Book Chapter – Part of the Methods in Molecular Biology book series (MIMB, volume 2668)
Published in June 2023
Summary: Extracellular vesicles (EVs) are a growing area of research, especially in intercellular communication. Commonly used purification methods are typically unable to be scaled and can lead to EVs with poor integrity. Here, we have proposed a novel purification method of EVs, utilizing tangential flow filtration and multimodal chromatography to isolate EVs at high purity from large volumes.
Abstract: Extracellular vesicle (EV) isolation from conditioned cell culture medium has been a challenging topic. It is particularly difficult to obtain pure and intact EVs at a large scale. The commonly used methods such as differential centrifugation, ultracentrifugation, size exclusion chromatography, polyethylene glycol (PEG) precipitation, filtration, and affinity-based purification each have their advantages and limitations. Here, we present a tangential-flow filtration (TFF) based, multi-step purification protocol that combines filtration, PEG precipitation, and Capto Core 700 multimodal chromatography (MMC) to isolate EVs at high purity from large volumes of cell culture conditioned medium.
Research paper published in Journal of Extracell Vesicles (JEV)
Published first online in May 2023
Summary: Extracellular vesicles (EVs) are growing as a topic of research, as they play roles in intercellular communication. A main challenge in EV research is heterogeneity, where different cells from the same cell line can produce a variety of different EVs. Isolation of EVs provides different challenges and limitations, which can impact the study of them. Here, we found that utilizing heparin chromatography can isolate EVs into two subpopulations, either heparin binding or non-heparin binding.
Abstract: Purifying extracellular vesicles (EVs) has been challenging because EVs are heterogeneous in cargo yet share similar sizes and densities. Most surface marker-based affinity separation methods are limited to research or diagnostic scales. We report that heparin chromatography can separate purified EVs into two distinct subpopulations as ascertained by MS/MS: a non-heparin-binding (NHB) fraction that contains classical EV markers such as tetraspanins and a heparin-binding (HB) fraction enriched in fibronectins and histones. Both fractions were similarly fusogenic but induced different transcriptional responses in endothelial cells. While EVs that were purified by conventional, non-affinity methods alone induced ERK1/2 phosphorylation and Ki67, the NHB fraction did not. This result suggests heparin chromatography as an additional novel fractionation step that is inherently scalable, does not lead to loss of material, and separates inflammatory and pyrogenic EVs from unreactive EVs, which will improve clinical applications.
Congratulation Gaby on your research!