Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

Summary

This protocol details a method to isolate extracellular vesicles (EVs), small membranous particles released from cells, from as little as 10 μl serum samples. This approach circumvents the need for ultracentrifugation, requires only a few minutes of assay time, and enables the isolation of EVs from samples of limited volumes.

Abstract

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain nucleic acid and protein cargo and are increasingly recognized as a means of intercellular communication utilized by both eukaryote and prokaryote cells. However, due to their small size, current protocols for isolation of EVs are often time consuming, cumbersome, and require large sample volumes and expensive equipment, such as an ultracentrifuge. To address these limitations, we developed a paper-based immunoaffinity platform for separating subgroups of EVs that is easy, efficient, and requires sample volumes as low as 10 μl. Biological samples can be pipetted directly onto paper test zones that have been chemically modified with capture molecules that have high affinity to specific EV surface markers. We validate the assay by using scanning electron microscopy (SEM), paper-based enzyme-linked immunosorbent assays (P-ELISA), and transcriptome analysis. These paper-based devices will enable the study of EVs in the clinic and the research setting to help advance our understanding of EV functions in health and disease.

Introduction

Extracellular vesicles (EVs) are heterogeneous membranous particles that range in size from 40 nm to 5,000 nm and are released actively by many cell types via different biogenesis routes^1-9. They contain unique and selected subsets of DNA, RNA, proteins, and surface markers from parental cells. Their involvement in a variety of cellular processes, such as intercellular communication¹⁰, immunity modulation¹¹, angiogenesis¹², metastasis¹², chemoresistance¹³, and the development of eye diseases⁹, is increasingly recognized and has spurred a great interest in their utility in diagnostic, prognost....

Protocol

A general diagram of the operation procedure is provided in Figure 1. Using ethical practices, we collected blood samples from healthy subjects, and obtained aqueous humor samples from patients through the Taichung Veterans General Hospital (TCVGH), Taichung, Taiwan under IRB approved protocols (IRB TCVGH No. CF11213-1).

1. Fabrication of Paper Devices

1. Cut chromatography paper into circles of 5 mm in diameter to provide the same layout as a 96-well microtiter plate. Sandwich these paper pieces with two polystyrene sheets with registered through-holes, and laminate.
2. Modify paper devices using the chemical conjugation method....

Results

The ability of the paper device to isolate subgroups of EVs efficiently relies upon its sensitive and specific recognition of EV surface markers. The stable modification of paper fibers with capture molecules is achieved by using avidin-biotin chemistry as described elsewhere^28-30. The effectiveness of chemical conjugation and that of the physisorption method is assessed using fluorescence-based readouts. The paper test zones are prepared following the protocol step 1) except the capture molecule is replaced w.......

Discussion

The most critical steps for successful isolation of subgroups of extracellular vesicles are: 1) a good choice of paper; 2) stable and high coverage of capture molecules on the surface of the paper fibers; 3) proper handling of samples; and 4) general laboratory hygiene practice.

Porous materials have been utilized in many inexpensive and equipment-free assays. They may have tunable pore size, versatile functionality, low cost and high surface-to-volume ratio permitting passive wicking of fluid.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chromatography Paper	GE Healthcare Life Sciences	3001-861	Whatman® Grade 1 cellulose paper
(3-Mercaptopropyl) trimethoxysilane	Sigma Aldrich	175617	This chemical reacts with water and moisture and should be applied inside a nitrogen-filled glove bag. Avoid eye and skin contact. Do not breathe fumes or inhale vapors.
Ethanol	Fisher Scientific	BP2818	Absolute, 200 Proof, molecular biology grade
Bovine serum albumin (BSA)	BioShop Canada Inc.	ALB001	Often referred to as Cohn fraction V.
N-γ-maleimidobutyryloxy succinimide ester (GMBS)	Pierce Biotechnology	22309	GMBS is an amine-to-sulfhydryl crosslinker. GMBS is moisture-sensitive.
Avidin	Pierce Biotechnology	31000	NeutrAvidin has 4 binding sites for biotin and its pI value is 6.3, which is more neutral than native avidin
Biotinylated mouse anti-human anti-CD63	Ancell	215-030	clone AHN16.1/46-4-5
biotinylated annexin V	BD Biosciences	556418	Annxin V has a high affinity for phosphatidylserine (PS)
Primary anti-CD9 and secondary antibody	System Biosciences	EXOAB-CD9A-1	The secondary antibody is horseradish peroxidise-conjugated
Serum separation tubes	BD Biosciences	367991	Clot activator and gel for serum separation
Annexin V binding buffer	BD Biosciences	556454	10x; dilute to 1x prior to use.
TMB substrate reagent set	BD Biosciences	555214	The set contains hydrogen peroxide and 3,3’,5,5’-tetramethylbenzidine (TMB)
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RNA isolation kit	Life Technologies	AM1560	MirVana RNA isolation kit
Polyvinylpyrrolidone-based RNA isolation aid	Life Technologies	AM9690	Plant RNA isolation aid contains polyvinylpyrrolidone (PVP) that binds to polysaccharides.
RNA cleanup kit	Qiagen Inc.	74004	MinElute RNA cleanup kit is designed for purification of up to 45 μg RNA.
Plasma chamber	March Instruments	PX-250
Scanning electron microscope	Hitachi Ltd.	S-4300
Desktop scanner	Hewlett-Packard Company	Photosmart B110	8-bit color images were captured. Cameras and smart phones may be also used.
Image-record system	J&H Technology Co	GeneSys G:BOX Chemi-XX8	16-bit fluroscence images were captured. Fluroscence microscopes may be also used.