat and mass transport model with applications in cryobiology

83. A coupled heat and mass transport model with applications in cryobiology

Sperm storage for cancer patients in the UK: a review of current practice

An increasing number of cancer patients can now hope to have a full and normal life due to significant improvements in treatment outcomes and survival rates. The application of cryobiology to store fertile gametes before sterilizing treatments has been a natural progression. Greater awareness has markedly increased the worldwide demand for long-term storage of sperm, and has prompted the UK Human Fertilization and Embryology Authority to extend the period of storage permitted by their regulations to 55 years. Other patients undergoing sterilizing chemotherapy and/or radiotherapy such as haemoglobinopathies requiring bone marrow transplantation and autoimmune disorders such as rheumatoid arthritis may further increase the indications for sperm storage. Most adult and adolescent patients and their relatives/spouses/parents/guardians value this service even though very few eventually use the sperm. There is an urgent need to develop national and international guidelines for the provision, organization, maintenance and management of the cryopreservation services.

The application of cryobiology to the microbiological assay of nystatin.

Improvements in the reproducibility of nystatin agar diffusion assays have been achieved by the use of liquid nitrogen stored inocula and deep frozen standard stock solutions. The overall percentage variability of the assay has been reduced from over 5% with daily prepared standards and inocula to around 1% with a frozen inocula and to 0.6% with a combination of frozen inocula and standards. The implications of these improvements in the standardization of nystatin assays, and microbiological assays generally are discussed.

Cryoconservation of South African plant genetic diversity

South Africa has a rich flora which exhibits among the highest species density in the world, distributed across nine biomes that support an impressive diversity of animal life. However, a variety of human actions, invasion by alien species, natural disturbances and climate change collectively impact negatively on the great diversity of both plant and animal species. In situ conservation has long been practised, primarily in nature reserves, complemented by ex situ conservation in national botanic gardens, but in vitro plant conservation is not common. In the context of animal biodiversity conservation, the Wildlife Biological Resource Centre of the National Zoological Gardens utilises cryobanking as one of its major focuses and is now poised to expand as the repository for the cryoconservation of plant germplasm, particularly for indigenous recalcitrant-seeded and poor-seeding species. However, there are particular problems associated with successful germplasm cryostorage of such tropical and subtropical plants. As we see the science and application of cryobiology and cryoconservation as cross-cutting and transdisciplinary, we have entrained formal networking among scientists offering a range of specialisations aimed at a deeper understanding of common problems and practical outcomes to facilitate both plant and animal biobanking. The endeavours are aimed at elucidating the basis of both successes and failures in our efforts to attain optimal outcomes. With focus on best practices, standard operating procedures, validation and risk management for cryopreserved and cold-stored plant and animal material, our ultimate aim is to facilitate restoration by the safe reintroduction of indigenous species.

Review of biomaterial thermal property measurements in the cryogenic regime and their use for prediction of equilibrium and non-equilibrium freezing applications in cryobiology

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 °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 °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.