Isolation and Flow Cytometry Characterization of Extracellular-Vesicle Subpopulations Derived from Human Mesenchymal Stromal Cells.

This unit describes protocols for isolating subpopulations of extracellular vesicles (EVs) purified from human adipose tissue-derived mesenchymal stromal cells by density gradient centrifugation and for characterizing them by flow cytometry (FCM). Determining the optimal strategy for isolating EVs is a critical step toward retrieving the maximal amount while ensuring the recovery of different vesicular subtypes. The first protocol details density gradient centrifugation to isolate both exosomes and microvesicles. In the second protocol, characterization of EV subpopulations by FCM is depicted, taking advantage of non-conventional modalities, in accordance with the latest technical indications. The procedures described here can be easily reproduced and can be employed regardless of the cell type used to obtain EVs. © 2019 by John Wiley & Sons, Inc.

Gorgun C ，Reverberi D ，Rotta G ，Villa F ，Quarto R ，Tasso R ... -  《-》

A Method for Isolating and Characterizing Mesenchymal Stromal Cell-derived Extracellular Vesicles.

The unit describes protocols for isolating and characterizing extracellular vesicles (EVs) derived from human adipose tissue-derived mesenchymal stromal cells (MSCs). EVs are a mixed population of membrane-surrounded structures with overlapping composition and size. Advances made in recent years have led to a better understanding of the biological role of EVs. In particular, they can be considered key factors responsible for MSC-paracrine activity, mediating their anti-inflammatory effects towards innate immune cells, such as macrophages. The topics comprise description of the MSC-conditioned medium containing vesicles preparation, EV isolation, and characterization mainly by specifically set up flow cytometry and electron microscopy approaches, and in vitro methodologies involved in testing the EV anti-inflammatory capacity. The procedures described here can be easily reproduced and can be employed regardless of the type of progenitor cells used to secrete EVs. © 2018 by John Wiley & Sons, Inc.

Lo Sicco C ，Reverberi D ，Pascucci L ，Tasso R ... -  《-》

Isolation and Characterization of Exosomes from Cultures of Tissue Explants and Cell Lines.

Exosomes are specialized, nanometer-sized extracellular vesicles of endosomal origin actively secreted into the extracellular space by a variety of cells under normal and pathological conditions. Exosomes have recently emerged as important intercellular communicators and modulators of diverse mechanisms and cellular responses. Characterization of their composition and function will open possibilities for new diagnostic methods and promising therapeutic approaches based on nanobiology. This unit provides a standard isolation procedure for purification of exosomes based on density gradient ultracentrifugation with sucrose. The process of isolating exosomes relies on obtaining proper source fluids/supernatants as well as qualitative and quantitative assessment of the isolated vesicles. The methodological procedures here can be divided in three parts: (1) pre-isolation procedures aiming to obtain fluids containing exosomes, with a focus on protocols for organ explants and cell cultures; (2) a procedure for exosome isolation with several gradient alternatives; and (3) post-isolation procedures for estimating the purity and yield of the exosomal fraction. © 2016 by John Wiley & Sons, Inc.

Mincheva-Nilsson L ，Baranov V ，Nagaeva O ，Dehlin E ... -  《-》

A Protocol for Isolation and Proteomic Characterization of Distinct Extracellular Vesicle Subtypes by Sequential Centrifugal Ultrafiltration.

Xu R ，Simpson RJ ，Greening DW 《-》

Analysis of individual extracellular vesicles by imaging flow cytometry.

Virtually all cells release extracellular vesicles (EVs) into their environment, such as exosomes and microvesicles. EVs can mediate intercellular communication processes in a targeted manner. Representing their cell of origin, EVs contain cell type specific signatures, qualifying them as a novel class of biomarkers. Furthermore, according to their tropism to certain target cells, EVs provide promising aspects to be used as drug delivery vehicles. Depending on their origin, certain EVs contain the potential to modulate physiological and pathophysiological processes. Although the EV field provides many interesting aspects, the methodology in EV research is limited. For now, EVs are mainly analyzed by nanoparticle tracking analysis and bulk molecular analysis, regularly Western Blot. These technologies cannot dissect the heterogeneity of EVs observed by electron microscopy (EM). Although EM technologies help to demonstrate the heterogeneity within EV samples, EM technologies are not appropriate to perform more complex and quantitative EV analyses. Flow cytometry (FCM) is a traditional method for dissecting the heterogeneity of given cell populations in a quantitative and complex manner. However, classical FCM regularly fails to detect objects in the size range of small EVs (sEVs) that typically is in the range between 70 and 150nm. Recently, we and others demonstrated the potential of imaging FCM for the analyses of small EVs at the single vesicle level. Here, at the example of sEVs harvested from supernatants of human mesenchymal stromal cells (MSCs), we share a protocol for studying the expression of the tetraspanins CD9, CD63 and CD81 on single EVs.

Tertel T ，Görgens A ，Giebel B 《-》