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Abstract In the last decades, gut microbiota and its role in mammal host development and health have been increasingly investigated. Metabolites produced by gut microbiota can affect intestinal homeostasis and immune system maturity and activation, and in turn, they can influence the health and growth performance of livestock. Therefore, a better understanding of the functional metabolic capability of the gut microbiota would be appreciated by the scientific community. In this study, the BiologTM Ecoplates technology was applied for studying the metabolic potential of the aerotolerant microbial community of pig fecal samples, evaluating the interference of different storage conditions and cell concentrations. The length of time for which a fecal sample maintained detectable and unchanged microbial metabolic activity was also investigated. Two assays aimed to evaluate differences in the metabolic activities between fresh and snap-frozen fecal samples at different dilutions and at different lengths of times of preservation at −80°C were carried out. The biodiversity and the predicted functionality of the entire bacterial community through a targeted metagenomic approach were also explored. The results highlighted that snap freezing of fecal samples preserved the metabolic activity of the microbial community when compared to fresh feces. Sample storage at −80 °C did not significantly affect the metabolic activity of the microbial community, which was stable for 150 days. Furthermore, the highest metabolic activity was detected with 1:2 to 1:5 dilutions of the stock suspension. BiologTM Ecoplates technology is a rapid and useful method to explore microbial communities’ metabolism in animal fecal samples contributing to investigate host animal physiology. Key points • Freezing of samples can preserve the functional activity of the aerotolerant microbial community for 150 days. • The concentration of microbial cells strongly influences metabolic activity detection. • Sequencing coupled with the BiologTMEcoplates could be a strategy to evaluate the metabolic potential of the microbiota of the fecal sample. Graphical abstract

Objective: Although transcriptomic assessments of small samples using high-throughput techniques are usually performed on fresh or frozen tissues, there is a growing demand for those performed on stained cellular specimens already used for diagnostic purposes. Study Design: The possibility of detecting mRNAs and microRNAs (miRNAs) from routinely processed cytological samples using nCounter® technology was explored. Fresh samples from pleural and peritoneal effusions were analyzed using 2 parallel methods: samples were smeared and routinely stained using the May-Grünwald-Giemsa or Diff-Quik® method and mounted using conventional methods, and they were also studied following a snap freezing method, in which samples were maintained at −80°C until use. mRNAs and miRNAs were assessed and compared after total RNA extraction from both routinely processed samples and their matched frozen controls. Results: A good concordance was found between the gene expression measured in routinely processed samples and their matched frozen controls for the majority of mRNAs and miRNAs tested. However, the standard deviation of low-expressed miRNA was high. Conclusions: Although nCounter® technology is a robust method to measure and characterize both mRNAs and miRNAs from routinely processed cytological samples, caution is recommended for the interpretation of low-expressed miRNA.

Background: High-throughput transcript sequencing plays an important role in the study of hepatocellular carcinoma (HCC) occurrence and development. High-quality biospecimens, especially high-quality RNA, are the most basic prerequisites for obtaining good transcript sequencing data. Our purpose was to explore the treatment conditions of in vitro ischemic tissue samples that can be used to obtain high-integrity RNA before freezing the samples in liquid nitrogen. Materials and Methods: Postoperative tumor tissues (T) and adjacent normal tissues (AN) from 50 HCC patients were randomly selected from May 5, 2017, to June 15, 2017. Postoperative tissue specimens from each HCC patient were stratified by tissue type (T or AN), ischemia time (minutes), and ischemia temperature (°C) into 16 groups: T-4°C-15 minutes, T-4°C-30 minutes, T-4°C-60 minutes, T-4°C-120 minutes, T-24°C-15 minutes, T-24°C-30 minutes, T-24°C-60 minutes, T-24°C-120 minutes, AN-4°C-15 minutes, AN-4°C-30 minutes, AN-4°C-60 minutes, AN-4°C-120 minutes, AN-24°C-15 minutes, AN-24°C-30 minutes, AN-24°C-60 minutes, and AN-24°C-120 minutes. RNA integrity was detected by RNA integrity number (RIN) and 1% agarose gel electrophoresis. Results: At an ischemia temperature of 4°C and ischemia time of >30 minutes, the RIN of T began to decrease. RIN also gradually decreased in T at an ischemia temperature of 4°C and in both T and AN at an ischemia temperature of 24°C for ischemia times 15, 30, 60, and 120 minutes. For an ischemia time ≤15 minutes and ischemia temperature 4°C or 24°C, the RINs of T and AN were significantly different. Furthermore, at ischemia temperature 4°C and ischemia time 30 and 60 minutes or ischemia temperature 24°C and ischemia time 30 minutes, the RIN of T was higher compared with AN. However, there was no significant difference in RIN between T and AN under other treatment conditions. Conclusions: Tissue quality is adversely affected by ischemia time and ischemia temperature. Therefore, temporary ischemia time (≤15 minutes) before snap freezing is key for maintaining high-integrity RNA in HCC tissues.

Sample preservation is a critical procedure in any research that relies on molecular tools and is conducted in remote areas. Sample preservation options include low and room temperature storage, which require freezing equipment and specific buffering solutions, respectively. The aim of the present study was to investigate whether DNA/RNA Shield 1x from Zymo Research and DESS (Dimethyl sulfoxide, Ethylenediamine tetraacetic acid, Saturated Salt) solution performed similarly to snap freezing in liquid nitrogen. Soil samples were stored for 1 month in each of the buffers and without any solution at a range of temperatures: -20, +4, and +23°C. All treatments were compared to the "optimal treatment", namely, snap freezing in liquid nitrogen. The quality and quantity of DNA were analyzed, and the microbial community structure was investigated in all samples. The results obtained indicated that the quantity and integrity of DNA was preserved well in all samples; however, the taxonomic distribution was skewed in samples stored without any solution at ambient temperatures, particularly when analyses were performed at lower taxonomic levels. Although both solutions performed equally well, sequencing output and OTU numbers in DESS-treated samples were closer to those snap frozen with liquid nitrogen. Furthermore, DNA/RNA Shield-stored samples performed better for the preservation of rare taxa.

Seminal oxidative stress (OS) is a major contributing factor to male infertility. Semen analysis cannot identify reactive oxygen species (ROS), which can be measured using a chemiluminescence assay. Measurement of redox potential provides a more comprehensive assessment of OS, although the test has yet to be fully validated. This study aimed to validate the MiOXsys analyser for measuring static oxidation-reduction potential (sORP). Results demonstrated that duplicate measurements must be taken, sensors must be batch tested, and sockets should be regularly changed to avoid inconsistency in measurement. Measurement of sORP using MiOXsys exhibited good reproducibility across different operators (p = 0.469), analysers (p = 0.963) and days (p = 0.942). It is not affected by mechanical agitation (p = 0.522) or snap freezing and thawing (p = 0.823). The stability of sORP over time requires further verification, particularly in samples with high initial sORP. Measurement is temperature sensitive between 2 and 37°C, significantly increasing with increasing temperature (p = 0.0004). MiOXsys is a more stable assay for assessing OS than chemiluminescence methods and permits greater flexibility for sample handling. MiOXsys could be implemented to complement semen analysis as part of routine diagnostic testing for male infertility and may be useful in identifying contributing factors to idiopathic infertility.

Metabolomic technologies including imaging mass spectrometry (IMS; also called mass spectrometry imaging, MSI, or matrix-assisted laser desorption/ionization-mass spectrometry imaging, MALDI MSI) are important methods to evaluate levels of many compounds in brain with high spatial resolution, characterize metabolic phenotypes of brain disorders, and identify disease biomarkers. ATP is central to brain energetics, and reports of its heterogeneous distribution in brain and regional differences in ATP/ADP ratios reported in IMS studies conflict with earlier studies. These discordant data were, therefore, analyzed and compared with biochemical literature that used rigorous methods to preserve labile metabolites. Unequal, very low regional ATP levels and low ATP/ADP ratios are explained by rapid metabolism during postmortem ischemia. A critical aspect of any analysis of brain components is their stability during and after tissue harvest so measured concentrations closely approximate their physiological levels in vivo. Unfortunately, the requirement for inactivation of brain enzymes by freezing or heating is not widely recognized outside the neurochemistry discipline, and procedures that do not prevent postmortem autolysis, including decapitation, brain removal/dissection, and 'snap freezing' are commonly used. Strong emphasis is placed on use of supplementary approaches to calibrate metabolite abundance in units of concentration in IMS studies and comparison of IMS results with biochemical data obtained by different methods to help identify potential artifacts.

Background Digital multiplex gene expression profiling is overcoming the limitations of many tissue-processing and RNA extraction techniques for the reproducible and quantitative molecular classification of disease. We assessed the effect of different skin biopsy collection/storage conditions on mRNA quality and quantity and the NanoString nCounter™ System’s ability to reproducibly quantify the expression of 730 immune genes from skin biopsies. Methods Healthy human skin punch biopsies (n = 6) obtained from skin sections from four patients undergoing routine abdominoplasty were subject to one of several collection/storage protocols, including: i) snap freezing in liquid nitrogen and transportation on dry ice; ii) RNAlater (ThermoFisher) for 24 h at room temperature then stored at − 80 °C; iii) formalin fixation with further processing for FFPE blocks; iv) DNA/RNA shield (Zymo) stored and shipped at room temperature; v) placed in TRIzol then stored at − 80 °C; vi) saline without RNAse for 24 h at room temperature then stored at − 80 °C. RNA yield and integrity was assessed following extraction via NanoDrop, QuantiFluor with RNA specific dye and a Bioanalyser (LabChip24, PerkinElmer). Immune gene expression was analysed using the NanoString Pancancer Immune Profiling Panel containing 730 genes. Results Except for saline, all protocols yielded total RNA in quantities/qualities that could be analysed by NanoString nCounter technology, although the quality of the extracted RNA varied widely. Mean RNA integrity was highest from samples that were placed in RNALater (RQS 8.2 ± 1.15), with integrity lowest from the saline stored sample (RQS 10,000 similar across extraction protocols. Conclusions A variety of processing methods can be used for digital immune gene expression profiling in mRNA extracted from skin that are comparable to snap frozen skin specimens, providing skin cancer clinicians greater opportunity to supply skin specimens to tissue banks. NanoString nCounter technology can determine gene expression in skin biopsy specimens with a high degree of sensitivity despite lower RNA yields and processing methods that may generate poorer quality RNA. The increased sensitivity of digital gene expression profiling continues to expand molecular pathology profiling of disease.

The objective of this study was to determine the combined impacts of initial sub-primal freezing with subsequent freezing of manufactured pork patties on quality attributes and oxidative stability. Patties were manufactured (n = 3 batches) from pork leg muscles (M. biceps femoris and M. semitendinosus) frozen at different methods including still-air freezing (SAF), blast freezing (BF), and cryogenic freezing (CF). Then, patties were subjected to additional freezing treatments. Frozen/thawed patties exhibited increased cooking loss, springiness, and chewiness, lipid and protein oxidation, and decreased protein solubility compared to unfrozen counterparts (P .05). However, patties from CF legs maintained similar protein solubility and lipid/protein oxidation compared to unfrozen controls (P .05), while significantly minimizing sub-primal thawing loss and oxidation compared to patties from SAF. The results of the present study suggest the importance of initial freezing rate of sub-primals with subsequent freezing on quality characteristics of frozen/thawed meat patties.

Aiming to increase the reproducibility of biomedical research results, biobanks obtain human tissues of the highest quality and carry out different storage methods adapted to the needs of analytical technique to be performed by the biomedical researchers. However, there is much controversy and little data concerning the real impact of different stabilization methods on tissue quality, integrity and functionality of derived biomolecules. The influence of four stabilization methods [RNAlater (RNL), snap freezing (SF), snap freezing using Optimal Cutting Tissue compound (SF-OCT) and formalin-fixed paraffin-embedded (FFPE)] on RNA quality and integrity was evaluated in paired samples of lung tissue. RNA integrity was evaluated through PCR-endpoint assays amplifying six fragments of different length of the HPRT1 gene and RNA Integrity Number (RIN). To evaluate the difference of tissue functionality among the stabilization methods tested, RT-qPCRs were performed focusing on the differential expression of the HPRT1, SNRPD3 and Jun genes. RNA from the samples preserved with the RNL or SF-OCT method showed better integrity compared to SF and FFPE, measured by PCR-endpoint and RTqPCR assays. However, only statistically significant differences were observed between the RNA from FFPE and other stabilization methods when gene expression of HPRT1, SNRPD3 and Jun housekeeping genes were determined by RT-qPCR. For the three mentioned genes, Cq and RIN values were highly correlated. The present work describes the fragility of SF samples, being critical the moment just before RNA extraction, although further experiments of tissue RNA are needed. Standardization pre-analytic workflow can lead to improved reproducibility between biomedical research studies. The present study demonstrated clear evidences about the impact of the stabilization method on RNA derived from lung human tissue samples., We thank all volunteers of Pulmonary Biobank Consortium for the participation and sample donation. We also thank Institut d'Investigacio Sanitaria de les Illes Balears (IdISBa) for publication funding (Liberi 2020 Program). This work was funded by the Ministerio de Ciencia, Innovacion y Universidades of Spain and Instituto de Salud Carlos III (PI16/00528) through the Spanish Biobank Network (PT17/0015/0001) and granted by Conselleria d'Innovacio, Recerca i Turisme del Govern de les Illes Balears (TEC/002/2017).

Background Human muscle biopsies are increasingly important for diagnosis, research, and to monitor therapeutic trials. We examined the use of a self-contained, vacuum-assisted biopsy system and a novel muscle freezing technique to improve, simplify, and standardize human muscle biopsy collection and cryopreservation in older adults. Methods The Vacora vacuum-assisted biopsy system was deployed in muscle biopsies of 12 individuals ranging in age from 57 to 80 years. This office-based approach was well tolerated as it is minimally invasive, uses only local anesthetic, and has a quick recovery. To maximize biopsy sample quality and reproducibility, we developed a novel muscle sample freezing protocol. Fresh human and mouse muscle biopsy samples were placed into readily available tissue cassettes followed by direct freezing in liquid nitrogen. After this modified snap freezing protocol, frozen muscle samples were enrobed in OCT for cryosectioning. We examined the effect of this freezing approach in histological sections of rodent and human muscle samples. ResultsThe Vacora Biopsy System provided as many as four skeletal muscle core samples from a single biopsy site. Biopsy samples from 12 older adults weighed an average of 147.5 ± 11 mg each and had a consistent size and shape. There were no complications, and the residual scar is less than 1cm. The freezing method using standard tissue cassettes with direct freezing in liquid nitrogen yielded high quality cryopreserved muscle tissue suitable for histological analysis without the need for isopentane and with little to no freeze-thaw damage. Conclusions These enhancements have streamlined and improved the consistency of our muscle biopsy protocol and provide sufficient high-quality sample for multi-dimensional downstream studies of human muscle in aging and disease.

Transcriptome analyses allow the distinguishing of pancreatic ductal adenocarcinoma (PDAC) subtypes, exhibiting different prognoses and chemotherapy responses. However, RNA extraction from pancreatic tissue is cumbersome and has been performed mainly from surgical samples, which are representative of &lt, 20% of cases. The majority of PDAC patients undergo endoscopic ultrasound (EUS)-guided tissue acquisition (EUS-TA), but RNA has been rarely extracted from EUS-TA with scanty results. Herein, we aimed to determine the best conditions for RNA extraction and analysis from PDAC EUS-TA samples in order to carry out molecular analyses. PDAC cases underwent diagnostic EUS-TA, with needles being a 25G fine needle aspiration (FNA) in all patients and then either a 20G lateral core-trap fine needle biopsy (FNB) or a 25G Franseen FNB, the conservation methods were either snap freezing, RNALater or Trizol. RNA concentration and quality (RNA integrity index, RIN) were analyzed and a panel of genes was investigated for tissue contamination and markers of molecular subtype and aggressivity through qRT-PCR. Seventy-four samples from 37 patients were collected. The median RNA concentration was significantly higher in Trizol samples (10.33 ng/uL) compared with snap frozen (0.64 ng/uL, p &lt, 0.0001) and RNALater (0.19 ng/uL, 0.0001). The RIN was similar between Trizol (5.15) and snap frozen samples (5.85), while for both methods it was higher compared with RNALater (2.7). Among the needles, no substantial difference was seen in terms of RNA concentration and quality. qRT-PCR analyses revealed that samples from all needles were suitable for the detection of PDAC subtype markers (GATA6 and ZEB1) and splice variants associated with mutational status (GAP17) as well as for the detection of contaminating tissue around PDAC cells. This is the first study that specifically investigates the best methodology for RNA extraction from EUS-TA. A higher amount of good quality RNA is obtainable with conservation in Trizol with a clear superiority of neither FNA nor FNB needles. RNA samples from EUS-TA are suitable for transcriptome analysis including the investigation of molecular subtype and splice variants expression.

To study the effect of different freezing methods on the quality changes of cuttlefish during the frozen storage of cuttlefish, fresh cuttlefish was treated with six freezing methods (refrigerator direct-freezing, saline solution impregnation freezing, flat freezing, tunnel type continuous freezing, air-blast freezing and liquid nitrogen freezing) and then stored at −18 °C for 90 days. The time to pass the maximum ice crystal generation zone for the above six freezing methods in this experiment was 165.5, 67.5, 34.5, 21.8, 20.4 and 1.5 min, respectively. In this study, water retention (thawing loss rate, centrifugal loss rate, and cooking loss), pH, malondialdehyde content, TVB-N value, and sulfhydryl content were measured to evaluate the quality after thawing. Protein secondary structure was measured by attenuated total reflection infrared spectroscopy (ATR-FTIR), water migration was determined by low-field NMR, and muscle microstructure was observed by scanning electron microscopy. The results showed that among the six freezing methods, liquid nitrogen freezing took the shortest time to pass through the maximum ice crystal generation zone. And it had the highest water retention, the lowest TVB-N content, the highest sulfhydryl content and the least irregular curling of protein secondary structure after 90 days of frozen storage. However, liquid nitrogen freezing can cause cracks and breakage in cuttlefish due to cryogenic fracture caused by ultra-low temperature, which affects its sensory evaluation. Although the freezing speed of flat freezing is faster than refrigerator direct-freezing and saline solution impregnation freezing, the muscle is extruded and deformed during the freezing process, and the damage is more serious, and the frozen storage quality is the worst. The comprehensive analysis results showed that the freezing speed of air- blast freezing was faster and the quality of cuttlefish in the freezing process was better, which was the more recommended freezing method, and this study provided some theoretical basis for the selection of freezing method in the actual production of cuttlefish.

The primary aim of the industrial use of sourdough is to improve bread quality and to replace additives. This study investigates the applicability of frozen stored barley sourdough in wheat bread. Sourdough was fermented with Weissella cibaria in the presence of sucrose and xylanase, and stored frozen for 4, 8, or 12 weeks. During storage, the number of viable cells in the sourdough declined. Softer dough and crumb were obtained after the sourdough was stored for a longer period (4 weeks), while bread specific volume was unaffected by the sourdough storage time. The highest bread specific volume, shape, crumb softness, and sensory liking were obtained with 20% of the sourdough. This study demonstrates the possibility for the industrial application of frozen barley sourdough, since it can improve the quality of bread compared to the native barley flour.

Introduction: Personalized treatment, supported by biomarkers, would improve survival of ovarian cancer patients. RNA molecules are potentially important biomarkers. The Danish CancerBiobank provides an infrastructure for handling and storage of biological material, including RNA, from Danish cancer patients. The aim of this study was to investigate the effects of handling-time and fresh-freezing versus RNAlater® fixation on RNA degradation in solid tissue from pelvic mass samples. Materials and Methods: We evaluated RNA quality in surgical tissue from patients with a pelvic mass. Corresponding samples were either fresh-frozen or fixed in RNAlater, at eight different time points after the surgery. Integrity was measured using a bioanalyzer, and the amount and quality were further investigated by quantitative reverse transcription-polymerase chain reaction measuring the expression of housekeeping genes B2M and HPRT1. Results: Our results show that tissue RNA is stable up to at least 180 minutes after the surgery, as the quality was comparable to the quality of RNA handled immediately. Likewise, patient RNA was of acceptable quality after both fresh-frezing and RNAlater fixation, but RNAlater fixation was slightly more effective for RNA preservation. Discussion and Conclusion: Our data suggest that RNA in pelvic mass samples is relatively stable. Knowledge about RNA stability is an important prerequisite for research in RNA biomarkers, where the challenge is to balance the need for careful RNA handling and storage with the need for effective large-scale biobanking in a busy clinical setting where patient treatment is the main priority.

A fresh and frozen high-quality patient bio-sample is required in molecular medicine for the identification of disease-associated mechanism at molecular levels. A common cooling procedure is immersing the tissue enclosed in a vial in a coolant such as liquid nitrogen. This procedure is not user friendly and is laborious as reducing the lag time from excision time to freezing depends on the logistic organizational structure within a hospital. Moreover snapfreezing must be done as soon as possible after tissue excision to preserve the tissue quality for molecular tests. Herein, we report an electrically powered snap freezing device as an alternative to quenching the vial in liquid nitrogen and therefore can be used directly at the location where the tissue is acquired. This device also facilitates the study of the effect of freezing conditions on the various molecular processes in the samples. Cooling experiments of a vial in the snap freezing device show that the cooling rates similar to or faster than quenching in liquid nitrogen are feasible. We performed experiments with several set point conditions and compared the results with a mathematical model.

Beef is nutritionally well-suited to elderly people but it has a relatively tough texture compared with many other foods. Strategies to develop meat products requiring reduced mastication effort could potentially enhance intake, and therefore nutritional status, in the elderly. Cooking steaks ‘sous-vide’ enhances tenderness and increase retention of natural juices. Freezing may also have a beneficial effect on tenderness due to cellular disruption through ice crystal formation. Five thermal treatments, including sous-vide cooking at 60 °C for 270 min (SV), cooking in a circulating water bath until core temperature of 70 °C was achieved (C), freezing at −20 °C for 48 h (F) and their combinations (i.e. SV, SVC, FC, FSV & FSVC) and a control, were applied to M. semitendinosus beef steaks. SV and FSVC significantly (P

Analysis of multiple analytes from biological samples can be challenging as different analytes require different preservation measures. Heat induced enzymatic inactivation is an efficient way to preserve proteins and their modifications in biological samples but RNA quality, as measured by RIN value, has been a concern in such samples. Here, we investigate the effect of heat stabilization compared with standard snap freezing on RNA quality using two RNA extraction protocols, QiaZol with and without urea pre-solubilization, and two RNA quality measurements: RIN value, as defined by the Agilent Bioanalyzer, and an alternative qPCR based method. DNA extraction from heat stabilized brain samples was also examined. The snap frozen samples had RIN values about 1 unit higher than heat stabilized samples for the direct QiaZol extraction but equal with stabilized samples using urea pre-solubilization. qPCR based RNA quality measurement showed no difference in quality between snap frozen and heat inactivated samples. The probable explanation for this discrepancy is that the RIN value is an indirect measure based on rRNA, while the qPCR score is based on actual measurement of mRNA quality. The DNA yield from heat stabilized brain tissue samples was significantly increased, compared to the snap frozen tissue, without any effects on purity or quality. Hence, heat stabilization of tissues opens up the possibility for a two step preservation protocol, where proteins and their modifications can be preserved in the first heat based step, while in a second step, using standard RNA preservation strategies, mRNA be preserved. This collection strategy will enable biobanking of samples where the ultimate analysis is not determined without loss of sample quality.

The effects of high pressure processing (HPP) on barramundi muscle at 0.1 MPa (control), 150 MPa, 200 MPa, 250 MPa and 300 MPa at 11 ± 2 °C for 3 min were studied before and after immediate blast freezing and storage at −18 °C for up to 18 weeks. Physicochemical measurements including colour, texture profile, drip loss, pH, free fatty acids and thiobarbituric acid (TBA) values were carried out. HPP at 200 MPa significantly increased hardness and delayed lipid oxidation during frozen storage (for up to 18 weeks) without resulting in a cooked appearance of barramundi muscle. However, drip loss of barramundi muscle pressurised at 200 MPa was similar to control samples and during frozen storage. Application of pressures higher than 250 MPa for 3 min resulted in the loss of visual freshness and increased drip loss of barramundi muscle. Thus, application of HPP at an appropriate level (150–200 MPa) before freezing could improve the quality of frozen barramundi muscle.

In the last two decades, over 100 studies have investigated the structure of the coral microbiome. However, as yet there are no standardized methods applied to sample preservation and preparation, with different studies using distinct methods. There have also been several comparisons made of microbiome data generated across different studies, which have not addressed the influence of the methodology employed over each of the microbiome datasets. Here, we assess three different preservation methods; salt saturated dimethyl sulfoxide (DMSO) – EDTA, snap freezing with liquid nitrogen and 4% paraformaldehyde solution, and two different preparation methodologies; bead beating and crushing, that have been applied to study the coral microbiome. We compare the resultant bacterial assemblage data for two coral growth forms, the massive coral Goniastrea edwardsi and the branching coral Isopora palifera. We show that microbiome datasets generated from differing preservation and processing protocols are comparable in composition (presence/absence). Significant discrepancies between preservation and homogenization methods are observed in structure (relative abundance), and in the occurrence and dominance of taxa, with rare (low abundance and low occurrence) phylotypes being the most variable fraction of the microbial community. Finally, we provide evidence to support chemical preservation with DMSO as effective as snap freezing samples for generating reliable and robust microbiome datasets. In conclusion, we recommend where possible a standardized preservation and extraction method be taken up by the field to provide the best possible practices for detailed assessments of symbiotic and conserved bacterial associations.

Barramundi (Lates calcarifer) fillets were subjected to two different ice forms (pre-freezing treatments: slurry ice [SI] and flake ice [FI]) for 0 (before freezing) and 1 h and 1, 2, 4, 8 and 16 days prior to blast freezing at −80C for 8 h. Fillets were then stored in a freezer at −20C for 20 days. Microbiological and physiochemical analysis were then carried out. Fillets that were exposed to pre-freezing treatments in FI demonstrated larger increase in total volatile base nitrogen, total viable count and pH; larger decrease in protein content; and smaller increase in moisture content compared with SI at the end of the frozen storage. Fillets exposed to pre-freezing treatments in FI are significantly (P

Background Placental dysfunction is related to many pregnancy complications, but collecting placental specimens for investigation in large scale epidemiologic studies is often infeasible. Standard procedures involving immediate collection after birth and snap freezing are often cost prohibitive. We aimed to collect pilot data regarding the feasibility and precision of a simpler approach, the collection of tissue samples following 24 hours of refrigeration of whole placentae at 4°C, as compared to the “gold standard” of snap freezing excised tissue within 40 minutes of delivery for the assessment of inflammatory cytokines. Methods Placentae were collected from 12 women after delivering live-born singleton babies via uncomplicated vaginal delivery. Two placentae were utilized to establish laboratory tissue processing and assay protocols. The other 10 placentae were utilized in a comparison of three tissue collection conditions. Specifically, key inflammatory cytokines were measured in 3 sections, representing three collection conditions. Sections 1 (full thickness) and 2 (excised prior to freezing) were obtained within 40 minutes of delivery and snap frozen in liquid nitrogen, and section 3 (full thickness) was obtained after refrigerating the placenta at 4°C for 24 hours. Results IL-6, IL-10, and IL-8 all had comparable concentrations and variability overall in all three section types. Levels of tumor necrosis factor alpha (TNF-α) were too low among samples to reliably measure using immunoassay. Conclusions Refrigeration of placentae prior to processing does not appear to compromise detection of these cytokines for purposes of large scale studies. These findings provide a framework and preliminary data for the study of inflammatory cytokines within the placenta in large scale and/or resource-limited settings.

International audience; Neuroblastoma (NB) is the most common type of extracranial solid tumor in children with a high prevalence in toddlers. For childhood cancer survivors, preservation of reproductive potential is an important factor for quality of life. The optimization of NB minimal residual disease (MRD) detection in testicular tissue is crucial to evaluate the risk of malignant cell reintroduction. The first step in the present study was to assess the accuracy of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to detect tyrosine hydroxylase (TH), paired-like homeobox 2b (PHOX2B) and doublecortin (DCX) mRNA expression in frozen/thawed testicular tissues of patients with non-obstructive azoospermia (NOA) contaminated (in vitro model) with an increasing number of IMR-32 and SK-N-SH NB cells. Testicular tissues were frozen by slow or snap freezing. The second step was to determine the expression levels of these markers in testicular samples from 4 pre-pubertal males (2 with stage IV NB and 2 with non-NB malignancy). The yield of extracted RNA was similar in testicular samples frozen by slow or snap freezing. In the in vitro model, TH and DCX transcripts were detected in uncontaminated testicular tissues, whereas PHOX2B mRNA was not detected. There was a strong positive association between the number of NB cells used for contamination and PHOX2B transcript levels. For IMR-32 and SK-N-SH NB cell lines, specificity and sensitivity rates of detection were 100% for PHOX2B following in vitro contamination with 10 tumor cells. In testicular samples from pre-pubertal males with and without NB, PHOX2B mRNA expression was not observed, but high expression levels of TH and DCX mRNA were detected, which were similar to expression detected in the in vitro model. Among the markers used in blood and bone marrow for NB MRD studies, the detection of PHOX2B transcripts by RT-qPCR may provide an accurate assessment of NB cells in testicular tissues from males who require fertility preservation.

Dry ice is commonly used as a cooling agent in the preclinical research environment. It permits rapid freezing of tissues and organs through liquid media. Laboratories depend on a constant supply of dry ice because it disintegrates in a matter of days. Here we tested whether commercially available pebble gravel could be used as dry ice replacement for snap freezing mouse brains in 2-methylbutane. We monitored 2-methylbutane temperature for one hour and tested different setups with the focus on creating a practical solution that can be used in every research laboratory. While gravel pebbles cannot replace dry ice in the laboratory entirely, they present a useful addition as readily available and reusable cooling agent.

[EN] Infusion of antioxidants into vegetables is a new food strategy managed by matrix processing. Raw and blanched apple were air- or freeze-dried. In the case of freeze-dried samples, different freezing methods were previously applied: conventional (¿28°C), blast freezing (¿30°C), and liquid N2 (¿196°C). Afterwards, air- and freeze-dried samples at different conditions were impregnated with a concentrated (40°Brix) tea extract and finally, air-dried for their stabilization. Total phenolic content (TPC), antioxidant capacity (AC), enzymatic activity, and microstructure were analyzed. Regardless of pretreatments, the impregnation and the further drying improved the antioxidant potential. Samples with the most porous microstructure free of degradative enzymes provided high AC (78.5¿±¿0.9¿mg Trolox/g dried matter) and TPC (16.7¿±¿0.2¿mg GAE/g dried matter)., The authors thank the Ministerio de Educacion, Cultura y Deporte of Spain for its financial support through fellowships from the Programa de Formacion de Profesorado Universitario del Programa Nacional de Formacion de Recursos Humanos de Investigacion and the subprogramas de Formacion y de Movilidad dentro del Programa Estatal de Promocion del Talento y su Empleabilidad, en el marco del Plan Estatal de Investigacion Cientifica y Tecnica y de Innovacion 2013-2016 en I+D+i. This research has also been supported by Unilever Research and Development Vlaardingen and by the Generalitat Valenciana through the project PROMETEOII/2014/005.

BACKGROUND Sample freezing is frequently used in oenological laboratories as a compromise solution to increase the number of samples that can be analysed, despite the fact that some grape characteristics are known to change after frozen storage. However, freezing is usually performed using standard freezers, which provide a slow freezing. The aim of this work was to evaluate whether blast freezing would decrease the impact of standard freezing on grape composition. RESULTS Grape quality parameters were assessed in fresh and in frozen stored samples that had been frozen using three different procedures: standard freezing and blast freezing using either a blast freezer or an ultra-freezer. The implications of frozen storage in grape samples reported in earlier research were observed for the three freezing methods evaluated. Although blast freezing improved repeatability for the most problematic parameters (tartaric acidity, TarA; total phenolics, TP), the improvement was not important from a practical point of view. However, TarA and TP were relatively repeatable among the three freezing procedures, which suggests that freezing had an effect on these parameters independently of the method used . According to our results, the salification potential of the must is probably implied in the changes observed for TarA, whereas for TP the precipitation of protoanthocyanins after association with cell wall material is hypothesized to cause the lack of repeatability between fresh and frozen grapes. CONCLUSIONS Blast freezing would not imply a great improvement if implemented in oenological laboratories, at least for the parameters included in this study. © 2013 Society of Chemical Industry

Air refrigerating systems offer a suitable alternative to vapour compression systems. Air can be used as working fluid in gas compression cycles for refrigeration, air conditioning and heat pump systems. This paper gives some results of theoretical investigation into improved air cycles for refrigeration systems and a comparison is made with their performances. The case-study relates to a dual purpose refrigerating plant. A blast freezing tunnel gives the coldest air stream and, in cascade, the air coming out from the tunnel is used in a channel network for feeding a lot of chilling cells. The case-study analysed indicates that there is a real possibility to build plants which will be equipped with air inverse cycles.

The objective of this study was to evaluate processing methods for frozen beef subprimals; the effects of freezing and thawing rates on tenderness, sensory properties, and retail display were evaluated. There were 6 treatments: fresh, never frozen 14 d wet aged (14D); fresh, never frozen 21 d wet aged (21D); blast frozen-fast thawed (BF); blast frozen-slow thawed (BS); conventionally frozen-fast thawed (CF); and conventionally frozen-slow thawed (CS). All frozen beef subprimals were aged for 14 d before freezing. Three beef subprimal cuts, rib eye roll (n=90), strip loin (n=90), and top sirloin butt (n=90), were used with 3 replications of 5 samples per treatment per week (total of 9 wk, n=270). Blast freezing occurred by placing spacers between the boxes of meat on pallets at -28°C with high air velocity for 3 to 5 d. Conventional freezing occurred with boxes of meat stacked on pallets and placed in a -28°C freezer with minimal air movement for at least 10 d. Fast thawing of subprimals (to an internal temperature of -1°C to 1°C) occurred by immersion in a circulating water bath ( 0.05). All LL and LT beef steaks had approximately 4 d to 40% discoloration, and all GM steaks had over 3 d to 40% discoloration. Steaks from the LL and LT began to discolor at about 3 d, and the GM began to discolor after 1 d. For all beef subprimals, purge loss during storage and thawing was significantly greater for the slow-thawed subprimals (P

Successful molecular analyses of human solid tissues require intact biological material with well-preserved nucleic acids, proteins, and other cell structures. Pre-analytical handling, comprising of the collection of material at the operating theatre, is among the first critical steps that influence sample quality. The aim of this study was to compare the experimental outcomes obtained from samples collected and stored by the conventional means of snap freezing and by PAXgene Tissue System (Qiagen). These approaches were evaluated by measuring rRNA and mRNA integrity of the samples (RNA Quality Indicator and Differential Amplification Method) and by gene expression profiling. The collection procedures of the biological material were implemented in two hospitals during colon cancer surgery in order to identify the impact of the collection method on the experimental outcome. Our study shows that the pre-analytical sample handling has a significant effect on the quality of RNA and on the variability of qPCR data. PAXgene collection mode proved to be more easily implemented in the operating room and moreover the quality of RNA obtained from human colon tissues by this method is superior to the one obtained by snap freezing.

Introduction: Metabolic profiling of intact tumor tissue by high-resolution magic angle spinning (HR MAS) MR spectroscopy (MRS) provides important biological information possibly useful for clinical diagnosis and development of novel treatment strategies. However, generation of high-quality data requires that sample handling from surgical resection until analysis is performed using systematically validated procedures. In this study, we investigated the effect of postsurgical freezing delay time on global metabolic profiles and stability of individual metabolites in intact tumor tissue. Materials and methods: Tumor tissue samples collected from two patient-derived breast cancer xenograft models (n = 3 for each model) were divided into pieces that were snap-frozen in liquid nitrogen at 0, 15, 30, 60, 90, and 120 min after surgical removal. In addition, one sample was analyzed immediately, representing the metabolic profile of fresh tissue exposed neither to liquid nitrogen nor to room temperature. We also evaluated the metabolic effect of prolonged spinning during the HR MAS experiments in biopsies from breast cancer patients (n = 14). All samples were analyzed by proton HR MAS MRS on a Bruker Avance DRX600 spectrometer, and changes in metabolic profiles were evaluated using multivariate analysis and linear mixed modeling. Results: Multivariate analysis showed that the metabolic differences between the two breast cancer models were more prominent than variation caused by freezing delay time. No significant changes in levels of individual metabolites were observed in samples frozen within 30 min of resection. After this time point, levels of choline increased, whereas ascorbate, creatine, and glutathione (GS) levels decreased. Freezing had a significant effect on several metabolites but is an essential procedure for research and biobank purposes. Furthermore, four metabolites (glucose, glycine, glycerophosphocholine, and choline) were affected by prolonged HR MAS experiment time possibly caused by physical release of metabolites caused by spinning or due to structural degradation processes. Conclusion: The MR metabolic profiles of tumor samples are reproducible and robust to variation in postsurgical freezing delay up to 30 min. © 2016 Haukaas, Moestue, Vettukattil, Sitter, Lamichhane, Segura, Giskeødegård and Bathen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Introduction: Metabolic profiling of intact tumor tissue by high resolution magic angle spinning (HR MAS) MR spectroscopy (MRS) provides important biological information possibly useful for clinical diagnosis and development of novel treatment strategies. However, generation of high-quality data requires that sample handling from surgical resection until analysis is performed using systematically validated procedures. In this study, we investigated the effect of postsurgical freezing delay time on global metabolic profiles and stability of individual metabolites in intact tumor tissue. Materials and methods: Tumor tissue samples collected from two patient-derived breast cancer xenograft models (n = 3 for each model) were divided into pieces that were snap-frozen in liquid nitrogen at 0, 15, 30, 60, 90, and 120 min after surgical removal. In addition, one sample was analyzed immediately, representing the metabolic profile of fresh tissue exposed neither to liquid nitrogen nor to room temperature. We also evaluated the metabolic effect of prolonged spinning during the HR MAS experiments in biopsies from breast cancer patients (n = 14). All samples were analyzed by proton HR MAS MRS on a Bruker Avance DRX600 spectrometer, and changes in metabolic profiles were evaluated using multivariate analysis and linear mixed modeling. Results: Multivariate analysis showed that the metabolic differences between the two breast cancer models were more prominent than variation caused by freezing delay time. No significant changes in levels of individual metabolites were observed in samples frozen within 30 min of resection. After this time point, levels of choline increased, whereas ascorbate, creatine, and glutathione (GS) levels decreased. Freezing had a significant effect on several metabolites but is an essential procedure for research and biobank purposes. Furthermore, four metabolites (glucose, glycine, glycerophosphocholine, and choline) were affected by prolonged HR MAS experiment time possibly caused by physical release of metabolites caused by spinning or due to structural degradation processes. Conclusion: The MR metabolic profiles of tumor samples are reproducible and robust to variation in postsurgical freezing delay up to 30 min.

Background. Biobanking plays an important role in translational cancer research. The impact of tissue ex-vivo ischemia time and storage period on RNA integrity is not well documented. Methods. Fresh-frozen colon tissues were collected in Taizhou Hospital of Zhejiang Province in China since 2004. Fifty-one colon cancer tissues with tumor cell content higher than 70 % and matched normal tissues during four storage periods (less than 15 months, 16–20 months, 21–25 months, and 26–40 months) were chosen to detect RNA quality. Fresh colon cancer tissues from 5 patients were cut into pieces and kept at room temperature or on ice for 0.5, 1, 2, and 4 h before snap freezing. RNA integrity was determined by microcapillary electrophoresis by the RNA integrity number (RIN) algorithm. Results. Sixty-seven percent of normal colon tissues and 94 % of colon cancer specimens yielded RNA with a RIN of C7. Matched colon cancer and normal tissues showed significant difference in RNA quality. RNA remained stable in colon cancer tissues kept at room temperature and on ice for up to 4 h, and long-term storage of banked colon specimens did not negatively influence RNA quality (RNA with RIN of C7 banked less than 15 months, 83 %; 16–20 months, 78 %; 21–25 months, 77 %; 26–40 months, 90 %). Conclusions. Frozen colon tissues yield high-quality RNA in approximately 80 % of specimens. Ex-vivo ischemia times and storage periods did not adversely affect RNA quality. This study showed that standard operation protocols and the maintenance of high-quality tissue repositories were the keys to translational medicine research.

This study investigated the effect of freezing method (slow or blast freezing) with or without blanching during storage at −20 °C on the levels of three polyacetylenes, falcarinol (FaOH), falcarindiol (FaDOH), falcarindiol-3-acetate (FaDOAc) in carrot disks. The quality of the carrot disks was also assessed using instrumental texture and colour measurements. Blast frozen carrot disks retained higher amounts of polyacetylenes compared to their slow frozen counterparts. Whilst the levels of retention of total polyacetylenes was higher in unblanched than blanched disks prior to freezing there was a sharp decrease in the levels of polyacetylenes in unblanched frozen carrots during the storage period for 60 days at −20 °C. FaDOH was observed to be the most susceptible to degradation during frozen storage of unblanched carrot disks, followed by FaOH and FaDOAc. The changes in the level of polyacetylenes during storage were adequately described by using Weibull model. The texture and colour were also found to decrease during frozen storage compared to fresh carrots.

Reliable stability and yield of RNA from breast cancer tissues are important for biobanking, clinical trials, and diagnostic testing.Aliquots of fresh primary tumor tissue from 17 surgically resected invasive breast cancers were placed into RNAlater at room temperature after tumor removal (baseline) and up to 3 hours thereafter or were snap frozen at baseline and 40 minutes thereafter. Samples were stored at -80°C until gene expression profiling with Affymetrix Human Gene U133A microarrays. We evaluated the effects of cold ischemic time (the time from tumor specimen removal to sample preservation) and sample preservation method on RNA yield, Bioanalyzer-based RNA integrity number, microarray-based 3'/5' expression ratios for assessing transcript integrity, and microarray-based measurement of single-gene and multigene expression signatures. The statistical significance of the effects was assessed using linear mixed effects regression models. All statistical tests were two-sided.Sample preservation in RNAlater statistically significantly improved RNA integrity compared with snap freezing as assessed by the RNA integrity number, which increased from 7.31 to 8.13 units (difference = 0.82 units, 95% confidence interval [CI] = 0.53 to 1.11 units, P.001), and RNA yield, which increased threefold from 8.9 to 28.6 μg (difference = 19.7 μg, 95% CI = 14.1 to 25.3 μg, P.001). Prolonged cold ischemic delay at room temperature before sample stabilization decreased the RNA integrity number by 0.12 units/h (95% CI = 0.02 to 0.23 units/h) compared with a projected average RNA integrity number of 7.39 if no delays were present (P = .008) and decreased the RNA yield by 1.5 μg/h (95% CI = 0 to 4 μg/h) from a baseline mean RNA yield of 33.5 μg if no delays were present (P = .019). Prolonged cold ischemia statistically significantly increased the 3'/5' ratio of control gene transcripts, particularly of STAT1 (P.001). Snap freezing statistically significantly increased the 3'/5' ratio of three control transcripts (ACTB, GAPDH, and 18S rRNA). Expression levels of single genes and multigene signatures for breast cancer were largely unaffected by sample preservation method or cold ischemia.Sample preservation in RNAlater improves RNA yield and quality, whereas cold ischemia increases RNA fragmentation as measured by the 3'/5' expression ratio of control genes. However, expression levels of single genes and multigene signatures that are of diagnostic relevance in breast cancer were mostly unaffected by sample preservation method or prolonged cold ischemic duration.

The objective of this study was to investigate the effect of blast freezing and blanching in combination followed by chilling, on the antioxidant activity (ARP), phenols, ascorbic acid and colour of broccoli, carrots and green beans. No significant changes (p > 0.05) in ARP of blanched frozen (BLFR) broccoli, carrot and green beans were observed. In contrast, UBFR (unblanched frozen) treatments caused a significant decrease (p

Consent regarding the correct processing and storage of blood microparticles is lacking and different protocols for the freeze-thaw cycle exist. Therefore, three different thawing procedures were evaluated regarding their influence on recovery and composition of microparticles. Microparticles were prepared by TRAP-6 or A23187 stimulation of platelet-rich plasma from smokers and nonsmokers (n = 8), from an endothelial cell line or directly obtained from platelet-free plasma of septic patients (n = 5). After snap-freezing in liquid nitrogen platelet-free samples were thawed at 37 degrees, at room temperature or on ice and staining of microparticles was carried out with Annexin V-Cy5 as well as fluorescein isothiocyanate (FITC) or phycoerythrin (PE) labelled antibodies or isotype controls. Microparticle concentrations were determined by means of Trucount tubes. Recovery of platelet microparticles was significantly reduced when samples were thawed on ice (P = 0.001 for all antigens) compared with the two other techniques (P = 0.6 for 37 degrees and P = 0.7 for room temperature, respectively) whereas microparticles of endothelial origin appeared to be less influenced. There was a strong trend towards altered microparticle composition as microparticle counts detected by CD41 staining showed a stronger decrease on ice than Annexin V enumeration (P = 0.07). For microparticle detection thawing of snap-frozen, platelet-free plasma samples should be carried out at room temperature or at 37 degrees C in a water bath but not on ice.

There is a need to simultaneously preserve evidence of interactions between the biological community and soil structural properties of a soil in as near an intact (natural) state as possible. Three dehydration techniques were implemented and assessed for their ability to minimise disruption of both biological and physical properties of the same arable soil sample. Dehydration techniques applied until samples were at constant weight were i) air-drying at 20 °C (AD); ii) −80 °C freeze for 24 h, followed by freeze-drying (−80FD); and iii) liquid nitrogen snap freeze, followed by freeze-drying (LNFD) and were compared to a moist control. Physical structure was determined and quantifled in three dimensions using X-ray computed tomography and microbial phenotypic community composition was assessed using phospholipid fatty acid (PLFA) proflling. This study conflrms that any form of dehydration, when preparing soil for simultaneous biological and physical analysis, will alter the soil physical properties, and cause some change in apparent community structure. Freeze-drying (both the LNFD and −80FD treatments) was found to minimise disruption (when compared to the moist control soil) to both the soil physical properties and the community structure and is a preferable technique to air-drying which markedly alters the size and character of the pore network, as well as the phenotypic proflle. The LNFD was the preferred treatment over the −80FD treatment as samples show low variability between replicates and a fast turn-around time between samples. Therefore snap freezing in liquid nitrogen, followed by freeze drying is the most appropriate form of dehydration when two sets of data, both physical and biological, need to be preserved simultaneously from a soil core.

Summary Macaroni cheese, poached fish, carrots and coq au vin were prepared and frozen by two methods, liquid nitrogen and blast freezing. The precooked frozen food was stored in the frozen state at – 18°C either for less than 18 days or more than 6 weeks. The products were reheated and rated by a taste panel for appearance, texture, odour and flavour; they were also assessed for degree of like or dislike. Products frozen by both methods were not significantly different except in respect of the texture and flavour of macaroni cheese which had been stored in the frozen state for more than 6 weeks. In these instances the liquid nitrogen product was rated higher than the blast frozen product. These results indicate therefore that for the products tested, liquid nitrogen freezing of multiportion packs of cooked products offers no appreciable advantage over freezing food in an efficient blast freezer, particularly when the frozen storage time is short.