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Cryobiology Feb 2022Cryopreservation is a widely used long-term preservation method to ensure the quality and vitality of microbes in laboratories and biological resource facilities....
Cryopreservation is a widely used long-term preservation method to ensure the quality and vitality of microbes in laboratories and biological resource facilities. However, freeze-thaw damage and osmotic pressure changes during cryopreservation adversely impacts microbial survival. Significant expenditure of resources and expertise are required to select the right cryoprotectant and optimize its concentration for maximum survival of diverse microorganisms. This work describes a cryopreservation method that obviates the need for cryoprotectants by exploiting the unique thermal characteristics of semi-spherical drops. Here, a plurality of these drops, each 10 μl in volume, created on a highly non-wetting flat-sheet substrate with holes and frozen at -70 °C. Deriving an f (x) metric as a measure of relative viability, storage in drops in the absence of cryoprotectants was found to improve the survivability of Staphylococcus epidermidis by 1.91 times compared with the same sample stored in larger 50-μl volumes in standard 1.5-ml tubes. This also compares well with a value of 2.33 obtained with standard preservation with cryoprotectant. The drop method allows high throughput aliquoting of the bacterial culture into multiple discrete drops using multichannel pipettes or automated liquid handlers and the edges of the holes provides a pinning action that holds the drop stably against gravitational roll-offs. It also allows samples to be removed in discrete small volumes, thus, reducing the number of freeze thaw cycles and associated cell damage. The flat-sheet architecture of the substrate reduces the amount of plastic waste generated and augments green laboratory practices.
Topics: Bacteria; Cryopreservation; Cryoprotective Agents; Freezing; Osmotic Pressure; Semen Preservation
PubMed: 34838822
DOI: 10.1016/j.cryobiol.2021.11.179 -
Journal of Diabetes Science and... Sep 2022Conflicting information is available regarding the stability of glucose concentrations in frozen plasma samples. Clinical trials could benefit from such long-term...
BACKGROUND
Conflicting information is available regarding the stability of glucose concentrations in frozen plasma samples. Clinical trials could benefit from such long-term storage because it would allow usage of a central laboratory with higher-quality laboratory analyzers in contrast to mobile analyzers in a decentralized setting.
METHODS
In this study, venous blood samples were collected in lithium-heparin gel tubes. Plasma was separated immediately after blood was drawn, and from each of the 21 plasma samples, 6 aliquots were prepared for measurement at 6 time points: immediately and after 2, 4, 6, 8, and 12 weeks. Between sampling and measurement, aliquots were stored at less than -20°C. Transport on dry ice was simulated by placing aliquots in a -80°C freezer for 5 days between weeks 8 and 12. Measurements were performed on a hexokinase-based laboratory analyzer.Average relative differences and corresponding 99% confidence intervals (CIs) were calculated between the stored aliquots' and the immediately measured aliquots' glucose concentrations. Glucose concentrations were deemed stable as long as average relative differences were ≤±2.5%.
RESULTS
Over the whole 12-weeks duration, the largest average relative difference was -1.82% (99% CI: -2.25% to -1.39%). Shorter storage durations tended to lead to less bias.
CONCLUSION
In this study, the stability of glucose concentrations in frozen plasma samples obtained with lithium-heparin gel tubes could be shown for up to 12 weeks. Future studies should be performed to assess whether this is independent of the glucose analyzer and the type of sampling tube used.
Topics: Blood Specimen Collection; Glucose; Hematologic Tests; Heparin; Humans; Lithium; Time Factors
PubMed: 33034207
DOI: 10.1177/1932296820963657 -
Frontiers in Chemistry 2020Understanding the stability of drugs in a forensic toxicology setting is critical for the evaluation of drug concentrations. Synthetic cathinones are new psychoactive...
Understanding the stability of drugs in a forensic toxicology setting is critical for the evaluation of drug concentrations. Synthetic cathinones are new psychoactive substances structurally derived from cathinone, the psychoactive component of ("khat"), a shrub that is indigenous to the Middle East and East Africa. Previous research has evaluated the stability of synthetic cathinones in biological matrices, including blood preserved with the combination of NaF and KCO used in gray-top tubes. However, it does not assess their stability in blood preserved with NaEDTA, used for some clinical samples. Further, stability in unpreserved urine samples was only studied for two weeks. This research evaluates the stabilities of four Schedule I synthetic cathinones: mephedrone, MDPV (3,4-methylenedioxypyrovalerone), naphyrone, and α-PVP (alpha-pyrrolidinopentiophenone) at 20°C (room temperature), 4°C (refrigerator), and -20°C (freezer). Stability was assessed in methanolic and acetonitrile solutions, as well as in NaEDTA-preserved blood and unpreserved urine. Solutions (1 mg/L) of each drug in each matrix stored in aliquots (100 μL, solvents; 1.2 mL, biological samples; = 12) at each of the three temperatures for triplicate analysis on days 3, 7, 14, and 30. On day 0 of each study, three additional aliquots of each solution were analyzed. Biological samples underwent solid-phase extraction before analysis. All samples were analyzed in full-scan by gas chromatography-mass spectrometry (GC-MS). The results of this study show that under room temperature and refrigerator storage conditions, mephedrone, naphyrone, and MDPV will degrade in methanol. This degradation starts are early as day 3. Additionally, all four drugs will degrade in NaEDTA-preserved human whole blood samples in at least one evaluated storage environment. However, when in acetonitrile-based working solutions and unpreserved urine samples, they proved to be more stable. Methanolic working solutions and samples of NaEDTA-preserved blood containing these cathinones should be stored in the freezer and used or tested with urgency to ensure that quantitative sample analysis is as accurate as possible in forensic casework.
PubMed: 33304885
DOI: 10.3389/fchem.2020.597726 -
Wei Sheng Yan Jiu = Journal of Hygiene... May 2022To evaluate the consistency of blood lipid test result under different storage conditions.
OBJECTIVE
To evaluate the consistency of blood lipid test result under different storage conditions.
METHODS
Blood samples from 140 subjects were collected, centrifuged and were divided into five aliquots. One aliquot was used to determine serum total cholesterol(TC), triglyceride(TG), high density lipoprotein cholesterol(HDL-C) and low density lipoprotein cholesterol(LDL-C) immediately, and the other four parts were stored at 4 ℃ for 4 h, 24 h, and frozen at-20 ℃ and-80 ℃ for 30 d, respectively, for the further measurement.
RESULTS
Immediate 0 h detection was compared with 4 ℃ for 4 h and 24 h, -20 ℃and-80 ℃ for 30 d: The difference Bland-Altman method was used to analyze TC and LDL-C data, and the mean TC difference values was 0.00, 0.00, 0.07 and 0.03 mmol/L, respectively. LDL-C difference value was-0.02, -0.04, -0.02 and-0.07 mmol/L, respectively, and 95% of the points were within the clinically acceptable conformance threshold. Ratio Bland-Altman method was used to analyze TG and HDL-C data. The mean TG ratios was 1.01, 1.00, 1.05, 1.05, and the mean HDL-C was 1.01, 1.03, 1.05 and 1.03, respectively. Ninety-five percent of the points were within the clinically acceptable consistency boundary. According to the 2021 Interlaboratory Quality Evaluation Standard of Clinical Laboratory Center of National Health Commission, the Bland-Altman analysis showed good consistency between the test result of different blood samples under different storage conditions and the immediate test result.
CONCLUSION
In large population epidemiological studies, the results of TC, LDL-C, HDL-C and TG in sera being refrigerated at 4 ℃ for 4 h and 24 h, and frozen at-20 ℃ and-80 ℃ for 30 d, were consistent with the result of immediate detection.
Topics: Cholesterol; Cholesterol, HDL; Cholesterol, LDL; Humans; Lipids; Triglycerides
PubMed: 35718916
DOI: 10.19813/j.cnki.weishengyanjiu.2022.03.024 -
ELife Nov 2021A group leader decided that his lab would share the fluorescent dyes they create, for free and without authorship requirements. Nearly 12,000 aliquots later, he reveals...
A group leader decided that his lab would share the fluorescent dyes they create, for free and without authorship requirements. Nearly 12,000 aliquots later, he reveals what has happened since.
Topics: Coloring Agents; Laboratories; Laboratory Personnel
PubMed: 34739373
DOI: 10.7554/eLife.74981 -
BMJ Open Ophthalmology Nov 2022NHS Blood and Transplant Tissue and Eye Services (TES) offer a serum eyedrop (SE) service to patients suffering from severe ocular surface disease. SE are prepared from...
INTRODUCTION
NHS Blood and Transplant Tissue and Eye Services (TES) offer a serum eyedrop (SE) service to patients suffering from severe ocular surface disease. SE are prepared from serum collected at blood donation sessions; the serum is diluted 1:1 with physiological saline. Formerly, 3ml aliquots of diluted serum were aliquoted into glass bottles in a Grade B clean room. Since this service was started, Meise Medizintechnik have developed an automatic closed filling system consisting of tubing-linked chains of squeezable vials. They can be heat-sealed closed, under sterile conditions, after the vials have been filled.
MATERIALS AND METHODS
TES R&D were asked to validate the Meise system to increase the efficiency and speed of SE production. Validation of the closed system consisted of a process simulation assessment, using bovine serum and simulating each step of the filling process, freezing to -80oC, checking the integrity of each vial and packing the vials into storage containers. They were then put into transport containers and shipped on a round-trip journey to simulate delivery to patients. On return the vials were thawed and the integrity of each vial re-checked visually and by squeezing in a plasma expressor.Subsequently a shelf-life study was carried out on three batches of fully consented human allogeneic SE. The serum was dispensed into vials, frozen as above and stored for set time points 0, 1, 3, 6 and 12 months in a standard domestic freezer set at -15-20oC to mimic a patient's freezer. At each time point, 10 random samples of vials were removed, and the outer containers were tested for damage or deterioration, the vials for integrity and their contents for sterility and stability. Stability was assessed by measuring serum albumin concentrations and sterility by testing for microbial contamination.
RESULTS
No structural damage or leakage was found in any of the vials, or the tubing evaluated, after thawing, at any time point. In addition, all samples tested negative for microbial contamination and serum albumin levels were always within the expected range (3 - 5 Dg/L) at each set time point.
CONCLUSION
These results demonstrate that Meise closed system vials can successfully dispense SE drops and the vials can be stored frozen without affecting integrity, sterility or stability. These vials have been in use in TES for 3 years saving clean room space and greatly increasing the numbers of patients that can use the SE service.
Topics: Humans; Ophthalmic Solutions; Drug Packaging; Freezing; Serum; Serum Albumin
PubMed: 37282689
DOI: 10.1136/bmjophth-2022-EEBA.28 -
Methods in Molecular Biology (Clifton,... 2021Biomimetic lipid bilayer systems are a useful tool for modeling specific properties of cellular membranes in order to answer key questions about their structure and... (Review)
Review
Biomimetic lipid bilayer systems are a useful tool for modeling specific properties of cellular membranes in order to answer key questions about their structure and functions. This approach has prompted scientists from all over the world to create more and more sophisticated model systems in order to decipher the complex lateral and transverse organization of cellular plasma membranes. Among a variety of existing biomembrane domains, lipid rafts are defined as small, dynamic, and ordered assemblies of lipids and proteins, enriched in cholesterol and sphingolipids. Lipid rafts appear to be involved in the development of Alzheimer's disease (AD) by affecting the aggregation of the amyloid-β (Aβ) peptide at neuronal membranes thereby forming toxic oligomeric species. In this review, we summarize the laboratory methods which allow to study the interaction of Aβ with lipid rafts. We describe step by step protocols to form giant (GUVs) and large unilamellar vesicles (LUVs) containing raft-mimicking domains surrounded by membrane nonraft regions. Using fluorescence microscopy GUV imaging protocols, one can design experiments to visualize micron-scale raft-like domains, to determine the micron-scale demixing temperature of a given lipid mixture, construct phase diagram, and photogenerate domains in order to assess the dynamics of raft formation and raft size distribution. LUV fluorescence spectroscopy protocols with proper data analysis can be used to measure molecular packing of raft/nonraft regions of the membrane, to report on nanoscale raft formation and determine nanoscale demixing temperature. Because handling of the Aβ requires dedicated laboratory experience, we present illustrated protocols for Aβ-stock aliquoting, Aβ aqueous solubilization, oligomer preparation, determination of the Aβ concentration before and after filtration. Thioflavin binding, dynamic light scattering, and transmission electron microscopy protocols are described as complementary methods to detect Aβ aggregation kinetics, aggregate sizes, and morphologies of observed aggregates.
Topics: Alzheimer Disease; Amyloid beta-Peptides; Animals; Biomimetics; Cell Membrane; Humans; Laboratories; Lipid Bilayers; Membrane Microdomains; Unilamellar Liposomes
PubMed: 32770501
DOI: 10.1007/978-1-0716-0814-2_4 -
Biopreservation and Biobanking Aug 2023The present study was conducted to observe the effects of removal of seminal plasma of Pantja buck semen and supplementation of bovine seminal plasma (BSP) in the...
The present study was conducted to observe the effects of removal of seminal plasma of Pantja buck semen and supplementation of bovine seminal plasma (BSP) in the extender before cryopreservation. In a preliminary experiment, different levels of BSP were supplemented (1, 3, 5, 7, and 9% v/v) in egg yolk (7.5% egg yolk)-tris (EYT) extender and used for cryopreservation of Pantja buck semen. Results in terms of motility, viability, plasma membrane integrity, acrosome integrity, and lipid peroxidation showed that 5% BSP was suitable for maintaining Pantja buck semen quality during cryopreservation. In the final experiment, pooled semen from four Pantja bucks was split into three aliquots (I, II, and III). Aliquot I was directly diluted in EYT extender and grouped as the control (C); aliquot II and III were washed separately with TALP solution and diluted as D1 (Washed semen with EYT extender) and D2 (Washed semen with EYT extender containing 5% BSP), respectively. Seminal attributes (sperm individual motility, viability, plasma membrane integrity, acrosome integrity, and total morphological abnormalities) were assessed at the postdilution, postequilibration, and post-thawing stages. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), malondialdehyde (MDA) concentration, and glutathione peroxidase (GSH-Px) activity were measured at post-thaw. Washed semen significantly improved ( < 0.05) seminal parameters at post-thaw compared with unwashed semen (control). A significant difference ( < 0.05) was observed in seminal attributes between freezing stages and between dilution groups. Significantly higher ( < 0.05) post-thaw sperm motility, viability, plasma membrane integrity, acrosome integrity, and GSH-Px activity, and significantly lower ( < 0.05) MDA concentration and extracellular release of enzymes (ALT, AST) were observed in group D2 compared with control and D1. The results of the present study demonstrated that cryopreservation of washed Pantja buck semen diluted with 5% BSP-supplemented EYT extender can improve post-thaw semen quality.
Topics: Male; Animals; Cattle; Semen; Spermatozoa; Semen Analysis; Egg Yolk; Sperm Motility; Cryoprotective Agents; Semen Preservation; Acrosome; Cryopreservation; Antioxidants; Dietary Supplements
PubMed: 35856825
DOI: 10.1089/bio.2022.0013 -
Biopreservation and Biobanking Aug 2021The aim of this work was to compare the effectiveness of ultra-rapid freezing (UF) and conventional slow freezing (CF) to cryopreserve buck sperm throughout the year....
The aim of this work was to compare the effectiveness of ultra-rapid freezing (UF) and conventional slow freezing (CF) to cryopreserve buck sperm throughout the year. During 1 year, semen from 10 adult Gabon bucks was collected by electroejaculation every 2 weeks. Before and after freezing, samples were selected by density gradient centrifugation, and after sperm selection, the sample was divided into two aliquots. One aliquot was CF with an extender based on Tris, citric acid, and glucose (TCG) +6% yolk +5% glycerol, and maintained at 5°C for 3 hours of equilibration before freezing. The other aliquot was frozen using an UF method with an extender based on TCG +6% yolk +100 mM sucrose, and maintained at 5°C for 30 minutes. The evaluations included the percentages of motile sperm, sperm with progressive motility, quality of sperm motility, and the percentages of sperm with functional membrane, live sperm, sperm with morphoabnormalities, and sperm with intact acrosome. The percentage of sperm with intact acrosome was higher using the conventional freezing method ( < 0.05). After thawing and at pre- and postselection stages, the quality of motility, and the percentages of motile sperm, progressive motile sperm, sperm with functional membrane, and with intact acrosome were greater using CF than UF ( < 0.005). Conventional freezing was more effective than UF to cryopreserve sperm from Gabon bucks, at least in our experimental conditions. Most differences in favor of CF were observed in the quality of motility, and the percentages of motile sperm, progressive motile sperm, sperm with functional membrane, and with intact acrosome during long periods of the year, or even remained throughout it.
Topics: Acrosome; Cryopreservation; Cryoprotective Agents; Freezing; Gabon; Humans; Male; Semen Preservation; Sperm Motility; Spermatozoa
PubMed: 33751902
DOI: 10.1089/bio.2020.0155 -
Journal of the American Chemical Society Feb 2021Carbocations are short-lived reactive intermediates in many organic and biological reactions that are difficult to observe. This field sprung to life with the discovery...
Carbocations are short-lived reactive intermediates in many organic and biological reactions that are difficult to observe. This field sprung to life with the discovery by Olah that a superacidic solution allowed the successful capture and nuclear magnetic resonance characterization of transient carbocations. We report here that water microdroplets can directly capture the fleeting carbocation from a reaction aliquot followed by its desorption to the gas phase for mass spectrometric detection. This was accomplished by employing desorption electrospray ionization mass spectrometry to detect a variety of short-lived carbocations (average lifetime ranges from nanoseconds to picoseconds) obtained from different reactions (e.g., elimination, substitution, and oxidation). Solvent-dependent studies revealed that aqueous microdroplets outperform organic microdroplets in the capture of carbocations. We provide a mechanistic insight demonstrating the survival of the reactive carbocation in a positively charged aqueous microdroplet and its subsequent ejection to the gas phase for mass spectrometric analysis.
PubMed: 33534557
DOI: 10.1021/jacs.0c12512