Method For Long-term Preservation Research Articles

Investigating cryopreserved PBMC functionality in an antigen-induced model of sarcoidosis granuloma formation

Sarcoidosis, a systemic inflammatory disease, poses challenges in understanding its etiology and variable clinical courses. Despite ongoing uncertainty about causative agents and genetic predisposition, granuloma formation remains its hallmark feature. To address this, we developed a validated in vitro human granuloma model using patient-derived peripheral blood mononuclear cells (PBMCs), offering a dynamic platform for studying early granuloma formation and sarcoidosis pathogenesis. However, a current limitation of this model is its dependence on freshly isolated PBMCs obtained from whole blood. While cryopreservation is a common method for long-term sample preservation, the biological effects of freezing and thawing PBMCs on granuloma formation remain unclear. This study aimed to assess the viability and functionality of cryopreserved sarcoidosis PBMCs within the granuloma model, revealing similar granulomatous responses to fresh cells and highlighting the potential of cryopreserved PBMCs as a valuable tool for studying sarcoidosis and related diseases.

Cryopreservation techniques in genipap and mangaba: challengers and perspectives

The genipap (Genipa americana L.) is a native species of Brazil, found in various regions of the country, from the North to the South, including Sergipe. On the other hand, the mangabeira (Hancornia speciosa Gomes), another Brazilian tropical plant, is recognized for its fruits rich in iron and vitamin C, widely exploited by the pulp, juice, and ice cream industry. However, it faces threats due to urban development in the coastal region. To preserve these species, techniques such as cryopreservation are fundamental. This approach, which maintains plant tissues at ultra-low temperatures, allows the conservation of genetic material indefinitely, contributing to the maintenance of biological diversity. Encapsulation technology, which involves enclosing plant material in a specific matrix, is also promising in this context, offering a long-term preservation method for plant explants and embryos. These methods represent significant advances in the field of plant species conservation, providing effective strategies to ensure the survival of these plants in the face of environmental challenges and human development. The objective of this review is to address some studies involving cryopreservation techniques for genipap and mangaba species. Through cryopreservation techniques, we can achieve ex situ conservation for the germplasm bank as well as for plant regeneration and reforestation of areas. It is evident that there is a need to increase studies on the conservation of both species.

Evaluation of long-term preservation methods for viral RNA in mosquitoes at room temperature

Mosquitoes are important vectors of various pathogenic viruses. Almost all viruses transmitted by mosquitoes are RNA viruses. Therefore, to detect viral genes, mosquito samples must be kept at low temperatures to prevent RNA degradation. However, prolonged transport from the field to laboratory can pose challenges for temperature control. The aim of this study was to evaluate methods for preserving viral RNA in mosquito bodies at room temperature. Virus-infected mosquito samples were immersed in ethanol, propylene glycol, and a commercially available nucleic acid preservation reagent at room temperature, and viral RNA stability was compared. As a result, for the two RNA viruses (San Gabriel mononegavirus and dengue virus 1) subjected to this experiment, no significant decrease in the viral RNA was observed for at least eight weeks after immersion in the reagents, and the amount of RNA remaining was equivalent to that of samples stored at − 80 °C. These results indicate that immersion storage in these reagents used in this study is effective in preserving viral RNA in mosquitoes under room temperature conditions and is expected to be implemented in epidemiologic surveillance that is not limited by the cold chain from the field to the laboratory.

Assessing ‘non-destructive’ DNA extraction method in small crustaceans kept in wet collections

Specimens in natural history museums are a valuable resource for biological research, such as taxonomic, biodiversity or evolutionary studies. However, the quality of DNA and even morphological characters can decrease over time, depending on previous fixation and long-term preservation methods. In recent years, advances in DNA extraction and sequencing techniques have allowed researchers to obtain DNA from museum specimens, even when the DNA was very fragmented. Extraction methods should ideally be morphologically non-destructive, leaving diagnostic characters intact for future taxonomic studies. Here, we assess whether the whole-body extraction widely used for several taxa would be destructive for small crustaceans kept in wet collections. We extracted the DNA from over 70 small (1-3 cm) and relatively fragile shrimps collected during the last 30 years by using: i) a piece of abdominal tissue and ii) from the entire remaining body of the animal. We photographed several samples before and after the lysis, focusing on taxonomically relevant characters. Although DNA concentration was higher in the whole-body extractions, the presence of intact DNA was not correlated to the amount of lysed tissue. The resulting genomic libraries had little to no difference in yield. The taxonomically relevant characters were primarily preserved in larger specimens, whereas smaller specimens (< 1.5 cm) became too fragile to handle or were damaged. We conclude that this method must be carried out carefully in smaller crustaceans, depending on size and taxon. We advise against using it with type specimens as the advantages do not outweigh the risks. Our experiment may provide future research with quantitative and qualitative evaluations to help scientists weigh their decisions when extracting DNA from wet collection material.

Primordial Germ Cell Cryopreservation and Revival of Drosophila Strains.

Drosophila strains must be maintained by the frequent transfer of adult flies to new vials. This carries a danger of mutational deterioration and phenotypic changes. Development of an alternative method for long-term preservation without such changes is therefore imperative. Despite previous successful attempts, cryopreservation of Drosophila embryos is still not of practical use because of low reproducibility. Here, we describe a protocol for primordial germ cell (PGC) cryopreservation and strain revival via transplantation of cryopreserved PGCs into agametic Drosophila melanogaster (D. melanogaster) host embryos. PGCs are highly permeable to cryoprotective agents (CPAs), and developmental and morphological variation among strains is less problematic than in embryo cryopreservation. In this method, PGCs are collected from approximately 30 donor embryos, loaded into a needle after CPA treatment, and then cryopreserved in liquid nitrogen. To produce donor-derived gametes, the cryopreserved PGCs in a needle are thawed and then deposited into approximately 15agametic host embryos. A frequency of at least 15% fertile flies was achieved with this protocol, and the number of progeny per fertile couple was always more than enough to revive the original strain (the average progeny number being 77.2 ± 7.1), indicating the ability of cryopreserved PGCs to become germline stem cells. The average number of fertile flies per needle was 1.1 ± 0.2, and 9 out of 26 needles produced two or more fertile progeny. It was found that 11 needles are enough to produce 6 or more progeny, in which at least one female and one male are likely included. The agametic host makes it possible to revive the strain quickly by simply crossing newly emerged female and male flies. In addition, PGCs have the potential to be used in genetic engineering applications, such as genome editing.

Effect of Lyoprotective Agents on the Preservation of Survival of a Bacillus cereus Strain PBG in the Freeze-Drying Process.

Lyophilization is a widely employed long-term preservation method in which the bacterial survival rate largely depends on the cryoprotectant used. Bacillus cereus strain PBC was selected for its ability to thrive in environments contaminated with arsenic, lead, and cadmium, tolerate 500 ppm of free cyanide, and the presence of genes such as ars, cad, ppa, dap, among others, associated with the bioremediation of toxic compounds and enterotoxins (nheA, nheB, nheC). Following lyophilization, the survival rates for Mannitol 2.5%, Mannitol 10%, and Glucose 1% were 98.02%, 97.12%, and 96.30%, respectively, with the rates being lower than 95% for other sugars. However, during storage, for the same sugars, the survival rates were 78.71%, 97.12%, and 99.97%, respectively. In the cake morphology, it was found that the lyophilized morphology showed no relationship with bacterial survival rate. The best cryoprotectant for the PBC strain was 1% glucose since it maintained constant and elevated bacterial growth rates during storage, ensuring that the unique characteristics of the bacterium were preserved over time. These findings hold significant implications for research as they report a new Bacillus cereus strain with the potential to be utilized in bioremediation processes.

INFLUENCE OF THE HEATING AGENT TEMPERATURE ON THE KINETICS OF THE CONVECTIVE DRYING PROCESS AND THE CONTENT OF BIOACTIVE COMPOUNDS IN APPLE POMACE

An environmentally ecological approach to the reuse of agro-industrial products in the food industry involves extracting biologically active substances or using them as supplementary products rich in dietary fibers. Apple pomace obtained after juice extraction presents a product rich in biologically active substances, soluble and insoluble fibers, organic acids, and minerals. Convective drying of apple pomace is an alternative method for long-term preservation by maintaining the biologically active substances. The research aimed to study the influence of temperature in the range of 60-80 °C on the drying of apple pomace to a final moisture content of 12.0±0.13% using the convective method, and the effect of temperature on the yield of bioactive compounds and antioxidant activity in Gold Delicious apple pomace. Changing the air temperature during convective drying from 60 to 80 °C reduced the drying time by 1.6 times and demonstrated a 1.4-fold increase in drying rate. The increase in the thermal agent's temperature led to an increase in the kinetic characteristic values; KI values increased by 4%, and KII values increased by 44%. Spectrophotometric analysis demonstrated that apple pomace dried at a temperature of 70 °C had the highest total content of polyphenols, tannins, and carotenoids. Thus, the convective drying method of apple pomace in the temperature range of 60-80 °C exerts a significant influence on the composition of biologically active compounds in the apple pomace composition.

Epigenomic analysis of formalin-fixed paraffin-embedded samples by CUT&Tag

For more than a century, formalin-fixed paraffin-embedded (FFPE) sample preparation has been the preferred method for long-term preservation of biological material. However, the use of FFPE samples for epigenomic studies has been difficult because of chromatin damage from long exposure to high concentrations of formaldehyde. Previously, we introduced Cleavage Under Targeted Accessible Chromatin (CUTAC), an antibody-targeted chromatin accessibility mapping protocol based on CUT&Tag. Here we show that simple modifications of our CUTAC protocol either in single tubes or directly on slides produce high-resolution maps of paused RNA Polymerase II at enhancers and promoters using FFPE samples. We find that transcriptional regulatory element differences produced by FFPE-CUTAC distinguish between mouse brain tumors and identify and map regulatory element markers with high confidence and precision, including microRNAs not detectable by RNA-seq. Our simple workflows make possible affordable epigenomic profiling of archived biological samples for biomarker identification, clinical applications and retrospective studies.

Long-Term Survival and Functional Recovery of Cryopreserved Vascularized Groin Flap and Below-the-Knee Rat Limb Transplants.

Effective cryopreservation of large tissues, limbs, and organs has the potential to revolutionize medical post-trauma reconstruction options and organ preservation and transplantation procedures. To date, vitrification and directional freezing are the only viable methods for long-term organ or tissue preservation, but are of limited clinical relevance. This work aimed to develop a vitrification-based approach that will enable the long-term survival and functional recovery of large tissues and limbs following transplantation. The presented novel two-stage cooling process involves rapid specimen cooling to subzero temperatures, followed by gradual cooling to the vitrification solution (VS) and tissue glass transition temperature. Flap cooling and storage were only feasible at temperatures equal to or slightly lower than the VS Tg (i.e., -135°C). Vascularized rat groin flaps and below-the-knee (BTK) hind limb transplants cryopreserved using this approach exhibited long-term survival (>30 days) following transplantation to rats. BTK-limb recovery included hair regrowth, normal peripheral blood flow, and normal skin, fat, and muscle histology. Above all, BTK limbs were reinnervated, enabling rats to sense pain in the cryopreserved limb. These findings provide a strong foundation for the development of a long-term large-tissue, limb and organ preservation protocol for clinical use.

Thiel embalming method used for anatomy dissection as an educational tool in teaching human anatomy, in research, and in training in comparison of different methods for long term preservation.

The use of human cadavers is beneficial in teaching gross human anatomy, training, and development in medicine. Fresh-frozen cadavers initially exhibit natural colour, tenderness, and flexibility. They also present innumerable difficulties, including the requirement of freezers for storage and a few weeks' work time because of fast decay. The risk of infection is also significant. A possible choice for fresh-frozen cadavers is embalmed cadavers. The requested characteristic needed for successful embalming is acceptable long-term structural preservation of organs and tissues with minimal deformity and prevention of outward aspects. Scientists are trying to develop an ideal embalming solution to preserve the human body. Such a solution renders the flexibility of internal organs, preventing dryness, and reducing fungal or bacterial growth. Cadavers should be in a condition comparable to that of an unembalmed cadaver, and the chemicals used in the solution should not be harmful to persons during manipulation. The promising results have been obtained by the use of different embalming techniques by many faculties of medicine as a tool to enhance the teaching of anatomy to students.

The effects of ozonated slurry ice treatment on microbial, physicochemical, and quality of large yellow croaker (Pseudosciaena crocea) during cold-chain circulation

Cold chains are effective in maintaining the quality of aquatic products and reducing food losses, especially for dynamic transportation. The purpose of the research was to evaluate the effects of different ice treatments on the microbial, physicochemical, and quality characteristics of large yellow croaker (Pseudosciaena crocea) during cold-chain circulation. Different ice treatments (flake ice (FI), slurry ice (SI), and ozonated slurry ice (OSI)) were used during circulation, respectively. Changes in microbial (total viable count (TVC), psychrophilic bacteria count (PBC), H2S-producing bacteria count, pseudomonas bacteria count), physicochemical (pH, K-value, total volatile basic nitrogen (TVB-N), thiobarbituric acid (TBA), biogenic amines (BAs) content, color difference, texture profile analysis (TPA)) was measured. Low-field nuclear magnetic resonance (LF-NMR) and magnetic resonance imaging (MRI) were also measured to evaluate the feasibility of preservation methods. The results demonstrated that compared with SI treatment, the growth of psychrophilic bacteria, H2S-producing bacteria, and pseudomonas bacteria was inhibited by OSI treatment on the 21st day (0.55, 0.24, and 0.73 log CFU/g reduction, respectively). Besides, OSI treatment exhibited inhibitory activity toward K-value and BAs during storage, along with texture softening. Compared with FI treatment, OSI could retard the water migration and extend the shelf-life of large yellow croaker (Pseudosciaena crocea) for another 6 days. Therefore, ozonated slurry ice will be a promising long-term preservation method for maintaining the quality of large yellow croaker.

Trehalose in Biomedical Cryopreservation-Properties, Mechanisms, Delivery Methods, Applications, Benefits, and Problems.

Cells and tissues are the foundation of translational medicine. At present, one of the main technological obstacles is their preservation for long-term usage while maintaining adequate viability and function. Optimized storage techniques must be developed to make them safer to use in the clinic. Cryopreservation is the most common long-term preservation method to maintain the vitality and function of cells and tissues. But, the formation of ice crystals in cells and tissues is considered to be the main mechanism that could harm cells and tissues during freezing and thawing. To reduce the formation of ice crystals, cryoprotective agents (CPAs) must be added to the cells and tissues to achieve the cryoprotective effect. However, conventional cryopreservation of cells and tissues often needs to use toxic organic solvents as CPAs. As a result, cryopreserved cells and tissues may need to go through a time-consuming washing process to remove CPAs for further applications in translational medicine, and multiple valuable cells are potentially lost or killed. Currently, trehalose has been researched as a nontoxic CPA due to its cryoprotective ability and stability during cryopreservation. Nevertheless, trehalose is a nonpermeable CPA, and the lack of an effective intracellular trehalose delivery method has become the main obstacle to its use in cryopreservation. This article illustrated the properties, mechanisms, delivery methods, and applications of trehalose, summarized the benefits and limits of trehalose, and summed up the findings and research direction of trehalose in biomedical cryopreservation.

Cryopreservation: A Comprehensive Overview, Challenges, and Future Perspectives.

Cryopreservation is the most prevalent method of long-term cell preservation. Effective cell cryopreservation depends on freezing, adequate storage, and correct thawing techniques. Recent advances in cryopreservation techniques minimize the cellular damage which occurs while processing samples. This article focuses on the fundamentals of cryopreservation techniques and how they can be implemented in a variety of clinical settings. The article presents a brief description of each of the standard cryopreservation procedures, such as slow freezing and vitrification. Alongside that, the membrane permeating and nonpermeating cryoprotectants are briefly discussed, along with current advancements in the field of cryopreservation and variables influencing the cryopreservation process. The diminution of cryoinjury incurred by the cell via the resuscitation process will also be highlighted. Inthe end applicationof cryopreservation techniques in many fields, with a special emphasis on stem cell preservation techniques and current advancements presented. Furthermore, the challenges while implementing cryopreservation and the futuristic scope of the fields are illustrated herein. The content of this review sheds light on various ways to enhance the output of the cell preservation process and minimize cryoinjury while improving cell revival.

Preservation of Viable Microorganisms in the Laboratory: An Overview of Basics, Methods and Practical Recommendations for Beginners

Preservation of viable microbial cultures in laboratory practice is necessary for performing both short-term and long-term projects with microorganisms in vitro. There is no universal method to preserve cultures of live microbial cells of different taxonomic groups. This brief review summarizes the basics of methods for short-term and long-term preservation of viable cultures of microorganisms in vitro. Practical recommendations are presented for the preservation of viable cultures of microorganisms, which can be useful in conventional microbiological laboratories.

ELEMENTAL ANALYSIS OF CERAMIC ARTEFACTS FROM TANJUNG SIMPANG MENGAYAU SHIPWRECK IN SABAH

This article discusses the scientific analysis of ceramic artefacts recovered from a shipwreck at Tanjung Simpang Mengayau in Kudat, Sabah. The main objective is to determine the chemical composition of these artefacts to identify the materials and technology used in the production of the ceramics. Understanding the ceramic materials and technology will assist archaeologists and conservators to find the best methods for long-term and sustainable preservation of the ceramics heritage in Sabah. In total, 16 ceramic jar and celadon bowl sherds recovered from the Tanjung Simpang Mengayau shipwreck were analysed using X-ray diffraction (XRD) and X-ray fluorescence (XRF). The results revealed the presence of quartz and mullite minerals in the jars while quartz, mullite and cristobalite minerals were found in the celadon bowls, suggesting that both the jars and celadon bowls were made of kaolinite clay with added sand and limestone, but fired at different temperatures of 1,345°C and 1,470°C, respectively. Data gathered from this study is important not only for finding out and comparing the ceramic materials and production methods, but also to identify the possible origins of the Tanjung Simpang Mengayau ceramics.

Isochoric freezing: An innovative and emerging technology for retention of food quality characteristics

Freezing is a well-established and long-term food preservation method. The most commonly used conventional freezing method follows isobaric (constant pressure) approach, which causes number of irreversible nutritional, mechanical, sensorial, and microstructural changes that make food unacceptable to consumer. The current pandemic has increased demand for food products with higher nutritional and physicochemical properties. These factors have led to the development of an isochoric freezing system. Isochoric freezing has great potential to retain the quality properties of food. Understanding the concept and application of the isochoric freezing system in the food industry is necessary to investigate its effect on various quality characteristics. Thus this review presents an outline of the working principle and impact of isochoric freezing on retention of nutrients, physicochemical and bioactive compounds of food products, and highlights current challenges and future needs by considering research published in previous years. A special section also shows the applications of isochoric freezing in medical field. Practical applications Isochoric (constant volume) freezing has emerging application in the food industry because of protects preserved food from ice damage maintains its quality properties, reduces up to 70% energy consumption, has less operating cost, and is environmentally friendly. Fewer temperature fluctuations will also experience in food products stored in the isochoric system as temperature increases continuously, resulting in ice melting inside the chamber rather than heating the food product. In conclusion, the isochoric freezing system has enormous potential in food industries in retaining nutrient and bioactive compounds compared to conventional freezing techniques.

Cell membrane fluidity and ROS resistance define DMSO tolerance of cryopreserved synovial MSCs and HUVECs

ObjectivesSynovial mesenchymal stem cells (MSCs) have high freeze–thaw tolerance, whereas human umbilical vein endothelial cells (HUVECs) have low freezing tolerance. The differences in cell type-specific freeze–thaw tolerance and the mechanisms involved are unclear. This study thus aimed to identify the biological and physical factors involved in the differences in freeze–thaw tolerance between MSCs and HUVECs.Materials and methodsFor biological analysis, MSC and HUVEC viability after freeze-thawing and alteration of gene expression in response to dimethyl sulfoxide (DMSO, a cryoprotectant) were quantitatively evaluated. For physical analysis, the cell membrane fluidity of MSCs and HUVECs before and after DMSO addition was assessed using a histogram for generalized polarization frequency.ResultsHUVECs showed lower live cell rates and higher gene expression alteration related to extracellular vesicles in response to DMSO than MSCs. Fluidity measurements revealed that the HUVEC membrane was highly fluidic and sensitive to DMSO compared to that of MSCs. Addition of CAY10566, an inhibitor of stearoyl-coA desaturase (SCD1) that produces highly fluidic desaturated fatty acids, decreased the fluidity of HUVECs and increased their tolerance to DMSO. The combination of CAY10566 and antioxidant glutathione (GSH) treatment improved HUVEC viability from 57 to 69%. Membrane fluidity alteration may thus contribute to pore-induced DMSO influx into the cytoplasm and reactive oxygen species production, leading to greater cytotoxicity in HUVECs, which have low antioxidant capacity.ConclusionsDifferences in freeze–thaw tolerance originate from differences in the cell membranes with respect to fluidity and antioxidant capacity. These findings provide a basis for analyzing cell biology and membrane-physics to establish appropriate long-term preservation methods aimed at promoting transplantation therapies.

Semen Collection, Cryopreservation and Artificial Insemination in Dogs

Today, the methods of artificial insemination, that have recently become a biotechnological phenomenon, have being used in dogs especially for those partners that are largely unable to mate because of differences in their body sizes. Artificial insemination is likely only by diluting the semen collected in order to protect it from the environmental conditions, by using some short- and long-term preservation methods and by transferring it into the females with proper techniques. Therefore, in this brief review, some data about the approach to the male at the time of semen collection, as well as the collection itself (digital manipulation, conic plastic rubber along with hand massage, and electrical stimulations (the electro-ejeculator)), dilution (Tes, Bes, Hepes, Pipes, Tris, Tes/Tris and commercial extenders, at 1:1-1:5 dilution rate), short (at +4 oC) - and long-term (in ampoule, pellet and straw) frozen storage (within liquid nitrogen, at -196 oC) of semen and artificial insemination methods (intravaginal catheter, Norwegian intrauterine catheter, endoscopic intrauterine and surgical intrauterine technique)) are given in dogs.

Aquaporins and Animal Gamete Cryopreservation: Advances and Future Challenges.

Simple SummaryCryopreservation is the method for the long-term preservation of gametes and embryos. In recent years, intensive research has focused on improving cryopreservation protocols for the determination of optimal freezing conditions and cryoprotective agents’ concentration for each cell type. The optimal cryopreservation protocol comprises the adequate balance between the freezing rate and the correct concentration of cryoprotective agents to achieve controlled cellular dehydration and minimal intracellular ice formation. Osmoregulation is, therefore, central in cryobiology. Water and some solutes can cross the plasma membrane, whereas facilitating transport takes a great part in intracellular/extracellular fluid homeostasis. Cells express water channels known as aquaporins that facilitate the transport of water and small uncharged solutes on their plasma membrane, including some cryoprotective agents. This review explores the expression and the function of aquaporins in gametes and embryos. In addition, the putative role of aquaporins for cryopreservation procedures is discussed.Cryopreservation is globally used as a method for long-term preservation, although freeze-thawing procedures may strongly impair the gamete function. The correct cryopreservation procedure is characterized by the balance between freezing rate and cryoprotective agents (CPAs), which minimizes cellular dehydration and intracellular ice formation. For this purpose, osmoregulation is a central process in cryopreservation. During cryopreservation, water and small solutes, including penetrating cryoprotective agents, cross the plasma membrane. Aquaporins (AQPs) constitute a family of channel proteins responsible for the transport of water, small solutes, and certain gases across biological membranes. Thirteen homologs of AQPs (AQP0-12) have been described. AQPs are widely distributed throughout the male and female reproductive systems, including the sperm and oocyte membrane. The composition of the male and female gamete membrane is of special interest for assisted reproductive techniques (ART), including cryopreservation. In this review, we detail the mechanisms involved in gamete cryopreservation, including the most used techniques and CPAs. In addition, the expression and function of AQPs in the male and female gametes are explored, highlighting the potential protective role of AQPs against damage induced during cryopreservation.

The review of fungi and omycota long-term storage methods

Fungi are extremly diverse and beneficial organisms both for environment and industry; thus we are obliged to secure it’s biodiversity. There are several laboratory methods for fungi longterm preservation. The main goal is to secure their vitality and biological properties. Some of them are simple but the same time laborious, other demand specific equipment. Among the most popular methods we find: freez-drying, cryopreservation at –80°C and in liquide nitrogen. The most reliable is cryopreservation in liquid nitrogen. However even this method is not universal and siutable for all cultures.