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PloS One 2022The objective of the COMET (COllection of MEtabolic Tissues) biobank project is to create a high-quality collection of insulin-sensitive tissues (liver, muscle, adipose...
BACKGROUND
The objective of the COMET (COllection of MEtabolic Tissues) biobank project is to create a high-quality collection of insulin-sensitive tissues (liver, muscle, adipose tissues, and epiploic artery) and blood sample derivatives (plasma, serum, DNA and RNA), collected from 270 grade 2-3 obese patients undergoing bariatric surgery. Relevant data on patient such as clinical/biological characteristics and sample handling are also collected. For this, our aim was to establish a Quality Management System (QMS) to meet the reliability and quality requirements necessary for its scientific exploitation.
MATERIALS AND METHODS
The COMET QMS includes: (1) Quality Assurance to standardize all stages of the biobanking process, (2) Quality Controls on samples from the first patients included in order to validate the sample management process and ensure reproducible quality; and 3) "in process" Quality Controls to ensure the reliability of the storage procedures and the stability of the samples over time.
RESULTS
For serum and plasma, several corrective actions, such as temperature handling and centrifugation conditions, were made to the protocol and led to improvement of the volume and quality of samples. Regarding DNA, all samples evaluated achieved a satisfactory level of purity and integrity and most of them yielded the required DNA quantity. All frozen tissue samples had RNAs of good purity. RNA quality was confirmed by RIN, achieving values in most cases over 7 and efficient amplification of housekeeping genes by RT-qPCR, with no significant differences among samples from the same tissue type. In the "in process" Quality Controls, DNA, RNA, and histological integrity of tissues showed no differences among samples after different preservation times.
CONCLUSION
Quality Control results have made it possible to validate the entire biobank process and confirm the utility of implementing QMS to guarantee the quality of a biospecimen collection.
Topics: Humans; Biological Specimen Banks; Reproducibility of Results; RNA; Preservation, Biological; Specimen Handling; DNA
PubMed: 36584180
DOI: 10.1371/journal.pone.0278780 -
PeerJ 2022The transport and storage of samples in temperatures of minus 80 °C is commonly considered as the gold standard for microbiome studies. However, studies conducting...
BACKGROUND
The transport and storage of samples in temperatures of minus 80 °C is commonly considered as the gold standard for microbiome studies. However, studies conducting sample collection at remote sites without a reliable cold-chain would benefit from a sample preservation method that allows transport and storage at ambient temperature.
METHODS
In this study we compare alpha diversity and 16S microbiome composition of 20 fecal sample replicates from Damaraland mole-rats () preserved in a minus 80 °C freezer and transported on dry ice to freeze-dried samples that were stored and transported in ambient temperature until DNA extraction.
RESULTS
We found strong correlations between relative abundances of Amplicon Sequence Variants (ASVs) between preservation treatments of the sample, no differences in alpha diversity measures between the two preservation treatments and minor effects of the preservation treatment on beta diversity measures. Our results show that freeze-drying samples can be a useful method for cost-effective transportation and storage of microbiome samples that yields quantitatively almost indistinguishable results in 16S microbiome analyses as those stored in minus 80 °C.
Topics: Feces; Freeze Drying; Preservation, Biological; Microbiota; Refrigeration
PubMed: 35310158
DOI: 10.7717/peerj.13095 -
International Journal of Molecular... Dec 2023Heart failure is a clinical syndrome associated with poor quality of life, substantial healthcare resource utilization, and premature mortality, in large part related to... (Review)
Review
Heart failure is a clinical syndrome associated with poor quality of life, substantial healthcare resource utilization, and premature mortality, in large part related to high rates of hospitalizations. The clinical manifestations of heart failure are similar regardless of the ejection fraction. Unlike heart failure with reduced ejection fraction, there are few therapeutic options for treating heart failure with preserved ejection fraction. Molecular therapies that have shown reduced mortality and morbidity in heart failure with reduced ejection have not been proven to be effective for patients with heart failure and preserved ejection fraction. The study of pathophysiological processes involved in the production of heart failure with preserved ejection fraction is the basis for identifying new therapeutic means. In this narrative review, we intend to synthesize the existing therapeutic means, but also those under research (metabolic and microRNA therapy) for the treatment of heart failure with preserved ejection fraction.
Topics: Humans; Quality of Life; Stroke Volume; Preservation, Biological; MicroRNAs; Heart Failure
PubMed: 38203612
DOI: 10.3390/ijms25010440 -
BMC Microbiology May 2021New developments in next-generation sequencing technologies and massive data received from this approach open wide prospects for personalised medicine and nutrition...
BACKGROUND
New developments in next-generation sequencing technologies and massive data received from this approach open wide prospects for personalised medicine and nutrition studies. Metagenomic analysis of the gut microbiota is paramount for the characterization of human health and wellbeing. Despite the intensive research, there is a huge gap and inconsistency between different studies due to the non-standardised and biased pipeline. Methodical and systemic understanding of every stage in the process is necessary to overcome all bottlenecks and grey zones of gut microbiota studies, where all details and interactions between processes are important.
RESULTS
Here we show that an inexpensive, but reliable iSeq 100 platform is an excellent tool to perform the analysis of the human gut microbiota by amplicon sequencing of the 16 S rRNA gene. Two commercial DNA extraction kits and different starting materials performed similarly regarding the taxonomic distribution of identified bacteria. DNA/RNA Shield reagent proved to be a reliable solution for stool samples collection, preservation, and storage, as the storage of faecal material in DNA/RNA Shield for three weeks at different temperatures and thawing cycles had a low impact on the bacterial distribution.
CONCLUSIONS
Altogether, a thoroughly elaborated pipeline with close attention to details ensures high reproducibility with significant biological but not technical variations.
Topics: Bacteria; DNA, Bacterial; Feces; Gastrointestinal Microbiome; High-Throughput Nucleotide Sequencing; Humans; Metagenomics; Preservation, Biological; RNA, Ribosomal, 16S
PubMed: 34051731
DOI: 10.1186/s12866-021-02233-y -
Journal of Biomedical Materials... Dec 2016Tissue engineering is increasingly being recognized as a new approach that could alleviate the burden of tissue damage currently managed with transplants or synthetic... (Review)
Review
Tissue engineering is increasingly being recognized as a new approach that could alleviate the burden of tissue damage currently managed with transplants or synthetic devices. Making this novel approach available in the future for patients who would potentially benefit is largely dependent on understanding and addressing all those factors that impede the translation of this technology to the clinic. Cell-associated factors in particular raise many challenges, including those related to cell sources, up- and downstream techniques, preservation, and the creation of in vitro microenvironments that enable cells to grow and function as far as possible as they would in vivo. This article highlights the main confounding issues associated with cells in tissue engineering and how these issues may hinder the advancement of therapeutic tissue engineering. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 3157-3163, 2016.
Topics: Animals; Humans; Preservation, Biological; Stem Cells; Tissue Engineering; Tissue and Organ Procurement
PubMed: 27502461
DOI: 10.1002/jbm.a.35856 -
Update on fertility preservation: new opportunities and challenges in the fight against infertility.La Clinica Terapeutica May 2022Infertility has been characterized as a disease by the World Health Organization (WHO) and reportedly affects about 10-12% of couples worldwide, while the incidence is...
Infertility has been characterized as a disease by the World Health Organization (WHO) and reportedly affects about 10-12% of couples worldwide, while the incidence is even higher in Italy, at about 15%. The issue of iatrogenic infertility arising from treatments that can compromise an individual's reproductive capacity, it is necessary to inform patients of the possible damage on their future fertility and on the possibilities to preserve it. The complexities inherent in the various techniques and approaches aimed at preserving fertility should be expounded upon thoroughly to the patients, who should also receive proper psychological assistance and counseling, which ought to take into account the ethical distinctive challenges and the possible misgivings that may be caused in patients. Ovarian Tissue Cryopreservation (OTC) and ovarian tissue transplantation (OTT) can constitute a valuable part of the clinical armamentarium for preserving fertility, although the data are still inconclusive, particularly in over-36 patients. The multidisciplinary nature of the healthcare teams involved in such interventions is of paramount importance to optimize results.
Topics: Cryopreservation; Fertility Preservation; Humans; Incidence; Infertility; Italy
PubMed: 35612336
DOI: 10.7417/CT.2022.2424 -
PloS One 2019The preservation of biological samples for an extended time period of days to weeks after initial collection is important for the identification, screening, and...
The preservation of biological samples for an extended time period of days to weeks after initial collection is important for the identification, screening, and characterization of bacterial pathogens. Traditionally, preservation relies on cold-chain infrastructure; however, in many situations this is impractical or not possible. Thus, our goal was to develop alternative bacterial sample preservation and transport media that are effective without refrigeration or external instrumentation. The viability, nucleic acid stability, and protein stability of Bacillus anthracis Sterne 34F2, Francisella novicida U112, Staphylococcus aureus ATCC 43300, and Yersinia pestis KIM D27 (pgm-) was assessed for up to 28 days. Xanthan gum (XG) prepared in PBS with L-cysteine maintained more viable F. novicida U112 cells at elevated temperature (40°C) compared to commercial reagents and buffers. Viability was maintained for all four bacteria in XG with 0.9 mM L-cysteine across a temperature range of 22-40°C. Interestingly, increasing the concentration to 9 mM L-cysteine resulted in the rapid death of S. aureus. This could be advantageous when collecting samples in the built environment where there is the potential for Staphylococcus collection and stabilization rather than other organisms of interest. F. novicida and S. aureus DNA were stable for up to 45 days upon storage at 22°C or 40°C, and direct analysis by real-time qPCR, without DNA extraction, was possible in the XG formulations. XG was not compatible with proteomic analysis via LC-MS/MS due to the high amount of residual Xanthomonas campestris proteins present in XG. Our results demonstrate that polysaccharide-based formulations, specifically XG with L-cysteine, maintain bacterial viability and nucleic acid integrity for an array of both Gram-negative and Gram-positive bacteria across ambient and elevated temperatures.
Topics: Bacteria; Cysteine; Microbial Viability; Polysaccharides; Polysaccharides, Bacterial; Preservation, Biological; Proteomics; Temperature
PubMed: 31490969
DOI: 10.1371/journal.pone.0221831 -
Microbiology Spectrum Oct 2021Field studies are central to environmental microbiology and microbial ecology, because they enable studies of natural microbial communities. Metaproteomics, the study of...
Field studies are central to environmental microbiology and microbial ecology, because they enable studies of natural microbial communities. Metaproteomics, the study of protein abundances in microbial communities, allows investigators to study these communities "," which requires protein preservation directly in the field because protein abundance patterns can change rapidly after sampling. Ideally, a protein preservative for field deployment works rapidly and preserves the whole proteome, is stable in long-term storage, is nonhazardous and easy to transport, and is available at low cost. Although these requirements might be met by several protein preservatives, an assessment of their suitability under field conditions when targeted for metaproteomic analyses is currently lacking. Here, we compared the protein preservation performance of flash freezing and the preservation solution RNA using the marine gutless oligochaete Olavius algarvensis and its symbiotic microbes as a test case. In addition, we evaluated long-term RNA storage after 1 day, 1 week, and 4 weeks at room temperature (22°C to 23°C). We evaluated protein preservation using one-dimensional liquid chromatography-tandem mass spectrometry. We found that RNA and flash freezing preserved proteins equally well in terms of total numbers of identified proteins and relative abundances of individual proteins, and none of the test time points was altered, compared to time zero. Moreover, we did not find biases against specific taxonomic groups or proteins with particular biochemical properties. Based on our metaproteomic data and the logistical requirements for field deployment, we recommend RNA for protein preservation of field-collected samples targeted for metaproteomic analyses. Metaproteomics, the large-scale identification and quantification of proteins from microbial communities, provide direct insights into the phenotypes of microorganisms on the molecular level. To ensure the integrity of the metaproteomic data, samples need to be preserved immediately after sampling to avoid changes in protein abundance patterns. In laboratory setups, samples for proteomic analyses are most commonly preserved by flash freezing; however, liquid nitrogen or dry ice is often unavailable at remote field locations, due to their hazardous nature and transport restrictions. Our study shows that RNA can serve as a low-hazard, easy-to-transport alternative to flash freezing for field preservation of samples for metaproteomic analyses. We show that RNA preserves the metaproteome equally well, compared to flash freezing, and protein abundance patterns remain stable during long-term storage for at least 4 weeks at room temperature.
Topics: Animals; Annelida; Bacteria; Bacterial Physiological Phenomena; Bacterial Proteins; Phylogeny; Preservation, Biological; Proteomics; Symbiosis
PubMed: 34704828
DOI: 10.1128/Spectrum.01429-21 -
Improved Method for Dental Pulp Stem Cell Preservation and Its Underlying Cell Biological Mechanism.Cells Aug 2023Dental pulp stem cells (DPSCs) are considered a valuable cell source for regenerative medicine because of their high proliferative potential, multipotency, and...
Dental pulp stem cells (DPSCs) are considered a valuable cell source for regenerative medicine because of their high proliferative potential, multipotency, and availability. We established a new cryopreservation method (NCM) for collecting DPSCs, in which the tissue itself is cryopreserved and DPSCs are collected after thawing. We improved the NCM and developed a new method for collecting and preserving DPSCs more efficiently. Dental pulp tissue was collected from an extracted tooth, divided into two pieces, sandwiched from above and below using cell culture inserts, and cultured. As a result, the cells in the pulp tissue migrated vertically over time and localized near the upper and lower membranes over 2-3 days. With regard to the underlying molecular mechanism, SDF1 was predominantly involved in cell migration. This improved method is valuable and enables the more efficient collection and reliable preservation of DPSCs. It has the potential to procure a large number of DPSCs stably.
Topics: Dental Pulp; Cryopreservation; Cancer Vaccines; Cell Culture Techniques; Stem Cells
PubMed: 37681870
DOI: 10.3390/cells12172138 -
Microbiology Spectrum Jun 2023Here, we aimed to compare the effects of different preservation methods on outcomes of fecal microbiota. We evaluated the effects of different preservation methods using... (Meta-Analysis)
Meta-Analysis
Here, we aimed to compare the effects of different preservation methods on outcomes of fecal microbiota. We evaluated the effects of different preservation methods using stool sample preservation experiments for up to 1 year. The stool samples from feces of healthy volunteers were grouped based on whether absolute ethanol was added and whether they were hypothermically preserved. Besides, we performed a systematic review to combine current fecal microbiota preservation evidence. We found that changed significantly and decreased significantly in the 12th month in the room temperature + absolute ethanol group. The four cryopreservation groups have more similarities with fresh sample in the 12 months; however, different cryopreservation methods have different effects on several phyla, families, and genera. A systematic review showed that the Shannon diversity and Simpson index of samples stored in RNAlater for 1 month were not statistically significant compared with those stored immediately at -80°C ( = 0.220 and = 0.123, respectively). The -80°C refrigerator and liquid nitrogen cryopreservation with 10% glycerine can both maintain stable microbiota of stool samples for long-term preservation. The addition of absolute ethanol to cryopreserved samples had no significant difference in the effect of preserving fecal microbial characteristics. Our study provides empirical insights into preservation details for future studies of the long-term preservation of fecal microbiota. Systematic review and meta-analysis found that the gut microbiota structure, composition, and diversity of samples preserved by storage methods, such as preservation solution, are relatively stable, which were suitable for short-term storage at room temperature. The study of gut bacteria has become increasingly popular, and fecal sample preservation methods and times need to be standardized. Here, we detail a 12-month study of fecal sample preservation, and our study provides an empirical reference about experimental details for long-term high-quality storage of fecal samples in the field of gut microbiology research. The results showed that the combination of -80°C/liquid nitrogen deep cryopreservation and 10% glycerol was the most effective method for the preservation of stool samples, which is suitable for long-term storage for at least 12 months. The addition of anhydrous ethanol to the deep cryopreserved samples did not make a significant difference in the preservation of fecal microbiological characteristics. Combined with the results of systematic reviews and meta-analyses, we believe that, when researchers preserve fecal specimens, it is essential to select the proper preservation method and time period in accordance with the goal of the study.
Topics: Humans; Gastrointestinal Microbiome; Preservation, Biological; Feces; Ethanol; Specimen Handling; Biodiversity; Nitrogen; RNA, Ribosomal, 16S
PubMed: 37093040
DOI: 10.1128/spectrum.04297-22