Technique For Long-term Preservation Research Articles

Recent advances in cryotolerance biomarkers for semen preservation in frozen form-A systematic review.

Spermatozoa cryopreservation has been practiced for decades and is a very useful technique for long-term preservation of sperm fertility. The capability for semen cryopreservation varies across species, seasons, latitudes, and even for different ejaculates from the same animal. This article summarizes research results on sperm cryotolerance biomarkers in several species, focusing on three areas: spermatozoa cryotolerance biomarkers, seminal plasma proteins cryotolerance biomarkers, and other cryotolerance biomarkers. We discovered that sperm cryoresistance biomarkers are primarily related to sperm plasma membrane stability, the presence of antioxidant substances in sperm or seminal plasma, sperm cell energy metabolism, water and small molecule transport channels in the sperm plasma membrane, and antistress substances in sperm or seminal plasma. The research conducted using diverse livestock models can be employed to enhance the basic and applied reproduction of other mammals through the study of sperm cryotolerance biomarkers, as well as the substantial similarities between livestock and other organisms, including endangered species.

Comparative Proteomics Reveals Prolonged Corneal Preservation Impaired Ocular Surface Immunity Accompanied by Fibrosis in Human Stroma.

Cornea transplantation is one of the most commonly performed allotransplantations worldwide. Prolonged storage of donor corneas leads to decreased endothelial cell viability, severe stromal edema, and opacification, significantly compromising the success rate of corneal transplantation. Corneal stroma, which constitutes the majority of the cornea, plays a crucial role in maintaining its shape and transparency. In this study, we conducted proteomic analysis of corneal stroma preserved in Optisol-GS medium at 4 °C for 7 or 14 days to investigate molecular changes during storage. Among 1923 identified proteins, 1634 were quantifiable and 387 were significantly regulated with longer preservation. Compared to stroma preserved for 7 days, proteins involved in ocular surface immunomodulation were largely downregulated while proteins associated with extracellular matrix reorganization and fibrosis were upregulated in those preserved for 14 days. The increase in extracellular matrix structural proteins together with upregulation of growth factor signaling implies the occurrence of stromal fibrosis, which may compromise tissue clarity and cause vision impairments. This study is the first to provide insights into how storage duration affects corneal stroma from a proteomic perspective. Our findings may contribute to future research efforts aimed at developing long-term preservation techniques and improving the quality of preserved corneas, thus maximizing their clinical application.

Controlled hydrogel-based encapsulation of macrophages determines cell survival and functionality upon cryopreservation

The development of novel cell-based therapies has increased the necessity to improve the long-term storage of cells. The current method of cryopreservation is far from optimal, causing ice-associated mechanical and osmotic damage to sensitive cells. Cell encapsulation is emerging as a new strategy to overcome those current limitations; however, few data are applicable to slow freezing, with conflicting results and multiple experimental conditions. The objective of this research work was to evaluate the impact of capsule size and encapsulation method on cell survival and functionality after a conventional freezing protocol. To this end, cells were encapsulated in alginate beads of different sizes, spanning the range of 200–2000 µm thanks to multiple extrusion techniques and conditions, and further cryopreserved using a slow cooling rate (−1°C/min) and 10 % DMSO as cryoprotectant. Our data show that there is a strong correlation between bead size and cell survival after a slow cooling cryopreservation process, with cell viabilities ranging from 7 to 70 % depending on the capsule size, with the smallest capsules (230 µm) achieving the highest level of survival. The obtained results indicate that the beads’ diameter, rather than their morphology or the technique used, plays a significant role in the post-thawing cell survival and functionality. These results show that a fine control of cell encapsulation in alginate hydrogels is required when it comes to overcoming the current limitations of long-term preservation techniques by slow cooling.

Cryobanking of Human Distal Lung Epithelial Cells for Preservation of Their Phenotypic and Functional Characteristics.

The epithelium lining airspaces of the human lung is maintained by regional stem cells, including basal cells of pseudostratified airways and alveolar type 2 (AT2) pneumocytes of the gas-exchange region. Despite effective techniques for long-term preservation of airway basal cells, procedures for efficient preservation of functional epithelial cell types of the distal gas-exchange region are lacking. Here we detail a method for cryobanking of epithelial cells from either mouse or human lung tissue for preservation of their phenotypic and functional characteristics. Flow cytometric profiling, epithelial organoid-forming efficiency, and single-cell transcriptomic analysis were used to compare cells recovered from cryobanked tissue with those of freshly dissociated tissue. AT2 cells within single-cell suspensions of enzymatically digested cryobanked distal lung tissue retained expression of the pan-epithelial marker CD326 and the AT2 cell surface antigen recognized by monoclonal antibody HT II-280, allowing antibody-mediated enrichment and downstream analysis. Isolated AT2 cells from cryobanked tissue were comparable with those of freshly dissociated tissue both in their single-cell transcriptome and their capacity for invitro organoid formation in three-dimensional cultures. We conclude that the cryobanking method described herein allows long-term preservation of distal human lung tissue for downstream analysis of lung cell function and molecular phenotype and is ideally suited for the creation of an easily accessible tissue resource for the research community.

Analyzing Consumer Perception on Quality and Safety of Frozen Foods in Emerging Economies: Evidence from Albania and Kosovo

Freezing technology is one of the most well established long-term preservation techniques for producing high-quality, nutritious foods with prolonged shelf-life. Frozen foods (FFs) are a significant section of the global food market experiencing rapid growth. It also represents an alternative to small producers in developing countries to add value to their products in a competitive market. However, unfairly, FFs are often perceived as less qualitative than fresh produce, although studies have shown that some FFs have higher nutritional values than fresh products. This study’s aim is to analyze consumers’ perceptions in the two Balkan countries towards FFs. A total of 380 questionnaires were completed in both countries (182 in Kosovo and 198 in Albania). Consumers’ perceptions towards FFs were measured through eleven items using a five-point Likert scale. The items addressed issues related to the quality and safety of FFs, information on FFs, and the impact of origin on the perception of FFs. The differences between populations were tested with the t-test and correlation analysis with the bootstrapping method for sociodemographic factors. The results show that Kosovo consumers generally show a higher positive attitude toward FFs than Albanian consumers. Albanian consumers prefer fresh foods over FFs. The lack of trust in food safety institutions was expressed with concern for the conditions of the frozen chain applied both on the imported and domestic frozen products. Similarly, the findings show that Albanian consumers are willing to pay more than the baseline price to obtain fresh products instead of frozen compared with Kosovo consumers. Additional studies are needed to explore whether the lack of trust in food safety institutions inhibits the successful development of FFs in Albania and Kosovo. In both countries, responsible authorities should help consumers to have a more profound knowledge of the quality of FFs and boost these activities to increase farmers’ incomes and play an active role in reducing food loss and waste.

Robust and Long-Term Cellular Protein and Enzymatic Activity Preservation in Biomineralized Mammalian Cells.

Preservation of evolved biological structure and function in robust engineering materials is of interest for storage of biological samples before diagnosis and development of vaccines, sensors, and enzymatic reactors and has the potential to avoid cryopreservation and its associated cold-chain issues. Here, we demonstrate that "freezing cells in amorphous silica" is a powerful technique for long-term preservation of whole mammalian cell proteomic structure and function at room temperature. Biomimetic silicification employs the crowded protein microenvironment of mammalian cells as a catalytic framework to proximally transform monomeric silicic acid into silicates forming a nanoscopic silica shell over all biomolecular interfaces. Silicification followed by dehydration preserves and passivates proteomic information within a nanoscale thin silica coating that exhibits size selective permeability (<3.6 nm), preventing protein leaching and protease degradation of cellular contents, while providing access of small molecular constituents for cellular enzymatic reaction. Exposure of dehydrated silicified cells to mild etchant or prolonged hydrolysis removes the silica, completely rerevealing biomolecular components and restoring their accessibility and functionality.

Caffeic Acid Phenethyl Ester Alleviates Cryodamage To Lung Cancer Cells During Cryopreservation

Cryopreservation is widely used technique for long-term preservation of viable cells at low temperature. In this process, considering the effects of cryodamage on cells, the application of safe and efficient cryoprotective agents is very important. Caffeic acid phenethyl ester (CAPE) is a natural biological compound which is found in propolis extract and possess beneficial effects such as anti-oxidant, antimicrobial, anti-inflammatory. In the current study was to investigate the cryoprotective effects of CAPE on human lung cancer cell line, A549. Firstly, cells were cryopreserved in freezing medium with/without different concentrations of CAPE (5, 10, and 20 μM). The cells were frozen slowly and kept in liquid nitrogen for one month. After thawing, the cryoprotective effects of CAPE were determined by cell viability, proliferation, colony formation, and gene expression levels. The results showed that 5μM CAPE supplemented freezing medium significantly increased the viability of post thaw A549 cells. 5μM CAPE treatment significantly increased cell proliferation after 24, 48 and 72h since thawing compared to control. 10 μM CAPE did not significantly affect cell viability compared to control group. Also, 5μM CAPE increased the number of A549 colonies compared to 10μM CAPE and control groups. Furthermore, markedly larger colonies were noticed in 5μM CAPE group. In addition, 5μM CAPE significantly increased apoptosis and proliferation-related genes, Akt, NFκB and Bcl-2, expression levels compared to 10μM CAPE and control groups. CAPE may be a potential cryoprotective agent for relieving cryodamage during cryopreservation.

Embalming Techniques for Long-Term Preservation of Bodies

Embalming Techniques for Long-Term Preservation of Bodies

A new low-cost method for long-term preservation of filamentous fungi

Abstract Preservation of fungi for commercial and research purposes for a long time, is a very tedious task. Cryopreservation and lyophilization have been extensively used as long-term preservation techniques. Here, we introduced a novel method for long preservation and fast revival of filamentous fungi using sterile cotton balls (CB). A 16 fungal genera and 47 species represented by 135 strains have been preserved on cotton balls moistened with Potato Dextrose Agar (PDA) and incubated at 25 ± 2 °C until giving sufficient growth, then preserved at 17–20 °C for 36 months. The revival of the fungal growth on PDA was monitored every 6 months. A 135 fungal strains have been revived by the CB method with an overall revival rate of 100% after two years of long-term preservation. While, 76 strains were revived after three years of preservation and the revival rate was 59%. The viability or the mycelial morphology was unaffected by the new method even after three years of preservation. It is an easy, cost-effective, and convenient method for preservation of filamentous fungi for long period without contamination risk. We believe that the CB method is a very convenient method for a cheap delivery of filamentous fungi between research institutes.

Long-term preservation of big data: prospects of current storage technologies in digital libraries

PurposeThe purpose of this paper is to investigate the prospects of current storage technologies for long-term preservation of big data in digital libraries.Design/methodology/approachThe study employs a systematic and critical review of the relevant literature to explore the prospects of current storage technologies for long-term preservation of big data in digital libraries. Online computer databases were searched to identify the relevant literature published between 2000 and 2016. A specific inclusion and exclusion criterion was formulated and applied in two distinct rounds to determine the most relevant papers.FindingsThe study concludes that the current storage technologies are not viable for long-term preservation of big data in digital libraries. They can neither fulfil all the storage demands nor alleviate the financial expenditures of digital libraries. The study also points out that migrating to emerging storage technologies in digital libraries is a long-term viable solution.Research limitations/implicationsThe study suggests that continuous innovation and research efforts in current storage technologies are required to lessen the impact of storage shortage on digital libraries, and to allow emerging storage technologies to advance further and take over. At the same time, more aggressive research and development efforts are required by academics and industry to further advance the emerging storage technologies for their timely and swift adoption by digital libraries.Practical implicationsThe study reveals that digital libraries, besides incurring significant financial expenditures, will suffer from potential loss of information due to storage shortage for long-term preservation of big data, if current storage technologies are employed by them. Therefore, policy makers and practitioners should meticulously choose storage technologies for long-term preservation of big data in digital libraries.Originality/valueThis type of holistic study that investigates the prospects of magnetic drive technology, solid-state drive technology, and data-reduction techniques for long-term preservation of big data in digital libraries has not been conducted in the field previously, and so provides a novel contribution. The study arms academics, practitioners, policy makers, and industry with the deep understanding of the problem, technical details to choose storage technologies meticulously, greater insight to frame sustainable policies, and opportunities to address various research problems.

Long-term cryopreservation of basidiomycetes.

Basidiomycetes have several biotechnological and industrial applications such as enzyme production, bioremediation, pharmaceutical and functional food production. Due to climatic features, the preservation of several basidiomycetes is threatened, and to guarantee the preservation of this genetic resource, the development of long-term preservation techniques is necessary once there is no universal protocol for the cryopreservation of basidiomycetes. Cryopreservation is a technique in which microorganisms are submitted to ultralow temperatures. Therefore, this study aimed to collect information on the main conditions for long-term cryopreservation of basidiomycetes in the last 20 years. Scientific articles on cryopreservation of basidiomycetes published from 1997 to 2016, were researched, and only the studies on two intervals of cryopreservation were considered: from 1 to 2 years and for longer than 2 years. The analyzed conditions of basidiomycete cryopreservation were: most studied genera, cryopreservation temperature, substrate, cryoprotectant (and preservation substrate), cryopreservation period, thawing temperature and cultivation medium after thawing, physiological and genetic stability of basidiomycetes after thawing in cryopreservation. In this review, the viability of the main cryopreservation conditions of basidiomycetes studied in the last 20 years are presented and discussed.

8. 糸状菌のプロトプラストの凍結保存法(昭和60年度第31回凍結及び乾燥研究会研究報告,故根井外喜男先生追悼号)

8. 糸状菌のプロトプラストの凍結保存法(昭和60年度第31回凍結及び乾燥研究会研究報告,故根井外喜男先生追悼号)

Tauroursodeoxycholic acid enhances the development of porcine embryos derived from in vitro-matured oocytes and evaporatively dried spermatozoa

Evaporative drying (ED) is an alternative technique for long-term preservation of mammalian sperm, which does not require liquid nitrogen or freeze-drying equipment, but offers advantages for storage and shipping at ambient temperature and low cost. However, the development of zygotes generated from these sperms was poor. Here, we demonstrated that the supplementation of tauroursodeoxycholic acid (TUDCA), an endogenous bile acid, during embryo culture improved the developmental competency of embryos derived from in vitro matured pig oocytes injected intracytoplasmically with boar ED spermatozoa by reducing the production of reactive oxygen species, the DNA degradation and fragmentation, and the expression of apoptosis-related gene Bax and Bak, and by increasing the transcription of anti-apoptosis gene Bcl-XL and Bcl-2. Furthermore, TUDCA treatment promoted the blastocyst quality manifested by the total cell numbers and the ratio of inner cell mass. Taken together, our data suggest that evaporative drying would be a potentially useful method for the routine preservation of boar sperm in combination with further optimization of subsequently embryo culture conditions.

Freezing dog semen using −80 °C ultra-freezer: Sperm function and in vivo fertility

Long term storage of canine frozen semen is conventionally performed in liquid nitrogen (LN2). However, previous works in freezing canine semen using a −80 °C ultra-freezer (−80°C-UF) showed no differences on sperm quality after thawing. The main objective of this study was to compare the effects of the freezing techniques using LN2 or -80°C-UF on sperm function and in vivo fertility of frozen–thawed dog semen. The sperm-rich fraction of the ejaculate was collected separately from five Chihuahua breed, and each one divided into two aliquots, and frozen and stored in LN2 or -80°C-UF. Sperm function was analyzed for motility and viability, acrosome integrity, mitochondrial function and phosphatidylserine translocation by flow cytometry before and after cryopreservation. A total of 10 bitches were intravaginal inseminated (IVAI; LN2 frozen-thawed semen = 5 and −80°C-UF frozen–thawed semen = 5). Pregnancy status was confirmed 30 d after IVAI by transabdominal ultrasonography and live born puppies at term were recorded. Sperm function parameters were affected for both freezing protocols. Differences (P < 0.05) were found between freezing and storage methods in most of the parameters of sperm function analyzed, except in the phosphatidylserine translocation. The percentages of pregnancies were not different between the two freezing and storage protocols used. Semen freezing and storage using −80 °C UF is an effective technique for long-term preservation of canine spermatozoa.

Development of a sperm cryopreservation protocol of Gangetic Mystus (Mystus cavasius)

Cryopreservation is considered as one of the most useful techniques for long-term preservation of genetic material specially sperm of fish. This study focused on the development of a sperm cryopreservation protocol for indigenous near threatened gulsha (Mystus cavasius) and a number of experiments were conducted for the purpose. To collect milt, male gulsha were sacrificed and milt was suspended in extenders. Different concentrations of NaCl were used to evaluate the activation of sperm motility and it decreased as the concentration of the extending media increased, therefore, motility was completely inhibited at 0.8% and 1.2% NaCl solution when sperm suspended in Kurokura-2 and Alsever’s solution, respectively. The toxicity of cryoprotectants to sperm were evaluated using two cryoprotectants, dimethyl sulfoxide (DMSO) and methanol along with the extenders, Alsever’s solution and Kurokura-2 solution. DMSO and methanol with 5% and 10% concentrations produced significantly higher motility during 5 and 10 min incubation and their 15% concentration found toxic to sperm. Alsever’s solution with 10% DMSO produced best equilibration (83.75±2.39%) as well as post-thaw motility (67.5±3.23%) while Kurokura-2 solution with DMSO produced similar equilibration motility (81.25±2.39%) but the post-thaw motility (50.0±6.12%) was significantly much lower than that of Alsever’s solution. Sperm preserved with Alsever’s solution plus DMSO produced highest fertilization, 72.5±7.5% and hatching, 56.8±5.6% while fresh sperm yielded 85.0±5.0% and 74.8±3.6% fertilization and hatching, respectively. The protocols that have been developed can be used for conservation of genetic materials of M. cavasius and other endangered fish species and new generations of them can be propagated using the cryopreserved sperm.Progressive Agriculture 27 (4): 517-529, 2016

Cryopreservation of hepatocyte (HepG2) cell monolayers: Impact of trehalose

A simple method to cryogenically preserve hepatocyte monolayers is currently not available but such a technique would facilitate numerous applications in the field of biomedical engineering, cell line development, and drug screening. We investigated the effect of trehalose and dimethyl sulfoxide (Me2SO) in cryopreservation of human hepatocellular carcinoma (HepG2) cells in suspension and monolayer formats. HepG2 cell monolayers were incubated for 24h at varying concentrations of trehalose (50–150mM) prior to cryopreservation to identify the optimum concentration for such preincubation. When trehalose alone was used as the cryoprotective agent (CPA), cells in monolayer format did not survive freezing while cells in suspension demonstrated 14% viability 24h after thawing. Only 6–13% of cells in monolayers survived freezing in cell culture medium supplemented with 10% Me2SO, but 42% of cells were recovered successfully if monolayers were preincubated with 100mM trehalose prior to freezing in the Me2SO supplemented medium. Interestingly, for cells frozen in suspension in presence of 10% Me2SO, metabolic activity immediately following thawing did not change appreciably compared to unfrozen control cells. Finally, Raman spectroscopy techniques were employed to evaluate ice crystallization in the presence and absence of trehalose in freezing solutions without cells because crystallization may alter the extent of injury observed in cell monolayers. We speculate that biomimetic approaches of using protective sugars to preserve cells in monolayer format will facilitate the development of techniques for long-term preservation of human tissues and organs in the future.

A simple technique for long-term preservation of leptospires

The viability of six serovars of Leptospira spp. was studied after long storage at -70°C. The bacteria were either preserved in Ellinghausen-McCullough-Johnson-Harris (EMJH) liquid growth medium or in sheep blood added as a cryoprotectant. The viability of the strains was observed on a monthly basis by dark-ground microscopy over a period of 20 months at -70°C. Addition of sheep blood was not significantly advantageous, since leptospires that were stored in EMJH showed a slight increase in number after recovery. The results suggest a very simple and useful technique for long-term preservation of such Leptospira.

96 THE EFFECT OF CHEMICAL DELIPIDATION ON CRYOPRESERVABILITY OF CAT EMBRYOS

Embryo cryopreservation is a desired technique for long-term preservation of embryos. However, the success rate of cryopreserved in vitro produced cat embryos is currently poor. Until recently, the mechanism underlying the cause of cryoinjury that occurs during cooling and cryopreservation is not well understood. This study aimed to examine the effect of chemical delipidation (forskolin) before cryopreservation of 4- to 8-cell cat embryos on post-thaw embryo survival and in vitro developmental capability. A total of 333 cumulus oocyte complexes (COCs) were matured and fertilized in vitro. At 24 h post-IVF, the presumptive embryos were randomly assigned into one of the following groups: 1) non-frozen control (n = 63); 2) forskolin treatment without freezing (n = 52); 3) freezing without forskolin (n = 77); and 4) freezing after forskolin treatment (n = 89). The embryos were cryopreserved using a programmable controlled-rate freezer. After freezing and thawing, the embryos were subsequently cultured in vitro for a further 6 days. The development competence was assessed by morula and blastocyst rates on Days 5 and 8 of their development, respectively. Percentages of cleaved embryos on Day 2 (IVF = Day 0) did not significantly differ among groups, indicating that there was no adverse effect of forskolin on cleavage rates. Furthermore, blastocyst formation rates of cat embryos treated with forskolin (53.5 ± 3.1) did not significantly differ when compared with non-treated controls (54 ± 9.3). Forskolin-treated embryos survived after cryopreservation at a higher rate than non-forskolin treatment, in terms of survival (93.1 ± 2.6 v. 88.2 ± 1.4), morula (56.9 ± 7.6 v. 40.8 ± 5.7), and blastocyst formation (47.6 ± 6.4 v. 35.6 ± 3.6) rates. It is concluded that partial delipidation of cat embryos before cryopreservation improves the cryopreservability of cat embryos. This study demonstrates that intracellular lipid has an impact on cryopreservability of cat embryos. Further study is required to examine in utero development of these delipidated embryos after embryo transfer. This study was financially supported by the Zoo organisation of Thailand and CHE-TRF Senior Research Scholars RTA-5080010.

The conservation of Ustilago tritici in infected seed

Traditional methods of maintaining strains of Ustilago tritici over long periods of time consume large amounts of labour and physical resources, with variable results. These methods generally involve the storage of teliospores of each isolate at temperatures of 2–4 oC. Very little long-term work has been conducted on the survival of mycelium of U. tritici in infected host seed. Our objective was to determine the survival of race T2 of U. tritici as mycelium in infected wheat seed stored at –15 oC for up to 32 years. There was a significant decline in the per cent viability of the inoculated wheat seed over time. Conversely, there was a significant increase in the per cent infection of the viable seed over time. This suggests that either the cold treatment enhances the survival of the mycelium of the pathogen, or the fungus enhances the survival of the wheat seed at low temperatures. Storage of U. tritici as mycelium in infected seed at –15 oC is an effective technique for long-term preservation of this pathogen.

The use of glycerol-preserved corneas in the developing world

Corneal opacity is the third leading cause of blindness in the developing world and encompasses a wide variety of infectious, inflammatory and degenerative eye diseases. Most caes of corneal blindness are treatable with partial or full-thickness keratoplasty, provided adequate corneal tissue and surgical skill is available. However, access to sightrestoring keratoplasty in developing countries is limited by the lack of developed eye banking networks and a critical shortage of tissue suitable for transplantation. Beyond the developed world, corneal transplantation using fresh corneal tissue (FCT) is further hindered by unreliable storage and transportation facilities, unorganized distribution networks, the cost-prohibitive nature of imported tissue, unreliable compliance with medications and follow-up instructions and inadequate health and education services. Glycerol-preserved corneas overcome many of these limitations inherent to the use of FCT. As surgical innovation in lamellar corneal surgery expands the potential use of acellular corneal tissue, long-term preservation techniques are being revisited as a way to increase availability of corneal tissue to corneal surgeons throughout the developing world. Herein, we discuss the advantages of using and the applications for glycerol-preserved corneal tissue throughout the developing world.