Cryoprotective Agents Solutions Research Articles

Monitoring of freezing patterns within 3D collagen-hydroxyapatite scaffolds using infrared thermography

The importance of cryopreservation in tissue engineering is unceasingly increasing. Preparation, cryopreservation, and storage of tissue-engineered constructs (TECs) at an on-site location offer a convenient way for their clinical application and commercialization. Partial freezing initiated at high sub-zero temperatures using ice-nucleating agents (INAs) has recently been applied in organ cryopreservation. It is anticipated that this freezing technique may be efficient for the preservation of both scaffold mechanical properties and cell viability of TECs. Infrared thermography is an instrumental method to monitor INAs-mediated freezing of various biological entities.In this paper, porous collagen-hydroxyapatite (collagen-HAP) scaffolds were fabricated and characterized as model TECs, whereas infrared thermography was proposed as a method for monitoring the crystallization-related events on their partial freezing down to −25 °C. Intra- and interscaffold latent heat transmission were descriptively evaluated. Nucleation, freezing points as well as the degree of supercooling and duration of crystallization were calculated based on inspection of respective thermographic curves. Special consideration was given to the cryoprotective agent (CPA) composition (Snomax®, crude leaf homogenate (CLH) from Hippophae rhamnoides, dimethyl sulfoxide (Me2SO) and recombinant type-III antifreeze protein (AFP)) and freezing conditions (‘in air’ or ‘in bulk CPA’).For CPAs without ice nucleation activity, thermographic measurements demonstrated that the supercooling was significantly milder in the case of scaffolds present in a CPA solution compared to that without them. This parameter (ΔT, °C) altered with the following tendency: 10 Me2SO (2.90 ± 0.54 (‘scaffold in a bulk CPA’) vs. 7.71 ± 0.43 (‘bulk CPA’, P < 0.0001)) and recombinant type-III AFP, 0.5 mg/ml (2.65 ± 0.59 (‘scaffold in a bulk CPA’) vs. 7.68 ± 0.34 (‘bulk CPA’, P < 0.0001)).At the same time, in CPA solutions with ice nucleation activity the least degree of supercooling and the longest crystallization duration (Δt, min) for scaffolds frozen ‘in air’ were documented for CLH from Hippophae rhamnoides (1.57 ± 0.37 °C and 21.86 ± 2.93 min) compared to Snomax, 5 μg/ml (2.14 ± 0.33 °C and 19.91 ± 4.72 min), respectively).Moreover, when frozen ‘in air’ in CLH from Hippophae rhamnoides, collagen-HAP scaffolds were shown to have the longest ice-liquid equilibrium phase during crystallization and the lowest degree of supercooling followed by alginate core-shell capsules and nanofibrous electrospun fiber mats made of poly ɛ-caprolactone (PCL) and polylactic acid (PLA) (PCL/PLA) blend.The paper offers evidence that infrared thermography provides insightful information for monitoring partial freezing events in TECs when using different freezing containers, CPAs and conditions. This may further TEC-specific cryopreservation with enhanced batch homogeneity and optimization of CPA compositions of natural origin active at warm sub-zero temperatures.

Infrared spectroscopic analysis of hydrogen-bonding interactions in cryopreservation solutions

BackgroundIn this study we investigated hydrogen bonding interactions in hydrated and frozen solutions of different cryoprotective agents (CPAs) including dimethyl sulfoxide, glycerol, ethylene glycol, propylene glycol, and trehalose. We also investigated the effect of CPAs on ice crystal growth during storage and correlated this with storage stability of liposomes. MethodsFTIR spectroscopy was used to study hydrogen bonding interactions in CPA solutions in H2O and D2O, and their thermal response was analyzed using van ’t Hoff analysis. The effect of CPAs on ice crystal growth during storage was investigated by microscopy and correlated with storage stability of liposomes encapsulated with a fluorescent dye. ResultsPrincipal component analyses demonstrated that different CPAs can be recognized based on the shape of the OD band region only. Chemically similar molecules such as glycerol and ethylene glycol closely group together in a principal component score plot, whereas trehalose and DMSO appear as condensed separated clusters. The OH/OD band of CPA solutions exhibits an overall shift to higher wavenumbers with increasing temperature and changed fractions of weak and strong hydrogen interactions. CPAs diminish ice crystal formation in frozen samples during storage and minimize liposome leakage during freezing but cannot prevent leakage during frozen storage. ConclusionsCPAs can be distinguished from one another based on the hydrogen bonding network that is formed in solution. DMSO-water mixtures behave anomalous compared to other CPAs that have OH groups. CPAs modulate ice crystal formation during frozen storage but cannot prevent liposome leakage during frozen storage.

Photothermal heating of titanium nitride nanomaterials for fast and uniform laser warming of cryopreserved biomaterials.

Titanium nitride (TiN) is presented as an alternative plasmonic nanomaterial to the commonly used gold (Au) for its potential use in laser rewarming of cryopreserved biomaterials. The rewarming of vitrified, glass like state, cryopreserved biomaterials is a delicate process as potential ice formation leads to mechanical stress and cracking on a macroscale, and damage to cell walls and DNA on a microscale, ultimately leading to the destruction of the biomaterial. The use of plasmonic nanomaterials dispersed in cryoprotective agent solutions to rapidly convert optical radiation into heat, generally supplied by a focused laser beam, proposes a novel approach to overcome this difficulty. This study focuses on the performance of TiN nanoparticles (NPs), since they present high thermal stability and are inexpensive compared to Au. To uniformly warm up the nanomaterial solutions, a beam splitting laser system was developed to heat samples from multiple sides with equal beam energy distribution. In addition, uniform laser warming requires equal distribution of absorption and scattering properties in the nanomaterials. Preliminary results demonstrated higher absorption but less scattering in TiN NPs than Au nanorods (GNRs). This led to the development of TiN clusters, synthetized by nanoparticle agglomeration, to increase the scattering cross-section of the material. Overall, this study analyzed the heating rate, thermal efficiency, and heating uniformity of TiN NPs and clusters in comparison to GNRs at different solution concentrations. TiN NPs and clusters demonstrated higher heating rates and solution temperatures, while only clusters led to a significantly improved uniformity in heating. These results highlight a promising alternative plasmonic nanomaterial to rewarm cryopreserved biological systems in the future.

Effectiveness of Clinical-Grade Chondroitin Sulfate and Ascorbic Acid in Mitigating Cryoprotectant Toxicity in Porcine Articular Cartilage

High concentrations of cryoprotective agents (CPAs) are required to achieve successful vitrification of articular cartilage; however, CPA cytotoxicity causes chondrocyte death. To reduce CPA toxicity, supplementation with research-grade additives, in particular chondroitin sulfate (CS) and ascorbic acid (AA), have previously been shown to improve chondrocyte recovery and metabolic function after exposure to CPAs at hypothermic conditions. However, it is necessary to evaluate the pharmaceutical equivalent clinical grade of these additives to facilitate the supplementation of additives into future vitrification protocols, which will be designed for vitrifying human articular cartilage in tissue banks. We sought to investigate the effectiveness of clinical-grade CS, AA, and N-acetylcysteine (NAC) in mitigating toxicity to chondrocytes during CPA exposure and removal, and determine whether a combination of two additives would further improve chondrocyte viability. We hypothesized that clinical-grade additives would exert chondroprotective effects comparable to those of research-grade additives, and that this protective effect would be enhanced if two additives were combined when compared with a single additive. The results indicated that both clinical-grade and research-grade additives significantly improved cell viability (p < 0.10) compared with the negative control (CPA with no additives). CS, AA, and NAC+AA increased cell viability significantly (p < 0.10) compared with the negative control. However, NAC, NAC+CS, and CS+AA did not improve cell viability when compared with the negative control (p > 0.10). We demonstrated that supplementation with clinical-grade CS or AA significantly improved chondrocyte viability in porcine cartilage subjected to high CPA concentrations, whereas supplementation with clinical-grade NAC did not benefit chondrocyte viability. Supplementation with clinical-grade additives in CPA solutions can mitigate CPA toxicity, which will be important in translating previously developed effective protocols for the vitrification of articular cartilage to human tissue banks.

Loading equine oocytes with cryoprotective agents captured with a finite element method model

Cryopreservation can be used to store equine oocytes for extended periods so that they can be used in artificial reproduction technologies at a desired time point. It requires use of cryoprotective agents (CPAs) to protect the oocytes against freezing injury. The intracellular introduction of CPAs, however, may cause irreversible osmotic damage. The response of cells exposed to CPA solutions is governed by the permeability of the cellular membrane towards water and the CPAs. In this study, a mathematical mass transport model describing the permeation of water and CPAs across an oocyte membrane was used to simulate oocyte volume responses and concomitant intracellular CPA concentrations during the exposure of oocytes to CPA solutions. The results of the analytical simulations were subsequently used to develop a phenomenological finite element method (FEM) continuum model to capture the response of oocytes exposed to CPA solutions with spatial information. FEM simulations were used to depict spatial differences in CPA concentration during CPA permeation, namely at locations near the membrane surface and towards the middle of the cell, and to capture corresponding changes in deformation and hydrostatic pressure. FEM simulations of the multiple processes occurring during CPA loading of oocytes are a valuable tool to increase our understanding of the mechanisms underlying cryopreservation outcome.

Cryopreservation of Canine Erythrocytes Using Dimethyl Sulfoxide, Polyethylene Glycol and Sucrose

Cryoprotective properties of combined media of permeable (dimethyl sulfoxide) and impermeable (polyethylene glycol with m. w. 1500) cryoprotective agents during rapid cooling in liquid nitrogen of canine erythrocytes using saline and sucrose-saline media have been investigated. It was found that the use of combined solutions of cryoprotective agents based on polyethylene glycol with m.w. 1500 (15%) and dimethyl sulfoxide (2.5–10%) in saline was not quite effective for cryopreservation of canine erythrocytes. Reducing the salt concentration and adding cell-impermeable sucrose to the cryopreservation medium increase the preservation of erythrocytes after warming. The best cryoprotective properties for canine erythrocytes were demonstrated by 10% dimethyl sulfoxide based on sucrose-saline medium, with high preservation of cells after freeze-warming, mechanical and osmotic stability of warmed erythrocytes. This indicates the possibility of a long-term storage and use of cryopreserved canine erythrocytes for transfusions.

Transport processes in equine oocytes and ovarian tissue during loading with cryoprotective solutions

BackgroundRational design of cryopreservation strategies for oocytes and ovarian cortex tissue requires insights in the rate at which cryoprotective agents (CPA) permeate and concomitant water transport takes place. The aim of the current study was to investigate possible differences in permeation kinetics of different CPAs (i.e., glycerol/GLY, ethylene glycol/EG, dimethyl sulfoxide/DMSO, and propylene glycol/PG), in equine oocytes as well as ovarian tissue. MethodsMembrane permeability of oocytes to water (Lp) and to CPAs (Ps) was inferred from video microscopic imaging of oocyte volume responses during perfusion with anisotonic and CPA solutions. CPA diffusion into ovarian tissue and tissue dehydration was monitored during incubation, using osmometer and weight measurements, to derive CPA diffusion coefficients (D). ResultsMembrane permeability of oocytes towards CPAs was found to increase in the order GLY < EG < DMSO<PG. Permeability towards water in anisotonic solutions was determined to be higher than in CPA solutions, indicating CPAs alter membrane permeability properties. CPA diffusion in ovarian tissue increased in the order GLY,PG < EG,DMSO. Tissue dehydration was found to increase with exposure to increasing CPA concentrations, which inversely correlated with CPA diffusivity. ConclusionsIn conclusion, it is shown here that the rate of CPA movement across membrane bilayers is determined by different physical barrier factors than those determining CPA movement in tissues. General significanceThe parameters presented in this study can be applied in models describing solute and water transport in cells and tissues, as well as in cryopreservation protocols.

Natural deep eutectic systems for nature‐inspired cryopreservation of cells

AbstractNatural deep eutectic systems (NADES) are emerging as potential cryoprotective agents (CPA) for cell preservation. In this investigation, we develop an optimized CPA formulation using trehalose‐glycerol NADES (T:G) diluted in Normosol‐R and supplemented with isoleucine. Differential scanning calorimetry (DSC) is used to define the thermophysical properties of NADES‐based solutions, and Raman spectroscopy is used to characterize the effect of NADES on ice formation and hydrogen bonding. Jurkat cells are cryopreserved in each solution, and post‐thaw cell recovery, apoptosis, and growth are quantified. Raman spectra and heat maps show that NADES suppresses both ice formation and dehydration of the nonfrozen region. Supplementing NADES with isoleucine does not affect the solution's thermophysical properties but significantly improves the cells' survival and proliferation post‐thaw. The study indicates that thermophysical properties of CPA solutions alone cannot predict optimal cell survival, suggesting that stabilization of biological structures by CPAs may play a role in successful cryopreservation.

Antioxidant additives reduce reactive oxygen species production in articular cartilage during exposure to cryoprotective agents

High concentrations of cryoprotective agents (CPA) are required during articular cartilage cryopreservation but these CPAs can be toxic to chondrocytes. Reactive oxygen species have been linked to cell death due to oxidative stress. Addition of antioxidants has shown beneficial effects on chondrocyte survival and functions after cryopreservation. The objectives of this study were to investigate (1) oxidative stress experienced by chondrocytes and (2) the effect of antioxidants on cellular reactive oxygen species production during articular cartilage exposure to high concentrations of CPAs. Porcine cartilage dowels were exposed to a multi-CPA solution supplemented with either 0.1 mg/mL chondroitin sulfate or 2000 μM ascorbic acid, at 4 °C for 180 min (N = 7). Reactive oxygen species production was measured with 5 μM dihydroethidium, a fluorescent probe that targets reactive oxygen species. The cell viability was quantified with a dual cell membrane integrity stain containing 6.25 μM Syto 13 + 9 μM propidium iodide using confocal microscopy. Supplementation of CPA solutions with chondroitin sulfate or ascorbic acid resulted in significantly lower dihydroethidium counts (p < 0.01), and a lower decrease in the percentage of viable cells (p < 0.01) compared to the CPA-treated group without additives. These results indicated that reactive oxygen species production is induced when articular cartilage is exposed to high CPA concentrations, and correlated with the amount of dead cells. Both chondroitin sulfate and ascorbic acid treatments significantly reduced reactive oxygen species production and improved chondrocyte viability when articular cartilage was exposed to high concentrations of CPAs.

Effect of Type I Antifreeze Proteins on the Freezing and Melting Processes of Cryoprotective Solutions Studied by Site-Directed Spin Labeling Technique.

Antifreeze proteins (AFPs) protect organisms living in subzero environments from freezing injury, which render them potential applications for cryopreservation of living cells, organs, and tissues. Cryoprotective agents (CPAs), such as glycerol and propylene glycol, have been used as ingredients to treat cellular tissues and organs to prevent ice crystal’s formation at low temperatures. To assess AFP’s function in CPA solutions, we have the applied site-directed spin labeling technique to a Type I AFP. A two-step process to prevent bulk freezing of the CPA solutions was observed by the cryo-photo microscopy, i.e., (1) thermodynamic freezing point depression by the CPAs; and (2) inhibition to the growth of seed ice crystals by the AFP. Electron paramagnetic resonance (EPR) experiments were also carried out from room temperature to 97 K, and vice versa. The EPR results indicate that the spin labeled AFP bound to ice surfaces, and inhibit the growths of ice through the bulk freezing processes in the CPA solutions. The ice-surface bound AFP in the frozen matrices could also prevent the formation of large ice crystals during the melting processes of the solutions. Our study illustrates that AFPs can play an active role in CPA solutions for cryopreservation applications.

Determination of Dielectric Properties of Cryoprotective Agent Solutions with a Resonant Cavity for the Electromagnetic Rewarming in Cryopreservation.

In the rewarming process during cryopreservation, preventing ice recrystallization and thermal stress is important, especially for large tissues and organs. Uniform and rapid heating is essential in ameliorating the problem and maintaining the viability of cryopreserved biological samples. Currently, the most promising method is heating by application of electromagnetic (EM) waves, the effectiveness of which is dependent on the dielectric properties (DP) of the cryopreserved materials. In this work, the cavity perturbation method was adopted to measure the DP of cryoprotectant solutions. Based on the values of DP, the cryoprotectant solutions most amenable to EM heating can be identified. A system composed of a rectangular resonant cavity, a network analyzer, and a fiber optic temperature meter was implemented for the measurement. The DP of three cryoprotectant solutions during cooling to -80°C were measured and presented. The data can be used to optimize the rewarming process with the numerical method. The results show that a cryoprotectant solution consisting of 41% (w/v) dimethyl sulfoxide and 6% (w/v) polyvinylpyrrolidone has the highest dielectric loss for EM rewarming among the tested solutions. In addition, the developed DP measurement system could not only improve the EM heating in cryopreservation but also benefit hyperthermia or other therapies associated with EM waves.

Formation of ice microparticles in cryoprotective solutions

The formation of ice microparticles in 0.2-mm thin layers of aqueous solutions of cryoprotective agents cooled to temperatures reaching–196°C was studied. The shape and size of ice microparticles were determined by cryomicroscopy. At temperatures below 0°C, the formation of solid ice masses was observed; further cooling caused ice fracturing induced by emerging thermomechanical stresses and the formation of microparticles. The shape and size of the particles depended on the composition of the frozen solution and on the rate of cooling. The components of cryoprotective solutions (cryoprotectants, egg yolk, sugars, and lipids) significantly changed the shape and size of the ice microparticles.

Measurement of Thermal Conductivities of Two Cryoprotective Agent Solutions for Vitreous Cryopreservation of Organs at the Temperature Range of 77 K-300 K Using a Thermal Sensor Made of Microscale Enamel Copper Wire.

Biobanking of organs by cryopreservation is an enabling technology for organ transplantation. Compared with the conventional slow freezing method, vitreous cryopreservation has been regarded to be a more promising approach for long-term storage of organs. The major challenges to vitrification are devitrification and recrystallization during the warming process, and high concentrations of cryoprotective agents (CPAs) induced metabolic and osmotic injuries. For a theoretical model based optimization of vitrification, thermal properties of CPA solutions are indispensable. In this study, the thermal conductivities of M22 and vitrification solution containing ethylene glycol and dimethyl sulfoxide (two commonly used vitrification solutions) were measured using a self-made microscaled hot probe with enameled copper wire at the temperature range of 77 K-300 K. The data obtained by this study will further enrich knowledge of the thermal properties for CPA solutions at low temperatures, as is of primary importance for optimization of vitrification.

Effect of trehalose as an additive to dimethyl sulfoxide solutions on ice formation, cellular viability, and metabolism

Cryopreservation is the only established method for long-term preservation of cells and cellular material. This technique involves preservation of cells and cellular components in the presence of cryoprotective agents (CPAs) at liquid nitrogen temperatures (−196 °C). The organic solvent dimethyl sulfoxide (Me2SO) is one of the most commonly utilized CPAs and has been used with various levels of success depending on the type of cells. In recent years, to improve cryogenic outcomes, the non-reducing disaccharide trehalose has been used as an additive to Me2SO-based freezing solutions. Trehalose is a naturally occurring non-toxic compound found in bacteria, fungi, plants, and invertebrates which has been shown to provide cellular protection during water-limited states. The mechanism by which trehalose improves cryopreservation outcomes remains not fully understood. Raman microspectroscopy is a powerful tool to provide valuable insight into the nature of interactions among water, trehalose, and Me2SO during cryopreservation. We found that the addition of trehalose to Me2SO based CPA solutions dramatically reduces the area per ice crystals while increasing the number of ice crystals formed when cooled to −40 or −80 °C. Differences in ice-formation patterns were found to have a direct impact on cellular viability. Despite the osmotic stress caused by addition of 100 mM trehalose, improvement in cellular viability was observed. However, the substantial increase in osmotic pressure caused by trehalose concentrations above 100 mM may offset the beneficial effects of changing the morphology of the ice crystals achieved by addition of this sugar.

Ethylene glycol and glycerol loading and unloading in porcine meniscal tissue

The development of a long-term storage method for meniscus, a complex tissue of the knee prone to injury, would improve the procedure and outcomes of meniscus transplantation. Cryopreservation uses cryoprotective agents (CPAs) including ethylene glycol (EG) and glycerol to preserve a variety of live tissues, and understanding of the CPA permeation kinetics will be critical in designing a vitrification protocol for meniscus.The purpose of this preliminary study was to understand the loading and unloading behaviours of EG and glycerol in meniscus by observing their efflux. For the main experiment, lateral and medial porcine menisci were incubated with CPA for 24 h at three temperatures (i.e., 4, 22, and 37 °C). Then, the menisci were immersed in 25 ml of X-VIVO™10 and CPA efflux was recorded by monitoring the molality of two consecutive washout solutions at different time points. In a subsequent experiment, menisci were incubated in the CPA solutions for 48 h at 22 °C, and the results were compared to those obtained at 22 °C in the main experiment.Results showed a rapid efflux of CPA from meniscus at the beginning of each wash. With increasing temperature, the amount of CPA efflux (and hence loading) increased. Using 24 h incubation, EG loaded the menisci more completely than glycerol. But after 48 h of incubation, both EG and glycerol achieved approximately the same degree of meniscus loading.This study provides preliminary data that will facilitate future design of experiments aimed at development of meniscus permeation studies.

57. A transmembrane mega highway for cryoprotective agent delivery via self-assembling organic nanopores with tunable function

Cryoprotective agents (CPAs) are critical additives that improve the post-thaw viability of cryopreserved biological systems by preventing ice crystal nucleation and growth. Membrane permeable CPAs also prevent osmotic shrinkage of the cells and reduce the volume of available water by penetrating and equilibrating across the cell membrane. All known CPAs exhibit various levels of cytotoxicity at their effective concentration which may be decreased by reducing the CPA loading temperature and exposure time. However, most CPAs become effectively impermeable at sub-zero temperatures. This is largely associated with the dysfunction of transmembrane protein channels at low temperature that serve as mass transport gates. An unbalanced gradient of CPA and salt across cell membrane leads to detrimental cell shrinkage as water efflux out of the cell. This could be detrimental to the fate of large, complex solid tissues/organs by damaging cell–cell and cell–matrix junctions. To facilitate the intracellular delivery and transmembrane equilibration of the CPA at sub-zero temperatures, we propose size and function tunable organic nanopores as a “mega highway” to deliver CPAs across cell membrane which allows a significant decrease in both the CPA exposure time and temperature during freezing. This method may be particularly effective when CPA loading in tissues using the “liquidus tracking” or step-wise methods where increasingly concentrated solutions of CPA are loaded in the tissue/organ at progressively decreasing temperatures. Recent work in Gong’s group has demonstrated the reliability of constructing well-defined nanotubular assemblies via the enforced stacking of shape-persistent macrocycles based on the interplay of multiple hydrogen-bonding, dipole-dipole, and aromatic π - π stacking interactions. The resultant nanotubes have modifiable surfaces and non-collapsible inner pores of a uniform diameter defined by the constituent macrocycles. The robust self-assembling nanopores have been found to act as transmembrane channels which can mediate highly selective transmembrane ion transport with high stability, unprecedented for synthetic ion channels, as well as exhibiting highly efficient transmembrane water permeability, which is comparable to aquaporin. Aquaporins have been found to efficiently assist transport of water and glycerol during freezing to prevent cell rupture under osmotic pressure. In this proposal, selected organic nanopores are delivered into a target system at physiological temperature (37 °C), followed by CPA loading at hypothermic temperature (e.g. 1 °C). A high influx rate of CPAs through shape-persistent nanopores can be maintained during cooling as a function of the concentration gradient across cell membrane, thereby reducing the required time to reach vitrification concentrations. Upon rewarming, these organic nanopores will facilitate rapid removal of CPAs to reduce exposure time and the consequent toxic side effects. The proposed incorporation of synthetic nanopores can significantly reduce toxicity and cell injury due to osmotic shrinkage caused by CPAs and salt during both the cooling and rewarming processes via (1) reducing CPA exposure time and (2) enabling rapid CPA loading and unloading at lower temperatures. Moreover, versatile functional organic nanotubes of diverse sizes and properties by modifying inner macrocyclic cavities allow selective CPA transport by while preventing ion exchange. Incorporation of functional supramolecular assemblies to enhance membrane permeability of CPAs could lead to a revolutionary solution to cryopreserve complex biological systems.

Theoretical investigation of a novel microwave antenna aided cryovial for rapid and uniform rewarming of frozen cryoprotective agent solutions

The most challenging issue in cryopreservation of mass biomaterials is to rewarm the frozen sample in a fast and uniform manner, so that the dangerous devitrification and recrystallization may be avoided. In this study, a conceptual innovation is the design of a novel cryovial, and we investigate the effects of the microwave heating after embedding superparamagnetic nanoparticles on the rewarming processes of the cell suspensions encapsulated in the cryovial. The electromagnetic field and the heat transfer during the hybrid rewarming processes of frozen EC2 solution with temperature-dependent properties were calculated. During the rewarming process of the sample in the cryovial in a traditional 37 °C water bath, the rewarming rate was 72.15 °C/min and the maximum temperature gradient in the sample was 20.5 °C/mm. After a slot antenna was included in the cryovial, the rewarming rate was 83.71 °C/min without nanoparticles and 106.41 °C/min after nanoparticles are embedded, the maximum temperature gradient in the sample was 40.2 °C/mm without nanoparticles and 28.7 °C/mm after nanoparticles embedded, respectively. This indicates that the rewarming rate and the uniformity of the temperature distribution increased after embedding nanoparticles. This could be because nanoparticles homogeneously generate heat in the sample and improve the time-dependent parameters of the sample.

C-2022: Monitoring of the residual cryoprotectant concentration with electrical conductivity measurements during cryoprotectant removal

Cryoprotective agents (CPA) should be added to cell suspension before freezing and removed after thawing. Nowadays people mainly apply centrifugation for CPA removal. This method may cause many problems. Meanwhile, a fast, easy and cheap method to monitor the residual CPA concentration remains an unfilled need. In this work, real-time monitoring of residual CPA concentration with electrical conductivity (EC) measurements was proposed and tested. It was found that CPA concentration is correlated with the EC of the solution. The standard data of “CPA concentration—EC of the solution” for a few widely used CPA solutions were obtained. A “dilution–filtration” system based on a hollow fiber dialyzer, three peristaltic pumps and some well-designed tubing connections was applied to remove CPA. Me2SO solutions (Me2SO–NaCl–H2O solutions with or without 5% BSA) were selected as representative examples. The results showed that CPA can be removed effectively by the “dilution–filtration” system. The ECs of waste solution (filtration product) and “cell suspension” were almost identical after a short period of processing, which were validated by osmolality measurements with an osmometer. EC readings of waste solution can convey the instant residual CPA concentrations in cell suspension. This validates the feasibility of a safe (no contamination), easy and cheap way to real-time monitoring of CPA concentration in cell suspension by measuring the EC of the waste solution. Compared to other CPA removal methods that are applied traditionally (centrifugation) or proposed recently (microfluidic device), the “dilution–filtration” method may offer advantages of low time and labor consumption, low injury to the cells, high effectiveness, ease of controlling the final suspension volume and low risk of contamination. The configuration of the “dilution–filtration” system can also be modified easily to achieve CPA addition and volume control of cell suspension (concentration or dilution of the cell suspension).

Tolerance of apexes of coral Pocillopora damicornis L. to cryoprotectant solutions

In this study, we investigated the tolerance of Pocillopora damicornis apexes to treatments with solutions containing penetrating and non-penetrating cryoprotective agents (CPAs). CPAs were employed individually or in binary, tertiary or quaternary solutions. In some experiments apexes were treated successively with two CPA solutions with increasing total concentration. P. damicornis apexes withstood exposure for up to 30min to solutions containing 0.6–0.8M sucrose (Suc) or trehalose (Tre). When apexes were treated with binary cryoprotectant solutions containing Suc and ethylene glycol (EG), methanol (Meth), dimethyl sulfoxide (Me2SO) or glycerol (Gly), the CPAs employed in combination with Suc could be ranked in the following order of decreasing tolerance: EG>Meth>Me2SO>Gly. P. damicornis apexes tolerated exposure to complex CPA solutions containing Suc, Me2SO, EG and/or Meth with a total molarity of 2.45M. In experiments where two successive CPA solutions were employed, apexes withstood treatment with the second, more concentrated solution at 0°C for up to 10min. These preliminary results pave the way to the development of a cryopreservation protocol for P. damicornis apexes.

Optimal vitrification protocol for mouse ovarian tissue cryopreservation: effect of cryoprotective agents and in vitro culture on vitrified-warmed ovarian tissue survival

What is the optimal vitrification protocol according to the cryoprotective agent (CPA) for ovarian tissue (OT) cryopreservation? The two-step protocol with 7.5% ethylene glycol (EG) and 7.5% dimethyl sulfoxide (DMSO) for 10 min then 20% EG, 20% DMSO and 0.5 M sucrose for 5 min showed the best results in mouse OT vitrification. Establishing the optimal cryopreservation protocol is one of the most important steps to improve OT survival. However, only a few studies have compared vitrification protocols with different CPAs and investigated the effect of in vitro culture (IVC) on vitrified-warmed OT survival. Some recent papers proposed that a combination of CPAs has less toxicity than one type of CPA. However, the efficacy of different types and concentrations of CPA are not yet well documented. A total of 644 ovaries were collected from 4-week-old BDF1 mice, of which 571 ovaries were randomly assigned to 8 groups and vitrified using different protocols according to CPA composition and the remaining 73 ovaries were used as controls. After warming, each of the eight groups of ovaries was further randomly divided into four subgroups and in vitro cultured for 0, 0.5, 2 and 4 h, respectively. Ovaries of the best two groups among the eight groups were autotransplanted after IVC. The CPA solutions for the eight groups were composed of EDS, ES, ED, EPS, EF, EFS, E and EP, respectively (E, EG; D, DMSO; P, propanediol; S, sucrose; F, Ficoll). The IVC medium was composed of α-minimal essential medium, 10% fetal bovine serum and 10 mIU/ml follicle-stimulating hormone (FSH). Autotransplantation of vitrified-warmed OTs after IVC (0 to 4 h) using the EDS or ES protocol was performed, and the grafts were recovered after 3 weeks. Ovarian follicles were assessed for morphology, apoptosis, proliferation and FSH level. The percentages of the morphologically intact (G1) and apoptotic follicles in each group at 0, 0.5, 2 and 4 h of IVC were compared. For G1 follicles at 0 and 4 h of IVC, the EDS group showed the best results at 63.8 and 46.6%, respectively, whereas the EP group showed the worst results at 42.2 and 12.8%, respectively. The apoptotic follicle ratio was lowest in the EDS group at 0 h (8.1%) and 0.5 h (12.7%) of IVC. All of the eight groups showed significant decreases in G1 follicles and increases in apoptotic follicles as IVC duration progressed. After autotransplantation, the EDS 0 h group showed a significantly higher G1 percentage (84.9%) than did the other groups (42.4-58.8%), while only the ES 4 h group showed a significant decrease in the number of proliferative cells (80.6%, 87.6-92.9%). However, no significant differences in apoptotic rates and FSH levels were observed between the groups after autotransplantation. The limitation of this study was the absence of in vitro fertilization using oocytes obtained from OT grafts, which should be performed to confirm the outcomes of ovarian cryopreservation and transplantation. We compared eight vitrification protocols according to CPA composition and found the EDS protocol to be the optimal method among them. The data presented herein will help improve OT cryopreservation protocols for humans or other animals.