Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods.

Tamás Baranyai,Zoltán Giricz,Ágnes Kittel,Péter Ferdinandy,István Voszka,Nikolett Marton,Péter Nagy,Zsófia Onódi,Matthew J Wood,Károly Módos,Imre Mäger,György Nagy,Edit Irén Buzás,Zoltán Pálinkás,Kata Herczeg,Samir El Andaloussi,Vikas Kumar,Marco Rito-Palomares

doi:10.1371/journal.pone.0145686

Abstract

BackgroundExosomes are emerging targets for biomedical research. However, suitable methods for the isolation of blood plasma-derived exosomes without impurities have not yet been described.AimTherefore, we investigated the efficiency and purity of exosomes isolated with potentially suitable methods; differential ultracentrifugation (UC) and size exclusion chromatography (SEC).Methods and ResultsExosomes were isolated from rat and human blood plasma by various UC and SEC conditions. Efficiency was investigated at serial UC of the supernatant, while in case of SEC by comparing the content of exosomal markers of various fractions. Purity was assessed based on the presence of albumin. We found that the diameter of the majority of isolated particles fell into the size range of exosomes, however, albumin was also present in the preparations, when 1h UC at 4°C was applied. Furthermore, with this method only a minor fraction of total exosomes could be isolated from blood as deduced from the constant amount of exosomal markers CD63 and TSG101 detected after serial UC of rat blood plasma samples. By using UC for longer time or with shorter sedimentation distance at 4°C, or UC performed at 37°C, exosomal yield increased, but albumin impurity was still observed in the isolates, as assessed by transmission electron microscopy, dynamic light scattering and immunoblotting against CD63, TSG101 and albumin. Efficiency and purity were not different in case of using further diluted samples. By using SEC with different columns, we have found that although a minor fraction of exosomes can be isolated without significant albumin content on Sepharose CL-4B or Sephacryl S-400 columns, but not on Sepharose 2B columns, the majority of exosomes co-eluted with albumin.ConclusionHere we show that it is feasible to isolate exosomes from blood plasma by SEC without significant albumin contamination albeit with low vesicle yield.

Highlights

Extracellular vesicles (EVs) are small membrane-enclosed structures with various functions and different origins
We found that the diameter of the majority of isolated particles fell into the size range of exosomes, albumin was present in the preparations, when 1h UC at 4°C was applied
Apoptotic bodies are released by apoptotic cells, microvesicles are shed from plasma membrane, while exosomes are secreted through the endosome-multivesicular body complex by most cell types

Summary

Introduction

Extracellular vesicles (EVs) are small membrane-enclosed structures with various functions and different origins. Three major types of extracellular vesicles have been characterized extensively: apoptotic bodies (>1000 nm), microvesicles (100–1000 nm), and exosomes (30– 100 nm). Apoptotic bodies are released by apoptotic cells, microvesicles are shed from plasma membrane, while exosomes are secreted through the endosome-multivesicular body complex by most cell types (e.g., reticulocytes [1], dendritic cells [2], endothelial cells [3], tumor cells [4], or cardiomyocytes [5]; see for review: [6]). A continuously growing number of reports indicate that EVs, exosomes convey information to neighboring or remote cells by delivering RNAs and proteins and affecting various physiological and pathological signaling pathways [7,8,9,10]. Suitable methods for the isolation of blood plasma-derived exosomes without impurities have not yet been described

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