Reproducible and scalable purification of extracellular vesicles using combined bind-elute and size exclusion chromatography

Abstract

Extracellular vesicles (EVs) play a pivotal role in cell-to-cell communication and have been shown to take part in several physiological and pathological processes. EVs have traditionally been purified by ultracentrifugation (UC), however UC has limitations, including resulting in, operator-dependant yields, EV aggregation and altered EV morphology, and moreover is time consuming. Here we show that commercially available bind-elute size exclusion chromatography (BE-SEC) columns purify EVs with high yield (recovery ~ 80%) in a time-efficient manner compared to current methodologies. This technique is reproducible and scalable, and surface marker analysis by bead-based flow cytometry revealed highly similar expression signatures compared with UC-purified samples. Furthermore, uptake of eGFP labelled EVs in recipient cells was comparable between BE-SEC and UC samples. Hence, the BE-SEC based EV purification method represents an important methodological advance likely to facilitate robust and reproducible studies of EV biology and therapeutic application.

Large‐scale, cross‐flow based isolation of highly pure and endocytosis‐competent extracellular vesicles

“…In principle even higher enrichment is possible, since CFF can be fed continuously allowing for input volumes in the multi-litre range. This report supports the recent findings by Corso et al in which a cross-flow/large MW SEC approach can be used to isolate EV in high concentrations from adherent cells [55]. In our hands, we have used volumes up to 400 mL from lymphoma cells (BCBL-1) and human plasma to expand upon this.…”

Management of Tamm–Horsfall Protein for Reliable Urinary Analytics

“…In some approaches, reagents including dithiothreitol (DTT) or deuterated water to create sucrose cushion or gradient have been used. Other methods to rid THP include size exclusion chromatography or serial combination of DTT, centrifugation, centrifugal ultrafiltration, and size exclusion chromatography . Yet another alternative to eliminate THP artifacts is to induce THP polymerization with diatomatous earth filter .…”

Scalable Production and Isolation of Extracellular Vesicles: Available Sources and Lessons from Current Industrial Bioprocesses

“…UC‐based workflows are the most common isolation methods since they enable the recovery of relatively high EV yields at the laboratory scale, especially when compared to alternative methods, such as ultrafiltration (UF), size‐exclusion chromatography (SEC), immunoaffinity capture, and polymer precipitation . However, the implementation of UC on a large scale is challenging because of several reasons: i) it requires large and heavy rotors that demand a consistent amount of electrical power to be operated; ii) being a batch process, it has several dead times that reduce the overall productivity; iii) it often copurifies contaminants; iv) it involves high shear forces, causing aggregation and rupture of EVs; v) the composition of the isolated EV mixture is highly sensitive to a variety of experimental settings such as tube type and rotor type, leading to low consistency of EV mixtures obtained with different ultracentrifuges …”

“…Even though multistep downstream processes are industrially applied, a purification protocol that relies on a single operation would be clearly preferable since every step affects the yield, the processing time, and the costs . To pursue this goal, Corso et al combined bind‐elution and SEC, thus moving towards the direction of mixed‐mode chromatography. In their setup, proteins and impurities smaller than 700 kDa were captured in the pores of the stationary phase by positively charged octylamine ligands, whereas larger particles like EVs flowed through the column without interacting .…”

“…To pursue this goal, Corso et al combined bind‐elution and SEC, thus moving towards the direction of mixed‐mode chromatography. In their setup, proteins and impurities smaller than 700 kDa were captured in the pores of the stationary phase by positively charged octylamine ligands, whereas larger particles like EVs flowed through the column without interacting . Despite improved purification, this approach still requires a concentration step prior to the SEC column loading …”