Use Of Cryoprotectants Research Articles

Cryopreservation, cryoprotectants, and potential risk of epigenetic alteration.

The cryopreservation of gametes and embryos has increased notably over the past 20years and is now an essential part of assisted reproductive technologies (ARTs). However, because the cryopreservationprocess is un-physiological for human cells, gametes, and embryos, cryobiologists have suggested diverse methods to successfully cryopreserve human gametes and embryos in order to maintain their viability and assure successful pregnancy. During the first period of early development, major waves of epigenetic reprogramming-crucial for the fate of the embryo-occur. Recently, concerns relating to the increased incidence of epigenetic anomalies and genomic-imprinting disorders have been reported after ARTs and cryopreservation. Epigenetic reprogramming is particularly susceptible to environmental and un-physiological conditions such as ovarian stimulation, embryo culture, and cryopreservation that might collectively affect epigenetics dysregulation. Additionally, recent literature suggests that epigenetic and transcriptomic profiles are sensitive to the stress induced by vitrification, osmotic shock, oxidative stress, rapid temperature and pH changes, and cryoprotectants; it is therefore critical to have a more comprehensive understanding of the potential induced perturbations of epigenetic modifications that may be associated with vitrification. The aim of this paper is to present a critical evaluation of the association of gamete and embryo cryopreservation, use of cryoprotectants, and epigenetic dysregulations with potential long-term consequences for offspring health.

Reconsidering freeze-induced protein aggregation: Air bubbles as the root cause of ice-water interface stress

Freeze-induced stress causing aggregation of proteins has typically been primarily attributed to the ice-water interface. However, we hypothesize that the underlying observed and perceived detrimental effect of ice is, to some extent, attributed to air bubbles expelled from ice crystal lattices or to nanobubbles existing prior to freezing. The reduction of dissolved air was achieved via a deaeration process by placing samples in a reduced pressure chamber, while the reduction of nanobubbles was achieved by filtering samples via a syringe filter. The results showed that the reduction of both dissolved air molecules and stable colloidal nanobubbles in a bovine IgG solution prior to freezing led to a significant decrease in aggregation after thawing compared to untreated samples (∼6,000 vs. ∼ 40,000 particles/mL at a freezing rate of 100 K/s, respectively). The deaeration-filtration treatment works additively with cryoprotectants such as trehalose, further reducing the freeze-induced aggregation of IgG. The results also demonstrated that air–water interfacial aggregation of IgG in bulk liquid samples is a time-dependent process. The number of IgG subvisible particles increased with time and temperature, suggesting that random collisions of denatured molecules promoted the formation of aggregates with spherical morphology. In contrast, the IgG subvisible count after freeze-thawing had already reached its nominal value, suggesting a time-independent process where denatured protein molecules were compressed between ice crystals into filament-like aggregates. In summary, the findings shift the current paradigm from ice crystals being the main destabilizing factor during freezing to air bubbles, although the two are intertwined. From a translational aspect, this study underscores the value of deaeration-filtration as an essential supplemental process that can be applied in addition to formulation approaches such as the use of cryoprotectants to further reduce freezing stress on proteins and increase their stability.

Starch nanoparticle preparation by the nanoprecipitation technique: Effects of formulation parameters

This study investigates how key formulation parameters influence the formation of starch nanoparticles using the nanoprecipitation technique. When loaded with different compounds, starch nanoparticles present a promising option for controlled drug delivery in food, biomedical, and pharmaceutical applications. The work thoroughly examines factors such as polymer concentration, NaOH concentration, solvent ratios, stirring speed, injection flow, and properties of the non-solvent phase. Characterization is conducted using dynamic light scattering, laser Doppler electrophoresis, and scanning electron microscopy, followed by statistical analysis. Increasing the stirring speed notably decreases nanoparticle size, yielding dimensions of 161.5±2.1 nm, 157.6±2.5 nm, and 136.0±0.8 nm at 500, 700, and 900 rpm, respectively. Higher starch concentrations slightly increase the zeta potential of starch nanoparticles to values of −1.97±0.4 mV, −3.1±0.7 mV, and −3.9±1.5 mV for concentrations of 0.5 %, 1 %, and 2 %, respectively. This charge can be attributed to the hydroxyl functional groups in starch monomers. The study also assesses the stability of starch nanoparticles at different pH levels (4, 7.4, and 9) and explores the use of cryoprotectants during freeze-drying. Remarkably, nanoparticles show enhanced stability at pH 7.4, maintaining their initial sizes over six weeks. Cryoprotectants, even in small amounts, effectively preserve nanoparticle size post-freeze-drying. By adjusting formulation parameters, researchers can tailor the physicochemical characteristics of nanoparticles to achieve specific sizes and ensure stability.

Impact of the Use of Cryoprotectants in the Production of Freeze-Dried Soluble Coffee from Cold Brew Arabica Coffee

Cold brew is a method of coffee extraction that uses low temperature, preserving the volatile compounds of coffee. Freeze-drying allows the preservation of coffee features and nutritional value. The aim of this study was to evaluate the effects of different cryoprotectants in cold brew extracts as a basis for freeze-dried coffee production. Thus, the Coffea arabica extracts and the soluble coffee were characterized concerning caffeine content, antioxidant capacity, total phenolic compounds, and antimicrobial activity to verify the potential of this method. The extracts did not show antimicrobial activity with a high soluble solid content. It was observed that the cold extraction methods were efficient regarding the caffeine content, antioxidant capacity, and total phenolic compounds. Freeze-dried coffees also did not show antimicrobial activity, and they maintained the water and humidity activity standards. In general, cryoprotectants displayed an unfavorable influence on the extract and freeze-dried coffee in the analyses performed. The coffee extract without cryoprotectants had a higher antioxidant capacity (88.12%) and content of phenolic compounds (7.74 mg AG/mL of the coffee extract). Only for the analyses of soluble solids, the cryoprotectants mannitol and fructose showed promising results (14.03 °Brix, 14.40 °Brix, 11.33 °Brix, respectively). Thus, for the analyses conducted, the cryoprotectants did not lead to significant advantages for this process.

The Effect of Cryoprotectants and Storage Conditions on the Transfection Efficiency, Stability, and Safety of Lipid-Based Nanoparticles for mRNA and DNA Delivery.

Lipid-based nanoparticles have recently shown great promise, establishing themselves as the gold standard in delivering novel RNA therapeutics. However, research on the effects of storage on their efficacy, safety, and stability is still lacking. Herein, the impact of storage temperature on two types of lipid-based nanocarriers, lipid nanoparticles (LNPs) and receptor-targeted nanoparticles (RTNs), loaded with either DNA or messenger RNA (mRNA), is explored and the effects of different cryoprotectants on the stability and efficacy of the formulations are investigated. The medium-term stability of the nanoparticles was evaluated by monitoring their physicochemical characteristics, entrapment and transfection efficiency, every two weeks over one month. It is demonstrated, that the use of cryoprotectants protects nanoparticles against loss of function and degradation in all storage conditions. Moreover, it is shown that the addition of sucrose enables all nanoparticles to remain stable and maintain their efficacy for up to a month when stored at -80°C, regardless of cargo or type of nanoparticle. DNA-loaded nanoparticles also remain stable in a wider variety of storage conditions than mRNA-loaded ones. Importantly, these novel LNPs show increased GFP expression that can signify their future use in gene therapies, beyond the established role of LNPs in RNA therapeutics.

Методические аспекты разработки и доклинических исследований лекарственных препаратов в интересах арктической медицины

There is an inextricable link between exploration and development of the Arctic territories and emergence of associated problems of medical and biological nature. It is necessary to design and develop emergency care and prevention drugs and medical devices for use in the Arctic. This review presents an analysis of additional requirements for drugs intended for the Far North and compares methods of modeling extreme conditions in animals. We outline medical and biological problems of the region highlight key areas of Arctic pharmacology: choice of pharmaceutical form, use of cryoprotectants and design of adaptogens. The study mainly revolves around the search for information on modeling extreme environmental factors in animal experiments, as this is a key stage in preclinical studies of drugs for the Arctic medicine. We present the relevant directions of further work promoting the subject: development of the hypoxia and hypothermia assessment criteria, development of modeling methods employing large laboratory animals, improvement of the equipment used.

An improvement to capture and visualize the protein conformational ensembles in double electron-electron resonance measurements.

Over the past decades, pulsed ESR double electron-electron resonance (DEER) has emerged as an effective biophysical tool for revealing protein conformational ensembles. With a collection of numerous interspin distances by DEER, they can be utilized to reveal the dynamic structural ensembles as opposed to static structures from other techniques. In a continuing effort to improve the technique, we present a combined approach of experimental and simulation advances that allows one to capture and visualize protein conformational ensembles in the absence of cryoprotectant additives. First, we present a rapid freezing method using a flexible fused silica microcapillary, enabling DEER measurement without the use of cryoprotectants. Second, we present a Monte Carlo based algorithm that allows one to efficiently utilize the sparse DEER data for deriving ensemble structures of a target protein. The Bid protein, a highly glycerol-sensitive protein, is used as a model. We show that our rapid-freezing DEER method can capture the Bid ensembles in the absence of glycerol, which was not possible with the conventional DEER setup. With the newly developed algorithm, we can efficiently visualize the Bid conformational ensembles, saving a lot for performing advanced structural modeling. Our combined method advances DEER for capturing and visualizing protein conformational ensembles.

Sperm cryopreservation for impaired spermatogenesis.

Sperm cryopreservation for men with severely impaired spermatogenesis is one of the commonest reasons for short-term sperm storage, usually in advance of fertility treatment. Cryopreservation is generally very effective, although not all spermatozoa survive the process of freezing and thawing. This review considers various aspects of freezing sperm, including an overview of methods, appropriate use of cryoprotectants and practical considerations, as well as oxidative stress and mechanisms of cell cryodamage.

Blood Hypothermic Storage and Erythrocyte Cryopreservation in Dogs

This review presents the analysis of reported data on the current methods for blood hypothermic storage and erythrocyte cryopreservation in dogs. The relevance of designing and improving the techniques for canine erythrocyte cryopreservation, cryobanking, as well as the application of cryopreserved blood components for transfusion were substantiated. The features of canine blood hypothermic storage and specific use of cryoprotectants with various modes of their action were considered. The advantages of applying DMSO and combined cryopreservatives during low temperature storage of canine erythrocytes were specified.

CRIOPRESERVAÇÃO DE SEMENTES DA ESPÉCIE NATIVA DO BRASIL AROEIRA-DO-SERTÃO (Astronium urundeuva M. Allemão Engl.)

ABSTRACT Aroeira-do-sertão is a Brazilian native species that has been widely explored. Thus, the population of this species has been reduced and techniques for its preservation are essential, such as the conservation of seeds in liquid nitrogen (LN). The objective of this work was to evaluate different cryoprotectant solutions for cryopreservation of aroeira-do-sertão seeds in LN (-196 °C). The treatments used were: control (7.5±1.5 °C); LN without cryoprotectant; sucrose 0.4 mol L-1; sucrose 0.8 mol L-1; glycerol 1 mol L-1; glycerol 2 mol L-1; PVS1 (plant vitrification solution); PVS2; PVS2 + 1% phloroglucinol; and PVS3. The seeds remained frozen for 120 days. The seeds were evaluated for germination and water content before cryopreservation. Several germination parameters were evaluated on the seventh day and plant growth variables were evaluated after 150 days. The seeds presented 9.2% water content and 74% germination before cryopreservation. The germination in the control treatment was 55%, whereas it varied from 61% to 69% under cryopreservation, denoting the positive effect of cryopreservation, even without cryoprotectants (69%). The seeds presented a triphasic water absorption model; the LN accelerated the germination, which started within 56 hours, whereas the germination in the control treatment started after 66 hours. The plant parts presented satisfactory development after 150 days, as shown by the Dickson quality index. The use of cryoprotectants did not affect seed germination and initial growth of seedlings. Aroeira-do-sertão seeds with 9.2% water content can be cryopreserved in LN without cryoprotectants.

Изучение влияния способа внесения криопротекторов на сохранение семенного материала мяты длиннолистной при криоконсервации

Представленная статья посвящена оптимизации условий криохранения семенного материала мяты длиннолистной. Мята длиннолистная — эфиромасличное растение, активно применяемое в парфюмерной, фармацевтической и пищевой промышленности. В экспериментах по криохранению использованы следующие эндоцеллюлярные и экзоцеллюлярные криопротекторы: поливинилпирролидон (ПВП), глицерин и диметисульфоксид (ДМСО). Кроме того, исследовано влияние на сохранение ростовых характеристик семян при криоконсервации способа внесения криопротектора: при комнатной температуре и на ледяной бане. Исходная всхожесть семян мяты длиннолистной составила 76,3±2 %, а энергия прорастания — 70,3±1,8 %. При замораживании в жидком азоте без применения криопротекторов всхожесть составила 64,9±0,8 %, а энергия прорастания — 18,95±0,2 %. Следует отметить, что именно показатель энергии прорастания оказался чувствительным к депонированию семенного материала при экстремально низких температурах. Анализ результатов сохранения жизнеспособности семян мяты длиннолистной при замораживании их в криопротекторах различного состава в случае внесения их при комнатной температуре показал, что тенденция резкого снижения энергии прорастания наблюдается во всех вариантах эксперимента. Наилучшими вариантами криопротекторов для со-хранения ростовых характеристик семенного материала оказались ДМСО 2,5 % и ПВП 3 %, вносимые на ледяной бане. Всхожесть семян, сохраняемых в этих криопротекторах, составила 85,8±3,1 % и 87,6±2,3 % соответственно. Таким образом, выявлено, что оптимальным способом внесения криопротекторов при криоконсервации семян мяты длиннолистной является температура, близкая к 0 оС, в этом случае серьезно повышается как всхожесть, так и энергия прорастания семян.

Cross-validation of distance measurements in proteins by PELDOR/DEER and single-molecule FRET

Pulsed electron-electron double resonance spectroscopy (PELDOR/DEER) and single-molecule Förster resonance energy transfer spectroscopy (smFRET) are frequently used to determine conformational changes, structural heterogeneity, and inter probe distances in biological macromolecules. They provide qualitative information that facilitates mechanistic understanding of biochemical processes and quantitative data for structural modelling. To provide a comprehensive comparison of the accuracy of PELDOR/DEER and smFRET, we use a library of double cysteine variants of four proteins that undergo large-scale conformational changes upon ligand binding. With either method, we use established standard experimental protocols and data analysis routines to determine inter-probe distances in the presence and absence of ligands. The results are compared to distance predictions from structural models. Despite an overall satisfying and similar distance accuracy, some inconsistencies are identified, which we attribute to the use of cryoprotectants for PELDOR/DEER and label-protein interactions for smFRET. This large-scale cross-validation of PELDOR/DEER and smFRET highlights the strengths, weaknesses, and synergies of these two important and complementary tools in integrative structural biology.

Simple Cryoprotectant-Free Method to Advance Pulsed Dipolar ESR Spectroscopy for Capturing Protein Conformational Ensembles.

Double electron-electron resonance (DEER) is a powerful technique for studying protein conformations. To preserve the room-temperature ensemble, proteins are usually shock-frozen in liquid nitrogen prior to DEER measurements. The use of cryoprotectant additives is, therefore, necessary to ensure the formation of a vitrified state. Here, we present a simple modification of the freezing process using a flexible fused silica microcapillary, which increases the freezing rates and thus enables DEER measurement without the use of cryoprotectants. The Bid protein, which is highly sensitive to cryoprotectant additives, is used as a model. We show that DEER with the simple modification can successfully reveal the cold denaturation of Bid, which was not possible with the conventional DEER preparations. The DEER result reveals the nature of Bid folding. Our method advances DEER for capturing the chemically and thermally induced conformational changes of a protein in a cryoprotectant-free medium.

Advances in storage of poultry semen

Semen cryopreservation is a key biotechnological strategy used to preserve and protect genetic resources, which are subject to increasingly serious reductions in some species, and to protect animal biodiversity. Assisted reproductive techniques, however, are still not utilized to the same extent in avian species to the extent that occurs in mammals. The reasons for this situation are described in this review. The content of this paper is focused on current poultry preservation systems, published since 2010, and new strategies that are very promising for preserving avian genetic resources. Two major types of storage technologies which are utilized for avian sperm preservation, liquid storage and cryopreservation, are emphasized. The issues on which there is a focus includes supplementation of avian extenders with various compounds prior to the preservation period, use of cryoprotectants and fertility results when there were in vitro sperm evaluations. Results from recent studies indicate there are opportunities to improve the quality of bird semen after preservation. It is obvious that cryo-diluent composition may be the most important factor for development of efficacious cryopreservation methods for avian semen.

Cellular feasibility study of Acutodesmus obliquus

The development and validation of efficient microalgae conservation methods is essential for the establishment and constitution of long-term culture collections, as well as for programming for breeding and possible genetic modification of algae. However, each species of microalgae demonstrates responses considered unpredictable, and thus, making it difficult to standardize universal methods so far. The results presented in this study indicate time as a limiting factor for the conservation of A. obliquus, in the methods of conservation by refrigeration, freezing and freezing with the use of cryoprotectants, such as DMSO, Gli and Poly. Freezing with the use of cryoprotective substances demonstrated satisfactory efficiency in the conservation of microalgae, and thus, surprisingly, in conservation by refrigeration. As already known, conservation by freezing only showed low efficiency, since the cells are broken by the formation of ice crystals in their interior, and thus, making the microalgae conservation unfeasible for a longer period. The study effectively showed the conservation of A. obliquus by refrigeration and freezing with the use of cryoprotective substances mentioned in this work.

​Ice Cream and Frozen Yoghurt - A Suitable Carrier for Probiotics: A Review

Nowadays there has been a significant interest in the development of innovative functional food products conferring customized benefits to the consumers viz., physical and mental well-being, dental health, gastro-intestinal functions, etc. Among the dairy products with live cultures, probiotic ice cream and fermented frozen desserts such as frozen yoghurt are the emerging functional foods. Ice creams and frozen yoghurt are food products showing potential for use as probiotic vehicles and are highly popular with the consumers. The problem to be tackled relates to the loss of viability of probiotic cells in frozen dairy desserts which can occur during product formulation, processing, freezing and storage. The development of probiotic frozen dairy desserts containing live probiotic bacteria necessitates certain technological interventions. The means used to attain higher survivability of probiotic cells in such frozen products include use of selected probiotic strains, use of prebiotics along with probiotics, encapsulation of probiotic cells, use of cryoprotectants, etc.

Overview on the Antioxidants, Egg Yolk Alternatives, and Mesenchymal Stem Cells and Derivatives Used in Canine Sperm Cryopreservation.

Simple SummaryCanine sperm cryopreservation is a method commonly used in veterinary clinics and laboratories. The present article reviews the antioxidants used in canine sperm cryopreservation, the egg yolk alternatives used for preventing cross-contamination, and the application of mesenchymal stem cells (MSCs) and their derivatives in dog sperm cryopreservation.Sperm cryopreservation is a widely used assisted reproductive technology for canine species. The long-term storage of dog sperm is effective for the breeding of dogs living far apart, scheduling the time of artificial insemination that suits the female, and preventing diseases of the reproductive tract. However, spermatozoa functions are impaired during the freeze–thaw processes, which may decrease reproductive performance. Numerous attempts have been made to restore such impairments, including the use of cryoprotectants to prevent the damage caused by ice crystal formation, and supplementation of antioxidants to reduce reactive oxygen species generation due to osmotic stress during the procedure. Egg yolk derivatives, antioxidants, and, more recently, mesenchymal stem cells (MSCs) and their derivatives have been proposed in this research field. This review article will summarize the current literature available on the topic.

Potentialities of using cryoprotectants in gluten-free frozen dough and microwave baking as an emerging technology

Disorders related to human health involving proteins that form the gluten network are increasingly in society, justifying the need to offer bakery products for this market niche. This study aimed to gather important information about the challenges involving technological production in the gluten-free bakery market. The approach encompasses the development of frozen dough for gluten-free baking, the use of cryoprotectants to maintain cell viability during the freeze-thawing step, the application of emulsifiers to improve the quality of the bread and the baking by an unconventional method using the microwaves. The introduction of frozen dough in the gluten-free bakery sector represents an interesting tool. However, these bread formulations require a higher amount of water compared to a traditional bread formulation (70 to 120 %), which makes the freezing and thawing steps extremely challenging, especially when it comes to the biopreservation of yeast cell viability. Cryopreservation with isolated and/or combined application of penetrating (glycerol) and non-penetrating (sucrose, fructo-oligosaccharide, trehalose, proteins and hydrocolloids) cryoprotectants promotes changes in the viscosity of the medium and is an excellent alternative for the maintenance of the microorganism responsible for the fermentation process. The appropriate choices of cryoprotectants, as well as the control of the freezing and thawing and baking processes, are important steps in the production line, to guarantee products with adequate technological and sensory qualities.

Crystallization of Proteins on Chip by Microdialysis for In Situ X-ray Diffraction Studies.

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins on-chip with the dialysis method and allowing in situ single-crystal or serial crystallography experiments at room temperature. The protocol details the fabrication process of the microchips, the manipulation of the on-chip crystallization experiments and the treatment of the in situ collected X-ray diffraction data for the structural elucidation of the protein sample. The main feature of this microfabrication procedure lies on the integration of a commercially available, semipermeable regenerated cellulose dialysis membrane in between two layers of the chip. The molecular weight cut-off of the embedded membrane varies depending on the molecular weight of the macromolecule and the precipitants. The device exploits the advantages of microfluidic technology, such as the use of minute volumes of samples (<1 µL) and fine tuning over transport phenomena. The chip coupled them with the dialysis method, providing precise and reversible control over the crystallization process and can be used for investigating phase diagrams of proteins at the microliter scale. The device is patterned using a photocurable thiolene-based resin with soft imprint lithography on an optically transparent polymeric substrate. Moreover, the background scattering of the materials composing the microchips and generating background noise was evaluated rendering the chip compatible for in situ X-ray diffraction experiments. Once protein crystals are grown on-chip up to an adequate size and population uniformity, the microchips can be directly mounted in front of the X-ray beam with the aid of a 3D printed holder. This approach addresses the challenges rising from the use of cryoprotectants and manual harvesting in conventional protein crystallography experiments through an easy and inexpensive manner. Complete X-ray diffraction data sets from multiple, isomorphous lysozyme crystals grown on-chip were collected at room temperature for structure determination.

Crystallization of Proteins on Chip by Microdialysis for &lt;i&gt;In Situ&lt;/i&gt; X-ray Diffraction Studies

This protocol describes the manufacturing of reproducible and inexpensive microfluidic devices covering the whole pipeline for crystallizing proteins on-chip with the dialysis method and allowing in situ single-crystal or serial crystallography experiments at room temperature. The protocol details the fabrication process of the microchips, the manipulation of the on-chip crystallization experiments and the treatment of the in situ collected X-ray diffraction data for the structural elucidation of the protein sample. The main feature of this microfabrication procedure lies on the integration of a commercially available, semipermeable regenerated cellulose dialysis membrane in between two layers of the chip. The molecular weight cut-off of the embedded membrane varies depending on the molecular weight of the macromolecule and the precipitants. The device exploits the advantages of microfluidic technology, such as the use of minute volumes of samples (<1 µL) and fine tuning over transport phenomena. The chip coupled them with the dialysis method, providing precise and reversible control over the crystallization process and can be used for investigating phase diagrams of proteins at the microliter scale. The device is patterned using a photocurable thiolene-based resin with soft imprint lithography on an optically transparent polymeric substrate. Moreover, the background scattering of the materials composing the microchips and generating background noise was evaluated rendering the chip compatible for in situ X-ray diffraction experiments. Once protein crystals are grown on-chip up to an adequate size and population uniformity, the microchips can be directly mounted in front of the X-ray beam with the aid of a 3D printed holder. This approach addresses the challenges rising from the use of cryoprotectants and manual harvesting in conventional protein crystallography experiments through an easy and inexpensive manner. Complete X-ray diffraction data sets from multiple, isomorphous lysozyme crystals grown on-chip were collected at room temperature for structure determination.