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, prognostic, therapeutic, and basic biology applications.

EVs can be classically categorized as exosomes, microvesicles, apoptotic bodies, oncosomes, ectosomes, microparticles, telerosomes, prostatosomes, cardiosomes, and vexosomes, etc., based on their biogenesis or cellular origin. For example, exosomes are formed in multivesicular bodies, whereas microvesicles are generated by budding directly from plasma membrane and apoptotic vesicles are from apoptotic or necrotic cells. However, the nomenclature is still under refined, partly due to a lack of thorough understanding and characterization of EVs. Several methods have been developed to purify EVs, including ultracentrifugation¹⁴, ultrafiltration¹⁵, magnetic beads¹⁶, polymeric precipitation^17-19, and microfluidic techniques^20-22. The most common procedure to purify EVs involves a series of centrifugations and/or filtration to remove large debris and other cellular contaminants, followed by a final high-speed ultracentrifugation, a process that is expensive, tedious, and nonspecific^14,23,24. Unfortunately, technological need for rapid and reliable isolation of EVs with satisfactory purity and efficiency is not yet met.

We have developed a paper-based immunoaffinity device that provides a simple, time- and cost-saving, yet efficient way to isolate and characterize subgroups of EVs²². Cellulose paper cut into a defined shape can be arranged and laminated using two plastic sheets with registered through-holes. In contrast to the general strategy to define the fluid boundary in paper-based devices by printing hydrophobic wax or polymers^25-27, these laminated paper patterns are resistant to many organic liquids, including ethanol. Paper test zones are chemically modified to provide stable and dense coverage of capture molecules (e.g., target-specific antibodies) that have high affinity to specific surface markers on EV subgroups. Biological samples can be pipetted directly onto the paper test zones, and purified EVs are retained after rinse steps. Characterization of isolated EVs can be performed by SEM, ELISA, and transcriptomic analysis.

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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 described in the following steps²⁸.
   1. Treat paper devices with oxygen plasma (100 mW, 1% oxygen, 30 sec) in a plasma chamber.
      CAUTION: Special caution must be taken when working with 3-mercaptopropyl trimethoxysilane and N-γ-maleimidobutyryloxy succinimide ester (GMBS), since they are both moisture sensitive and toxic. Wear protective gloves and avoid inhalation or contact with skin and eyes. Keep the stock bottle tightly closed and allow it to equilibrate to RT before opening to avoid water condensation. Alternatively, open the stock bottle inside a nitrogen-filled glove bag or box. Aliquot into small vials to avoid frequent opening of the stock bottle.
   2. Immediately incubate treated paper devices in a 4% (v/v) solution of 3-mercaptopropyl trimethoxysilane in ethanol (200 proof) for 30 min.
   3. Rinse paper devices with ethanol and incubate them with 0.01 μmol/ml GMBS in ethanol for 15 min.
   4. Rinse with ethanol (200 proof) and incubate paper devices with 10 µg/ml avidin solution in phosphate buffered saline (PBS) for 1 hr at 4 °C. Store at 4 °C if needed, and use within 4 weeks.
3. Wet each paper test zones with 10 μl PBS containing 1% (w/v) bovine serum albumin (BSA) for 3 × 10 min before spotting 10 μl biotinylated capture molecules for 3 × 10 min. Use anti-CD63 antibody (20 μg/ml in PBS containing 1% (w/v) BSA and 0.09% (w/v) sodium azide) or annexin V (1:20, v/v in annexin V binding buffer) as capture molecules.
4. Rinse off unbound anti-CD63 antibody or annexin V molecules using 10 μl corresponding PBS containing 1% (w/v) BSA or annexin V binding buffer solutions for 3 × 1 min, respectively.

2. Serum and Aqueous Humor Sample Collection and Processing

1. Serum collection.
   1. Collect 10 ml of peripheral blood by venipuncture in serum separation tubes and gently invert the tube five times. Set the tube in a vertical position and wait for 30 min.
   2. Centrifuge at 1,200 × g for 15 min. Transfer the serum from the top layer to a clean tube, and centrifuge again at 3,000 × g for 30 min.
   3. Pass the supernatant through a 0.8 μm filter. Keep the sample at -80 °C until use.
2. Aqueous humor collection: collect aqueous humor samples from patients diagnosed by an ophthalmologist directly through invasive procedures and store samples at -80 °C until use.

3. Isolation of Extracellular Vesicles

1. Spot samples onto each paper test zone at a rate of 5 μl/min.
2. Rinse off unbound EVs with 10 μl corresponding PBS containing 1% (w/v) BSA or annexin V binding buffer solutions for 3 × 1 min for paper devices functionalized with anti-CD63 antibody or annexin V molecules, respectively. Perform the following downstream assays.

4. Downstream Assay Example 1: Scanning Electromicrographs

1. Fix EVs captured on functionalized paper test zone using 10 μl 0.5× Karnovsky’s fixative for 10 min.
2. Rinse the samples with ample PBS for 2 × 5 min.
3. Dehydrate the samples with subsequent 35% ethanol for 10 min, 50% ethanol for 2 × 10 min, 70% ethanol for 2 × 10 min, 95% ethanol for 2 × 10 min, and 100% ethanol for 4 × 10 min.
4. Critical dry and sputter coat the samples with palladium/gold, and examine using an scanning electron microscope operated at low electron acceleration voltage (~ 5 kV).

5. Downstream Assay Example 2: Paper-based ELISA

1. Add a 5 μl solution containing anti-CD9 primary antibody at 1:1,000 dilution in PBS to each test zone containing captured EVs. Wait 1  min.
2. Rinse the samples with 30 ml PBS shaken at 100 rpm for 30 sec.
3. Add a 5 μl solution containing horseradish peroxidase (HRP)-linked secondary antibody at 1:1,000 dilution in PBS and wait 1 min.
4. Rinse the samples with 30 ml PBS shaken at 100 rpm for 30 sec.
5. Add a 5 μl colorimetric substrate containing 1:1 volume ratio of hydrogen peroxide and 3,3’,5,5’-tetramethylbenzidine (TMB) and scan using a desktop scanner.

6. Downstream Assay Example 3: RNA Isolation

1. Immerse the samples in 35 μl of polyvinylpyrrolidone-based RNA isolation aid and 265 μl of lysis/binding buffer to lyse EVs captured. Vortex vigorously.
2. Add 30 μl homogenate provided in the isolation kit to the lysate. Vortex vigorously and put on ice for 10 min.
3. Extract the RNA using acid phenol-chloroform separation described in the following steps.
   1. Take 330 μl of phenol-chloroform from the bottom layer of the bottle and add to the homogenate/lysate. Vortex vigorously.
   2. Centrifuge at 10,000 x g for 5 min. Transfer the aqueous (upper) phase to a clean tube and note the volume removed.
4. Precipitate the total RNA with 100% ethanol of the volume that is 1.25 times the volume of the aqueous phase removed in the previous step and collect the RNA in the elution solution preheated to 95 °C.
5. Concentrate and further purify the RNA using an RNA cleanup kit according to the manufacture’s protocol.

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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...

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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...

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Materials

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.