Conventional Cryoprotectants Research Articles

Adenosine monophosphate boosts the cryoprotection of ultrasound-assisted freezing to frozen surimi: Insights into protein structures and gelling behaviors

Ultrasound-assisted freezing (UAF) is a clean technique for meat cryoprotections; however, its effectiveness is still limited compared to conventional cryoprotectants, e.g., sugars, polyols, especially at high dosages. To resolve this problem, a synergistic cryoprotection strategy was developed in this study. Adenosine monophosphate (AMP), an adenosine-type food additive, was introduced into frozen surimi at a considerably reduced content (0.08%), yet substantially enhanced the efficiency of UAF to comparable levels of commercial cryoprotectant (4% sucrose with 4% sorbitol). Specifically, UAF/AMP treatment retarded denaturation of surimi myofibrillar protein (MP) during 60-day frozen storage, as evidenced by its increased solubility, Ca2+-ATPase activity, sulfhydryl content, declined surface hydrophobicity, particle size, and stabilized protein conformation. Gels of UAF/AMP-treated surimi also demonstrated more stabilized microstructures, uniform water distributions, enhanced mechanical properties and water-holding capacities. This study provided a feasible approach to boost the cryoprotective performance of UAF, thus expanding its potential applications in frozen food industry.

Improving vitrification efficiency of human in vitro matured oocytes by the addition of LEA proteins.

Can the addition of late embryogenesis-abundant (LEA) proteins as a cryoprotective agent during the vitrification cryopreservation of in vitro matured oocytes enhance their developmental potential after fertilization? LEA proteins improve the developmental potential of human in vitro matured oocytes following cryopreservation, mostly by downregulating FOS genes, reducing oxidative stress, and inhibiting the formation of ice crystals. Various factors in the vitrification process, including cryoprotectant toxicity, osmotic stress, and ice crystal formation during rewarming, can cause fatal damage to oocytes, thereby affecting the oocytes developmental potential and subsequent clinical outcomes. Recent studies have shown that LEA proteins possess high hydrophilicity and inherent stress tolerance, and can reduce low-temperature damage, although the molecular mechanism it exerts protective effects is still unclear. Two LEA proteins extracted and purified by us were added to solutions for vitrification-warming of oocytes at concentrations of 10, 100, and 200 µg/mL, to determine the optimal protective concentration for each protein. Individual oocyte samples were collected for transcriptomic analysis, with each group consisting of three sample replicates. Immature oocytes were collected from patients who were undergoing combined in vitro fertilization (IVF) treatment and who had met the designated inclusion and exclusion criteria. These oocytes underwent in vitro maturation (IVM) culture for experimental research. A fluorescence microscope was used to detect the levels of mitochondrial membrane potential (MMP), reactive oxygen species (ROS), and calcium in the mitochondria of vitrified-warmed human oocytes treated with different concentrations of LEA proteins, and the protective effect of the protein on mitochondrial function was assessed. The levels of intracellular ice recrystallization inhibition (IRI) in human oocytes after vitrification-warming were characterized by the cryomicroscope, to determine the LEA proteins inhibitory effect on recrystallization. By analyzing transcriptome sequencing data to investigate the potential mechanism through which LEA proteins exert their cryoprotective effects. The secondary structures of AfrLEA2 and AfrLEA3m proteins were shown to consist of a large number of α-helices and the proteins were shown to be highly hydrophilic, in agreement with previous reports. Confocal microscopy results showed that the immunofluorescence of AfrLEA2-FITC and AfrLEA3m-FITC-labeled proteins appeared to be extracellular and did not penetrate the cell membrane compared with the fluorescein isothiocyanate (FITC) control group, indicating that both AfrLEA2 and AfrLEA3m proteins were extracellular. The group treated with 100 µg/mL AfrLEA2 or AfrLEA3mprotein had more uniform cytoplasmic particles and fewer vacuoles compared to the 10 and 200 µg/mL groups and were closest to the fresh group. In the 100 µg/mL groups, MMPs were significantly higher while ROS and calcium levels were significantly lower than those in the control group and were closer to the levels observed in fresh oocytes. Meanwhile, 100 µg/mL of AfrLEA2 or AfrLEA3m protein caused smaller ice crystal formation in the IRI assay compared to the control group treated with dimethylsulphoxide (DMSO) and ethylene glycol (EG); thus, the recrystallization inhibition was superior to that with the conventional cryoprotectants DMSO and EG. Further results revealed that the proteins improved the developmental potential of human oocytes following cryopreservation, likely by downregulating FOS genes and reducing oxidative stress. The in vitro-matured metaphase II (IVM-MII) oocytes used in the study, due to ethical constraints, may not accurately reflect the condition of MII oocytes in general. The AfrLEA2 and AfrLEA3m proteins are recombinant proteins and their synthetic stability needs to be further explored. LEA proteins, as a non-toxic and effective cryoprotectant, can reduce the cryoinjury of oocytes during cryopreservation. It provides a new promising method for cryopreservation of various cell types. This work was supported by the National Key Research and Development Program of China (2022YFC2703000) and the National Natural Science Foundation of China (52206064). The authors declare no competing interest. N/A.

Dimethylglycine Can Enhance the Cryopreservation of Red Blood Cells by Reducing Ice Formation and Oxidative Damage

The cryopreservation of red blood cells (RBCs) holds great potential for ensuring timely blood transfusions and maintaining an adequate RBC inventory. The conventional cryoprotectants (CPAs) have a lot of limitations, and there is an obvious need for novel, efficient, and biocompatible CPAs. Here, it is shown for the first time that the addition of dimethylglycine (DMG) improved the thawed RBC recovery from 11.55 ± 1.40% to 72.15 ± 1.22%. We found that DMG could reduce the mechanical damage by inhibiting ice formation and recrystallization during cryopreservation. DMG can also scavenge reactive oxygen species (ROS) and maintain endogenous antioxidant enzyme activities to decrease oxidative damage during cryopreservation. Furthermore, the properties of thawed RBCs were found to be similar to the fresh RBCs in the control. Finally, the technique for order performance by similarity to ideal solution (TOPSIS) was used to compare the performance of glycerol (Gly), hydroxyethyl starch (HES), and DMG in cryopreservation, and DMG exhibited the best efficiency. This work confirms the use of DMG as a novel CPA for cryopreservation of RBCs and may promote clinical transfusion therapy.

Freeze Drying of Polymer Nanoparticles and Liposomes Exploiting Different Saccharide-Based Approaches.

Biodegradable nanocarriers represent promising tools for controlled drug delivery. However, one major drawback related to their use is the long-term stability, which is largely influenced by the presence of water in the formulations, so to solve this problem, freeze-drying with cryoprotectants has been proposed. In the present study, the influence of the freeze-drying procedure on the storage stability of poly(lactide-co-glycolide) (PLGA) nanoparticles and liposomes was evaluated. In particular, conventional cryoprotectants were added to PLGA nanoparticle and liposome formulations in various conditions. Additionally, hyaluronic acid (HA), known for its ability to target the CD44 receptor, was assessed as a cryoprotective excipient: it was added to the nanocarriers as either a free molecule or conjugated to a phospholipid to increase the interaction with the polymer or lipid matrix while exposing HA on the nanocarrier surface. The formulations were resuspended and characterized for size, polydispersity index, zeta potential and morphology. It was demonstrated that only the highest percentages of cryoprotectants allowed the resuspension of stable nanocarriers. Moreover, unlike free HA, HA-phospholipid conjugates were able to maintain the particle mean size after the reconstitution of lyophilized nanoparticles and liposomes. This study paves the way for the use of HA-phospholipids to achieve, at the same time, nanocarrier cryoprotection and active targeting.

Cryopreservation of tissues by slow-freezing using an emerging zwitterionic cryoprotectant

Cryopreservation of tissues is a tough challenge. Cryopreservation is categorized into slow-freezing and vitrification, and vitrification has recently been recognized as a suitable method for tissue cryopreservation. On the contrary, some researchers have reported that slow-freezing also has potential for tissue cryopreservation. Although conventional cryoprotectants have been studied well, some novel ones may efficiently cryopreserve tissues via slow-freezing. In this study, we used aqueous solutions of an emerging cryoprotectant, an artificial zwitterion supplemented with a conventional cryoprotectant, dimethyl sulfoxide (DMSO), for cell spheroids. The zwitterion/DMSO aqueous solutions produced a better cryoprotective effect on cell spheroids, which are the smallest units of tissues, compared to that of a commercial cryoprotectant. Cryopreservation with the zwitterion/DMSO solutions not only exhibited better cell recovery but also maintained the functions of the spheroids effectively. The optimized composition of the solution was 10 wt% zwitterion, 15 wt% DMSO, and 75 wt% water. The zwitterion/DMSO solution gave a higher number of living cells for the cryopreservation of mouse tumor tissues than a commercial cryoprotectant. The zwitterion/DMSO solution was also able to cryopreserve human tumor tissue, a patient-derived xenograft.

Proline-conditioning and chemically-programmed ice nucleation protects spheroids during cryopreservation.

Spheroids mimic 3-D tissue niches better than standard cell cultures. Cryopreserving spheroids, however, remains challenging as conventional cryoprotectants do not mitigate all damage mechanisms. Here chemically-programmed extracellular ice nucleation is used to prevent supercooling, alongside proline pre-conditioning, which are found to synergystically improve post-thaw recovery of spheroids. This validates the need to identify compounds and materials to address both biochemical and biophysical damage pathways beyond standard cryoprotectants.

Evaluation of chromosome organization and microtubule arrangement in goat (capra aegragrus) oocytes after vitrification, in vitro maturation and fertilization, and early embryo development

Objective: Evaluate the use of Ethylene Glycol (EG), Dimethyl Sulfoxide (DMSO), Sucrose and Fetal Bovine Serum (FBS) as cryoprotectants and their effect on the organization of chromosomes and the arrangement of microtubules, during the vitrification process in goat oocytes matured in vitro and in the development of preimplantation embryos produced in vitro.
 Design/methodology/approach: In vitro matured oocytes were divided into 3 groups (control group, cryoprotectant exposed group, vitrified group). A mixture of 15% EG, 15% DMSO, 0.4 M sucrose and 20% FBS was used for the vitrification using the Cryotop device. In vitro matured oocytes were warmed and afterwards each group was divided into two more groups. Both groups were subjected to immunofluorescence, the first group to observe the damage produced to the chromosomes and microtubules and the second group to observe the effect on the in vitro embryo development.
 Results: The combined use of 15% EG, 15% DMSO, 0.4 M Sucrose and 20% FBS during vitrification did not prevent cryoinjuries in goat oocytes and in vitro produced embryos, since embryo development was disrupted before the blastocyst stage by stopping cleavage at the morula stage. This disruption was associated with chromosome decondensation and the absence of a microtubule network, thereby hindering chromosomal segregation.
 Limitations on study/implications: The effect of conventional cryoprotectants on chromosomes and microtubules arrangement on vitrified goat oocytes and in vitro embryo production.
 Findings/conclusions: The combined use of 15% EG, 15% DMSO, 0.4 M sucrose and 20% FBS as vitrification cryoprotectants did not prevent cryoinjuries in caprine oocytes and did not improve caprine embryo development in vitro.

Influence of Konjac oligo-glucomannan as cryoprotectant on physicochemical and structural properties of silver carp surimi during fluctuated frozen storage

The aim of this study was to analyze the effect of Kanjoc oligo-glucomannan (KOG) on functional and structural changes in silver carp surimi during fluctuated (FLUC) frozen storage. During this study, KOG was prepared and added 1 and 3% in surimi and myofibrillar protein (MP) and compared with a positive control (PC) of sucrose and sorbitol mixture added samples. The results showed that the water holding capacity (WHC), textural and structural properties decreased during FLUC-I and FLUC-II, indicating that it could be due to protein denaturation induced by the formation of irregular crystallizations and recrystallization. Moreover, it was noted that the addition of KOG (3%) enhanced the stability of α-helix content (58–47%) during FLUC-I and (60–50%) during FLUC-II by inhibiting the hydrophobic residue exposure and change in aromatic amino acids. Meanwhile, KOG (3%) enhanced the textural properties by inhibiting the freeze induced protein denaturation. During FLUC frozen storage, the KOG (3%) was found to be a more effective concentration than conventional cryoprotectant mixture (PC). At industrial level, KOG (3%) could be used as a potential alternative to conventional cryoprotectants to attain prolonged commercial and economic values of silver carp.

Macromolecular cryoprotectants for the preservation of mammalian cell culture: lessons from crowding, overview and perspectives.

Cryopreservation is a process used for the storage of mammalian cells at a very low temperature, in a state of 'suspended animation.' Highly effective and safe macromolecular cryoprotectants (CPAs) have gained significant attention as they obviate the toxicity of conventional CPAs like dimethyl sulfoxide (DMSO) and reduce the risks involved in the storage of cultures at liquid nitrogen temperatures. These agents provide cryoprotection through multiple mechanisms, involving extracellular and intracellular macromolecular crowding, thereby impacting the biophysical and biochemical dynamics of the freezing medium and the cryopreserved cells. These CPAs vary in their structures and physicochemical properties, which influence their cryoprotective activities. Moreover, the introduction of polymeric crowders in the cryopreservation media enables serum-free storage at low-DMSO concentrations and high-temperature vitrification of frozen cultures (-80 °C). This review highlights the need for macromolecular CPAs and describes their mechanisms of cryopreservation, by elucidating the role of crowding effects. It also classifies the macromolecules based on their chemistry and their structure-activity relationships. Furthermore, this article provides perspectives on the factors that may influence the outcomes of the cell freezing process or may help in designing and evaluating prospective macromolecules. This manuscript also includes case studies about cellular investigations that have been conducted to demonstrate the cryoprotective potential of macromolecular CPAs. Ultimately, this review provides essential directives that will further improve the cell cryopreservation process and may encourage the use of macromolecular CPAs to fortify basic, applied, and translational research.

Role of Ovalbumin/β-Cyclodextrin in Improving Structural and Gelling Properties of Culter alburnus Myofibrillar Proteins during Frozen Storage

This study aimed to analyze the cryoprotective effect of a ovalbumin (OVA) and β-cyclodextrin (βCD) mixture (3:1, OVA/βCD) on the structure, rheology and gelling properties of myofibrillar proteins (MPs) during 90 days of frozen storage. A mixture of OVA/βCD at different concentrations (0, 2, 4, and 6%) was added to MPs and stored at −18 °C for 90 days. The addition of OVA/βCD significantly decreased the sulfhydryl contents while it increased the surface hydrophobicity, which was closely connected with tertiary structural changes. Circular dichroism analysis showed that the addition of OVA/βCD enhanced the stability of the secondary structure by inhibiting the decline in the α-helix. Rheological properties analysis indicated that 6% OVA/βCD treatment showed better storage modulus (G’) and loss modulus (G”). In addition, treatment of OVA/βCD showed better gel forming properties than the control group (0%), helping to form a homogeneous and denser gel network. The results proved that 6% OVA/βCD could be act as a promising cryoprotectant, which can improve the structure and gel behavior of Culter alburnus MPs during frozen storage. Moreover, OVA/βCD could be a potential alternative to conventional cryoprotectants at the industrial level to increase the economic and commercial values of seafood products.

The role of trifunctional cryoprotectants in the frozen storage of aquatic foods: Recent developments and future recommendations.

Freeze-induced changes including protein denaturation, ice crystals formation and lipid oxidation are mainly responsible for the quality deterioration persistent in aquatic foods. Here, for the first time, the cryoprotectants with trifunctional properties have been suggested for aquatic food cryopreservation and have exhibited exceptional cryoprotective abilities. In this study, in-depth discussion of protein denaturation, ice crystal formation and lipid oxidation is added in order to understand their mechanism, emphasizing on the necessity and use of trifunctional cryoprotectants in aquatic foods during frozen storage. Trifunctional cryoprotectants have strong abilities to prevent the formation of malondihaldehyde and aldehydes resulting from lipid oxidation, which further interact with proteins, subsequently lead to protein denaturation. Besides these all cryoprotective properties, ice crystal binding abilities distinguish trifunctional cryoprotectants from conventional cryoprotectants. Moreover, this study added with recent advances in cryoprotectants including antifreeze proteins and protein hydrolysates with their role in retarded freeze-induced changes. This study concluded that trifunctional cryoprotectants are effective owing to their hydrophilic amino acid chains, radical scavenging, water entrapping abilities, as well as the hydroxyl groups, which interact at the functional sites of protein molecules. Furthermore, polysaccharides and protein hydrolysates are the potential ingredients with trifunctional cryoproperties. However, more scientific research is required for material optimization to attain the desired level of cryoprotection.

Effect of Carboxymethyl Chitosan Magnetic Nanoparticles Plus Herring Antifreeze Protein on Conformation and Oxidation of Myofibrillar Protein From Red Sea Bream (Pagrosomus major) After Freeze-Thaw Treatment

The effects on the quality of frozen red sea bream, which were pretreated by soaking in solutions containing trehalose, carboxymethyl chitosan magnetic nanoparticles (CCMN), and glycerin or 0.1% herring antifreeze protein (hAFP), were investigated. In this study, the viscoelastic properties of protein studied with the dynamic rheology, Raman and intrinsic fluorescence spectrometry were used to explore the conformation changes of MFP. Surface hydrophobicity, particle size, and zeta potential were carried out to analyze protein aggregation. Solubility, the content of total sulfhydryl/disulfide bond/carbonyl/dityrosine, and the Ca2+-ATPase activity were measured to explore the degree of protein oxidation. SDS-PAGE was conducted to analyze the protein denaturation. Results showed that the pretreatment of red sea bream with soak solutions could improve the viscoelasticity of fillets protein, stabilize the secondary and tertiary conformation of the MFP, inhibit the protein aggregation and oxidation, and decrease the degree of protein denaturation compared with the control (4 °C thawing), especially the soak solutions containing hAFP could minimize the freeze-thaw damage. To summarize, the hAFP helped to retain the above characteristics of frozen fillets much better than that of conventional cryoprotectants (glycerin) to improve the quality of the frozen product after thawing.

The Effect of Helium on Cryopreservation of HeLa and L929 Cells

The effect of helium on cell survival during cryopreservation was studied using the HeLa and L929 cell lines. Cell suspensions were incubated in an atmosphere of helium, nitrogen, or air and frozen in the presence or absence of glycerol as a cryoprotectant. After thawing, the cell viability was evaluated by the Trypan Blue exclusion test and culture development for 18 h. Helium was found to provide better preservation of cell suspensions compared with nitrogen and air. The positive effect of helium was the greatest in the case of freezing without cryoprotectants (the HeLa cell survival increased by a factor of 1.5–2) and somewhat lower in the case of freezing in the presence of low glycerol concentrations (the L929 cell survival increased by a factor of 1.2–1.5 in the presence of 3% glycerol). Use of helium for cell suspensions may improve cryopreservation methods by making it possible to reduce the concentrations of conventional cryoprotectants, which are generally highly toxic and undesirable to use for cryopreservation of biological material for medical applications.

Bio-inspired solute enables preservation of human oocytes using minimum volume vitrification.

The ability to cryopreserve human oocytes has significant potential for fertility preservation. Current cryopreservation methods still suffer from the use of conventional cryoprotectants, such as dimethyl sulphoxide (DMSO), causing loss of viability and function. Such injuries result from the toxicity and high concentration of cryoprotectants, as well as mechanical damage of cells due to ice crystal formation during the cooling and rewarming processes. Here we report the preservation of human oocytes following vitrification using an innovative bio-inspired cryoprotectant integrated with a minimum volume vitrification approach. The results demonstrate that the recovered human oocytes maintained viability following vitrification and rewarming. Moreover, when this approach was used to vitrify mouse oocytes, the recovered oocytes preserved their viability and function following vitrification and rewarming. This bio-inspired approach substitutes DMSO, a well-known toxic cryoprotectant, with ectoine, a non-toxic naturally occurring solute. The bio-inspired vitrification approach has the potential to improve fertility preservation for women undergoing cancer treatment and endangered mammal species.

Small molecule ice recrystallization inhibitors mitigate red blood cell lysis during freezing, transient warming and thawing.

During cryopreservation, ice recrystallization is a major cause of cellular damage. Conventional cryoprotectants such as dimethyl sulfoxide (DMSO) and glycerol function by a number of different mechanisms but do not mitigate or control ice recrystallization at concentrations utilized in cryopreservation procedures. In North America, cryopreservation of human red blood cells (RBCs) utilizes high concentrations of glycerol. RBC units frozen under these conditions must be subjected to a time-consuming deglycerolization process after thawing in order to remove the glycerol to <1% prior to transfusion thus limiting the use of frozen RBC units in emergency situations. We have identified several low molecular mass ice recrystallization inhibitors (IRIs) that are effective cryoprotectants for human RBCs, resulting in 70–80% intact RBCs using only 15% glycerol and slow freezing rates. These compounds are capable of reducing the average ice crystal size of extracellular ice relative to a 15% glycerol control validating the positive correlation between a reduction in ice crystal size and increased post-thaw recovery of RBCs. The most potent IRI from this study is also capable of protecting frozen RBCs against the large temperature fluctuations associated with transient warming.

Effect of human sperm freezing-thawing process on vitrification and conventional freezing: evaluation of survival, motility, DNA oxidation and mitochondrial activity

Sperm Vitrification is a method for cryopreservation, without the use of conventional cryoprotectants, by plunging the sperm suspension directly into liquid nitrogen. We developed and evaluated a new method of vitrification, using a combination of 0,25 M trehalose-sucrose. This study aimed to compare the vitrification (V) with conventional freezing protocol (CF) on several sperm parameters: motility, vitality, mitocondryal activity and DNA oxidation.

20. The complexity of tissue and organ cryopreservation: A call for interdisciplinary research

The development of cryopreservation protocols for cell-based therapeutics have been based on established strategies to balance the “two-factors” of cryoinjury that influence cell recovery. By managing the competing effects of ice formation and solute composition changes on the cell through the use of carefully choreographed cryoprotectant addition/removal and cooling/thawing processes, successful protocols have been developed for many cell systems. Knowledge of cryoprotectant toxicity, ice formation and cell volume excursions, has allowed different cryopreservation strategies to be custom developed for the same cell system to meet the needs of the biobank or clinical program. The unique physical properties of tissues and organs have been shown to significantly affect the biological response to freezing and thawing. The diversity of cell types and cell densities as well as the morphological differences between constituent cells significantly affects the osmotic and thermal state of tissues and organs. This has dramatic implications on the cooling and thawing rates that can be attained and hence the response of the tissue to freezing and thawing. In addition, the requisite cell–cell and cell–matrix interactions in a tissue have been implicated in the poor survival of tissues following freezing. It is clear that the freezing response of tissues and organs, and the mechanism by which damage occurs are affected by the physical structures of these complex systems. In complex systems, ice crystallization becomes much more complicated and difficult to balance through the use of conventional cryoprotectants and cooling/thawing rates due to thermal and mass transfer limitations creating damaged cellular zones within the tissue or organ. Conventional biophysical approaches to modulate cell response to freezing and thawing may need to be supplemented with molecular strategies to directly modulate cell stress response. Vitrification, or the avoidance of ice crystallization, may address the direct effects of ice crystallization and solute concentrations, but challenges with cryoprotectant permeation and toxicity create new obstacles. Minimization of interstitial and extracellular ice in specific locations within tissues and organs may be more important and achievable than complete elimination. Developing tools to selectively control ice crystallization will be required. To advance the science of tissue and organ cryopreservation will require a concerted effort by a broad interdisciplinary field of scientists, engineers, and clinicians working to develop new strategies and tools. The ideas presented at this conference will hopefully stimulate not only the search for improved techniques for the cryopreservation of tissues and organs, but more importantly, a desire to clearly understand the response of biological systems to low temperatures.

37. Designing innovative procedures for avoiding cryoprotectant toxicity

Cryopreservation of complex tissues and organs will undoubtedly require the use of cryoprotectants (CPAs) to either mitigate damage caused by ice formation, or to prevent ice formation altogether. The latter approach, known as vitrification, has demonstrated particular promise for cryopreservation of large three-dimensional systems such as tissues and organs. To completely suppress ice formation, high CPA concentrations are required. However, it is well known that CPAs can be cytotoxic, especially at high concentrations. We have recently been exploring the potential for rational design of minimally toxic CPA addition and removal procedures. Our approach is based on minimization of a toxicity cost function, a mathematical model that allows calculation of the cumulative cytotoxicity during a candidate CPA addition or removal procedure using predictions of cell membrane transport. We have used this new approach to mathematically optimize CPA addition and removal procedures for isolated cells and cell monolayers, resulting in optimized procedures that are theoretically much less toxic than conventional procedures. The predicted benefit of the toxicity-minimized procedures is especially pronounced during CPA addition. The optimized CPA addition procedures call for exposure to a hypotonic CPA solution that causes the cells to swell to the maximum volume limit, and the majority of CPA loading is accomplished while the cells are in the swollen state. This swelling strategy significantly deviates from conventional CPA addition methods, which focus on avoiding excessive cell shrinkage. Swelling is advantageous because it allows a given amount of CPA to be loaded while minimizing the intracellular CPA concentration, and hence the toxicity. We recently tested this swelling strategy using experiments with cultured endothelial cells. Our results demonstrate that: (1) viability is significantly higher after addition and removal of 17 molal glycerol using a toxicity-minimized procedure (80%) in comparison to a traditional multistep procedure (5%); and (2) inducing swelling during CPA addition is required to obtain this improvement in viability. The potential for extending this mathematical optimization approach to more complex systems, including organs, will be discussed.

Peroxidation due to cryoprotectant treatment is a vital factor for cell survival in Arabidopsis cryopreservation

Cryopreservation can be a safe and cost-effective tool for the long-term storage of plant germplasm. In Arabidopsis, the ability to recover from cryogenic treatment was lost as growth progressed. Growth could be restored in 48-h seedlings, whereas 72-h seedlings died after cryogenic treatment. Why seedling age and survival are negatively correlated is an interesting issue. A comparative transcriptomics was performed to screen differentially expressed genes between 48- and 72-h seedlings after exposure to cryoprotectant. Among differentially expressed genes, oxidative stress response genes played important roles in cryoprotectant treatment, and peroxidation was a key factor related to cell survival. Seedlings underwent more peroxidation at 72-h than at 48-h. A comprehensive analysis indicated that peroxidation injured membrane systems leading to photophosphorylation and oxidative phosphorylation damage. Furthermore, the apoptosis-like events were found in cryogenic treatment of Arabidopsis seedlings. 48- and 72-h seedlings underwent different degrees of membrane lipid peroxidation during cryoprotectant treatment, and reducing the injury of oxidative stress was an important factor to successful cryopreservation. This study provided a novel insight of genetic regulatory mechanisms in cryopreservation, and established an excellent model to test and evaluate the effect of exogenous antioxidants and conventional cryoprotectants in plant cryopreservation.

High Osmolality Vitrification: A New Method for the Simple and Temperature-Permissive Cryopreservation of Mouse Embryos

Procedures for cryopreserving embryos vary considerably, each having its specific advantages and disadvantages in terms of technical feasibility, embryo survival yield, temperature permissibility and species- or strain-dependent applicability. Here we report a high osmolality vitrification (HOV) method that is advantageous in these respects. Cryopreservation by vitrification is generally very simple, but, unlike slow freezing, embryos should be kept at a supercooling temperature (below –130°C) to avoid cryodamage. We overcame this problem by using an HOV solution containing 42.5% (v/v) ethylene glycol, 17.3% (w/v) Ficoll and 1.0 M sucrose. This solution is more viscous than other cryopreservation solutions, but easy handling of embryos was assured by employing a less viscous equilibration solution before vitrification. Most (>80%) embryos cryopreserved in this solution survived at –80°C for at least 30 days. Normal mice were recovered even after intercontinental transportation in a conventional dry-ice package for 2–3 days, indicating that special containers such as dry shippers with liquid nitrogen vapor are unnecessary. The HOV solution could also be employed for long-term storage in liquid nitrogen, as with other conventional cryoprotectants. Finally, we confirmed that this new vitrification method could be applied successfully to embryos of all six strains of mice we have tested so far. Thus, our HOV method provides an efficient and reliable strategy for the routine cryopreservation of mouse embryos in animal facilities and biomedical laboratories, and for easy and cheap transportation.