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Abstract

Immunology and Infection

Isolation of Low Endotoxin Content Extracellular Vesicles Derived from Cancer Cell Lines

Published: February 17th, 2023

DOI:

10.3791/65062

1Department of Clinical Immunology, Jagiellonian University Medical College, 2Doctoral School of Medical and Health Sciences, Jagiellonian University, 3Institute of Veterinary Sciences, University Center of Veterinary Medicine JU-AU, University of Agriculture in Kraków

* These authors contributed equally

Abstract

Extracellular vesicles (EVs) are a heterogeneous population of membrane vesicles released by cells in vitro and in vivo. Their omnipresence and significant role as carriers of biological information make them intriguing study objects, requiring reliable and repetitive protocols for their isolation. However, realizing their full potential is difficult as there are still many technical obstacles related to their research (like proper acquisition). This study presents a protocol for the isolation of small EVs (according to the MISEV 2018 nomenclature) from the culture supernatant of tumor cell lines based on differential centrifugation. The protocol includes guidelines on how to avoid contamination with endotoxins during the isolation of EVs and how to properly evaluate them. Endotoxin contamination of EVs can significantly hinder subsequent experiments or even mask their true biological effects. On the other hand, the overlooked presence of endotoxins may lead to incorrect conclusions. This is of particular importance when referring to cells of the immune system, including monocytes, because monocytes constitute a population that is especially sensitive to endotoxin residues. Therefore, it is highly recommended to screen EVs for endotoxin contamination, especially when working with endotoxin-sensitive cells such as monocytes, macrophages, myeloid-derived suppressor cells, or dendritic cells.

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Keywords Extracellular Vesicles

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