Dataset Information

Extracellular vesicles protect glucuronidase model enzymes during freeze-drying.

ABSTRACT: Extracellular vesicles (EVs) are natural nanoparticles that play important roles in intercellular communication and are increasingly studied for biosignalling, pathogenesis and therapy. Nevertheless, little is known about optimal conditions for their transfer and storage, and the potential impact on preserving EV-loaded cargoes. We present the first comprehensive stability assessment of different widely available types of EVs during various storage conditions including -80?°C, 4?°C, room temperature, and freeze-drying (lyophilisation). Lyophilisation of EVs would allow easy handling at room temperature and thus significantly enhance their expanded investigation. A model enzyme, ?-glucuronidase, was loaded into different types of EVs derived from mesenchymal stem cells, endothelial cells and cancer cells. Using asymmetric flow field-flow fractionation we proved that the model enzyme is indeed stably encapsulated into EVs. When assessing enzyme activity as indicator for EV stability, and in comparison to liposomes, we show that EVs are intrinsically stable during lyophilisation, an effect further enhanced by cryoprotectants. Our findings provide new insight for exploring lyophilisation as a novel storage modality and we create an important basis for standardised and advanced EV applications in biomedical research.

SUBMITTER: Frank J

PROVIDER: S-EPMC6098026 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Extracellular vesicles protect glucuronidase model enzymes during freeze-drying.

Frank Julia J Richter Maximilian M de Rossi Chiara C Lehr Claus-Michael CM Fuhrmann Kathrin K Fuhrmann Gregor G

Scientific reports 20180817 1

Extracellular vesicles (EVs) are natural nanoparticles that play important roles in intercellular communication and are increasingly studied for biosignalling, pathogenesis and therapy. Nevertheless, little is known about optimal conditions for their transfer and storage, and the potential impact on preserving EV-loaded cargoes. We present the first comprehensive stability assessment of different widely available types of EVs during various storage conditions including -80 °C, 4 °C, room tempera ...[more]

PMID: 30120298

Similar Datasets

Project description:Purpose: The goal of this study was to determine the microRNA (miRNA) content of extracellular vesicles (EVs) derived from murine mesenchymal stem cells (mMSC), and evaluate reproducibility among distinct EV productions. We also aimed at assessing the effect of freeze-drying on EV miRNA content, by performing sequencing on freeze-dried EVs and calculating statistical difference between unmodified and freeze-dried EVs. Methods: mMSC-derived EVs were obtained from mMSC in culture in reduced serum medium Opti-MEM by differential centrifugation, with a final step at 100,000 g for 110 min at 4°C. EV pellets (freeze-dried (n=3) or not (n=2)) were resuspended in Qiazol lysis buffer and RNA was extracted following RNeasy Micro kit. cDNA libraries for sequencing were prepared using the TruSeq Small RNA Sample Preparation Kit. Amplified cDNA constructs were purified on 6 % PAGE gel and DNA molecules corresponding to 15–50 nucleotide transcripts were excised, eluted from gel, and concentrated. Image analyses and base calling were performed using the HiSeq Control Software and Real-Time Analysis component (Illumina). Before statistical analysis, genes with less than 15 reads (cumulating all the analysed samples) were filtered out. Differentially expressed miRNA were identified using three Bioconductor packages: edgeR, DESeq and DESeq2. Results: Considering miRNAs detected with at least 5 counts (in terms of normalised counts), 339 miRNAs were identified and miRNA content was highly conserved among the two batches tested, with 237 miRNAs out of the 339 present in both batches (70%). Statistical analysis did not evidence statistical difference between unmodified EVs (n=2) and freeze-dried EVs (n=3) (DESeq2, p<0.05). No statistical difference was found using other Bioconductor packages DESeq and edgeR. These results indicated conservation of miRNA content following freeze-drying. Conclusion: mMSC-EV miRNA content was comparable between the two EV productions analysed, indicating reproducibility. Some of the miRNAs identified were consistent with previously published results on MSC-derived EVs. Freeze-drying conserved miRNA content.

Project description:Vacuum freeze-drying of biological materials is one of the best methods of water removal, with final products of highest quality. The solid state of water during freeze-drying protects the primary structure and the shape of the products with minimal volume reduction. In addition, the lower temperatures in the process allow maximal nutrient and bioactive compound retention. This technique has been successfully applied to diverse biological materials, such as meats, coffee, juices, dairy products, cells, and bacteria, and is standard practice for penicillin, hormones, blood plasma, vitamin preparations, etc. Despite its many advantages, having four to ten times more energy requirements than regular hot air drying, freeze-drying has always been recognized as the most expensive process for manufacturing a dehydrated product. The application of the freeze-drying process to plant-based foods has been traditionally dedicated to the production of space shuttle goods, military or extreme-sport foodstuffs, and specialty foods such as coffee or spices. Recently, the market for 'natural' and 'organic' products is, however, strongly growing as well as the consumer's demand for foods with minimal processing and high quality. From this perspective, the market for freeze-dried plant-based foods is not only increasing but also diversifying. Freeze-dried fruits and vegetables chunks, pieces, or slices are nowadays majorly used in a wide range of food products such as confectionaries, morning cereals, soups, bakeries, meal boxes, etc. Instant drinks are prepared out of freeze-dried tea, coffee, or even from maple syrup enriched with polyphenol concentrated extracts from trees. The possibilities are endless. In this review, the application of freeze-drying to transform plant-based foods was analyzed, based on the recent research publications on the subject and personal unpublished data. The review is structured around the following related topics: latest applications of freeze-drying to plant-based foods, specific technological problems that could be found when freeze-drying such products (i.e., presence of cuticle; high sugar or lipid concentration), pretreatments and intensification technologies employed in freeze-drying of plant-based foods, and quality issues of these freeze-dried products.

Dataset Information

Extracellular vesicles protect glucuronidase model enzymes during freeze-drying.

Publications

Extracellular vesicles protect glucuronidase model enzymes during freeze-drying.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets