Concept: Food preservation
Development of techniques to isolate, culture, and transplant human spermatogonial stem cells (SSCs) has the future potential to treat male infertility. To maximize the efficiency of these techniques, methods for SSC cryopreservation need to be developed to bank SSCs for extended periods of time. Although, it has been demonstrated that SSCs can reinitiate spermatogenesis after freezing, optimal cryopreservation protocols that maximize SSC proliferative capacity post-thaw have not been identified. The objective of this study was to develop an efficient cryopreservation technique for preservation of SSCs. To identify efficient cryopreservation methods for long-term preservation of SSCs, isolated testis cells enriched for SSCs were placed in medium containing dimethyl sulfoxide (DMSO) or DMSO and trehalose (50 mM, 100 mM, or 200 mM), and frozen in liquid nitrogen for 1 week, 1 month, or 3 months. Freezing in 50 mM trehalose resulted in significantly higher cell viability compared to DMSO at all thawing times and a higher proliferation rate compared to DMSO for the 1 week freezing period. Freezing in 200 mM trehalose did not result in increased cell viability; however, proliferation activity was significantly higher and percentage of apoptotic cells was significantly lower compared to DMSO after freezing for 1 and 3 months. To confirm the functionality of SSCs frozen in 200 mM trehalose, SSC transplantation was performed. Donor SSCs formed spermatogenic colonies and sperm capable of generating normal progeny. Collectively, these results indicate that freezing in DMSO with 200 mM trehalose serves as an efficient method for the cryopreservation of SSCs.
Sequencing-based studies of the human faecal microbiota are increasingly common. Appropriate storage of sample material is essential to avoid the introduction of post-collection bias in microbial community composition. Rapid freezing to -80 °C is commonly considered to be best-practice. However, this is not feasible in many studies, particularly those involving sample collection in participants' homes. We determined the extent to which a range of stabilisation and storage strategies maintained the composition of faecal microbial community structure relative to freezing to -80 °C. Refrigeration at 4 °C, storage at ambient temperature, and the use of several common preservative buffers (RNAlater, OMNIgene.GUT, Tris-EDTA) were assessed relative to freezing. Following 72 hours of storage, faecal microbial composition was assessed by 16 S rRNA amplicon sequencing. Refrigeration was associated with no significant alteration in faecal microbiota diversity or composition. However, samples stored using other conditions showed substantial divergence compared to -80 °C control samples. Aside from refrigeration, the use of OMNIgene.GUT resulted in the least alteration, while the greatest change was seen in samples stored in Tris-EDTA buffer. The commercially available OMNIgene.GUT kit may provide an important alternative where refrigeration and cold chain transportation is not available.
Analysis of brain ultrastructure using electron microscopy typically relies on chemical fixation. However, this is known to cause significant tissue distortion including a reduction in the extracellular space. Cryo fixation is thought to give a truer representation of biological structures, and here we use rapid, high-pressure freezing on adult mouse neocortex to quantify the extent to which these two fixation methods differ in terms of their preservation of the different cellular compartments, and the arrangement of membranes at the synapse and around blood vessels. As well as preserving a physiological extracellular space, cryo fixation reveals larger numbers of docked synaptic vesicles, a smaller glial volume, and a less intimate glial coverage of synapses and blood vessels compared to chemical fixation. The ultrastructure of mouse neocortex therefore differs significantly comparing cryo and chemical fixation conditions.
Time of Flight secondary ion mass spectrometry (TOF-SIMS) has been used to explore the distribution of phospholipids in the plasma membrane of Tetrahymena pyriformis during cell division. The dividing cells were freeze dried prior to analysis followed by line scan and region of interest analysis at various stages of cell division. The results showed no signs of phospholipid domain formation at the junction between the dividing cells. Instead the results showed that the sample preparation technique had a great impact on one of the examined phospholipids, namely phosphatidylcholine (PC). Phosphatidylcholine and 2-aminoethylphosphonolipid (2-AEP) have therefore been evaluated in Tetrahymena cells that have been subjected to different sample preparation techniques: freeze drying ex situ, freeze fracture, and freeze fracture with partial or total freeze drying in situ. The result suggests that freeze-drying ex situ causes the celia to collapse and cover the plasma membrane.
Post-mortem interval (PMI) is frequently calculated using immature stages of carrion frequenting Calliphoridae (Diptera). This is based on identification to species level, followed by age estimation of the samples. These two processes depend on suitable preservation of insects for subsequent analyses, yet preservation methods for the pupal stage are poorly defined and inappropriate methods may result in discolouration or nucleic acid degradation. This study examined the effects of 21 common preservation methods on Calliphora vicina pupae of 4 and 7d old, assessing consequences of the various methods for DNA-based species identification, age estimation using morphological analyses, and differential gene expression (DGE) studies. Pupae were examined within two weeks of preservation and again after 6-8 months. Of the methods tested, hot-water-killing (HWK) followed by storage in 80% ethanol at -20°C or 4°C was the best treatment for external morphology and histological analyses respectively. DNA based species identification was possible following all methods. RNA integrity and amplification were best when pupae were stored at -80°C or in RNAlater (-20°C), however HWK and storage in 80% ethanol at -20°C was also acceptable, and thus the latter is proposed as a universal preservative method for pupae. This study proposes a preservation method for pupae that enables DNA-based species identification, internal and external morphological analysis for age estimation, and DGE study to be carried out on a single specimen, enabling a multidisciplinary approach to age estimation from a single pupa.
In spray freeze drying (SFD) solutions are frozen by spraying into a very cold environment and subsequently dried by sublimation. In contrast to conventional freeze drying, spray freeze drying has the possibility to produce flowable lyophilizates which offers a variety of new pharmaceutical applications. Here, a drop jet nozzle is proposed as liquid dispenser that is able to produce droplets with a very narrow size distribution compared to standard methods. The drop jet nozzle is mounted in a spray tower designed to prevent direct contact of the product with the freezing medium. Various formulations have been tested containing lysozyme as model protein and stabilizers such as bovine serum albumin, polyvinylpyrrolidone or dextran in various concentrations and mannitol. Excellent free flowing and nearly monodispersed, porous particles are produced where particle properties can be controlled by formulation and process conditions. The particle diameter varied between 231±3μm and 310±10μm depending on the formulation composition. The lysozyme activity was >94±5% for all formulations exhibiting a full preservation of enzyme activity. This new method is very promising for the production of nearly monodisperse particulate lyophilizates in various therapeutic applications.
Fruits serve as a source of energy, vitamins, minerals, and dietary fiber. One of the barriers in increasing fruit and vegetables consumption is time required to prepare them. Overall, fruit bars have a far greater nutritional value than the fresh fruits because all nutrients are concentrated and, therefore, would be a convenience food assortment to benefit from the health benefits of fruits. The consumers prefer fruit bars that are more tasted followed by proper textural features that could be obtained by establishing the equilibrium of ingredients, the proper choosing of manufacturing stages and the control of the product final moisture content. Fruit bar preparations may include a mixture of pulps, fresh or dried fruit, sugar, binders, and a variety of minor ingredients. Additionally to the conventional steps of manufacturing (pulping, homogenizing, heating, concentrating, and drying) there have been proposed the use of gelled fruit matrices, dried gels or sponges, and extruders as new trends for processing fruit bars. Different single-type dehydration or combined methods include, in order of increasing process time, air-infrared, vacuum and vacuum-microwave drying convective-solar drying, convective drying, and freeze drying are also suggested as alternative to solar traditional drying stage. The dehydration methods that use vacuum exhibited not only higher retention of antioxidants but also better color, texture, and rehydration capacity. Antioxidant activity resulting from the presence of phenolic compounds in the bars is well established. Besides this, fruit bars are also important sources of carbohydrates and minerals. Given the wide range of bioactive factors in fresh fruits that are preserved in fruit bars, it is plausible that their uptake consumption have a positive effect in reducing the risk of many diseases.
Applications for antimicrobials derived from the mangosteen (Garcinia mangostana L.) plant are presently restricted by high production costs. Extraction from cultivation or processing waste streams using a solvent free approach could lessen to permit commercial applications in food processing and preservation.
As liquid liposomal formulations are prone to chemical degradation and aggregation, these formulations often require freeze drying (e.g. lyophilization) to achieve sufficient shelf-life. However, liposomal formulations may undergo oxidation during lyophilization and/or during prolonged storage. The goal of the current study was to characterize the degradation of 1, 2-dilinolenoyl-sn-glycero-3-phosphocholine (DLPC) during lyophilization, and to also probe the influence of metal contaminants in promoting the observed degradation. Aqueous sugar formulations containing DLPC (0.01 mg/ml) were lyophilized, and DLPC degradation was monitored using HPLC/UV and GC/MS methods. The effect of ferrous ion and sucrose concentration, as well as lyophilization stage promoting lipid degradation, was investigated. DLPC degradation increased with higher levels of ferrous ion. After lyophilization, 103.1% ± 1.1%, 66.9% ± 0.8%, and 28.7% ± 0.7% DLPC remained in the sucrose samples spiked with 0.0 ppm, 0.2 ppm and 1.0 ppm ferrous ion, respectively. Lipid degradation predominantly occurs during the freezing stage of lyophilization. Sugar concentration and buffer ionic strength also influence the extent of lipid degradation, and DLPC loss correlated with degradation product formation. We conclude that DLPC oxidation during the freezing stage of lyophilization dramatically compromises the stability of lipid-based formulations. In addition, we demonstrate that metal contaminants in sugars can become highly active when lyophilized in the presence of a reducing agent.
The aims of this study were to determine the stability of Podoviridae coliphage CA933P during lyophilization and storage in different media, and to establish similarities between the results obtained and those expected through mechanisms described for proteins stabilization during freeze-drying. PBS and SM buffer were assayed as lyophilization media. The effect of inorganic salts concentration as well as the addition of disaccharides on phage stability during freeze-drying and storage was also studied. The addition of low sucrose concentration (0.1 mol l(-1)) to SM buffer stabilized phage during freezing and drying steps of the lyophilization process, but higher sugar concentrations were detrimental to phage stability during freeze-drying. Sucrose stabilized phage during storage for at least 120 days. The lyoprotective effect of low concentrations of disaccharides during the drying step of the lyophilization of proteins as well as the stabilization of the freeze-dried product in time correlated with the results obtained for phage CA933P.