This work was supported in part by the Taiwan National Science Council grants- NSC 99-2320-B-007-005-MY2 (CC) and NSC 101-2628-E-007-011-MY3 (CMC), and the Veterans General Hospitals and University System of Taiwan Joint Research Program (CC).

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
<ol>
	<li>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.</li>
	<li>Modify paper devices using the chemical conjugation method described in the following steps28.
	<ol>
		<li>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-&#947;-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.</li>
		<li>Immediately incubate treated paper devices in a 4% (v/v) solution of 3-mercaptopropyl trimethoxysilane in ethanol (200 proof) for 30 min.</li>
		<li>Rinse paper devices with ethanol and incubate them with 0.01 &#956;mol/ml GMBS in ethanol for 15 min.</li>
		<li>Rinse with ethanol (200 proof) and incubate paper devices with 10 &#181;g/ml avidin solution in phosphate buffered saline (PBS) for 1 hr at 4 &#176;C. Store at 4 &#176;C if needed, and use within 4 weeks.</li>
	</ol>
	</li>
	<li>Wet each paper test zones with 10 &#956;l PBS containing 1% (w/v) bovine serum albumin (BSA) for 3 &#215; 10 min before spotting 10 &#956;l biotinylated capture molecules for 3 &#215; 10 min. Use anti-CD63 antibody (20 &#956;g/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.</li>
	<li>Rinse off unbound anti-CD63 antibody or annexin V molecules using 10 &#956;l corresponding PBS containing 1% (w/v) BSA or annexin V binding buffer solutions for 3 &#215; 1 min, respectively.</li>
</ol>
2. Serum and Aqueous Humor Sample Collection and Processing
<ol>
	<li>Serum collection.
	<ol>
		<li>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.</li>
		<li>Centrifuge at 1,200 &#215; g for 15 min. Transfer the serum from the top layer to a clean tube, and centrifuge again at 3,000 &#215; g for 30 min.</li>
		<li>Pass the supernatant through a 0.8 &#956;m filter. Keep the sample at -80 &#176;C until use.</li>
	</ol>
	</li>
	<li>Aqueous humor collection: collect aqueous humor samples from patients diagnosed by an ophthalmologist directly through invasive procedures and store samples at -80 &#176;C until use.</li>
</ol>
3. Isolation of Extracellular Vesicles
<ol>
	<li>Spot samples onto each paper test zone at a rate of 5 &#956;l/min.</li>
	<li>Rinse off unbound EVs with 10 &#956;l corresponding PBS containing 1% (w/v) BSA or annexin V binding buffer solutions for 3 &#215; 1 min for paper devices functionalized with anti-CD63 antibody or annexin V molecules, respectively. Perform the following downstream assays.</li>
</ol>
4. Downstream Assay Example 1: Scanning Electromicrographs
<ol>
	<li>Fix EVs captured on functionalized paper test zone using 10 &#956;l 0.5&#215; Karnovsky&#8217;s fixative for 10 min.</li>
	<li>Rinse the samples with ample PBS for 2 &#215; 5 min.</li>
	<li>Dehydrate the samples with subsequent 35% ethanol for 10 min, 50% ethanol for 2 &#215; 10 min, 70% ethanol for 2 &#215; 10 min, 95% ethanol for 2 &#215; 10 min, and 100% ethanol for 4 &#215; 10 min.</li>
	<li>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).</li>
</ol>
5. Downstream Assay Example 2: Paper-based ELISA
<ol>
	<li>Add a 5 &#956;l solution containing anti-CD9 primary antibody at 1:1,000 dilution in PBS to each test zone containing captured EVs. Wait 1&#160; min.</li>
	<li>Rinse the samples with 30 ml PBS shaken at 100 rpm for 30 sec.</li>
	<li>Add a 5 &#956;l solution containing horseradish peroxidase (HRP)-linked secondary antibody at 1:1,000 dilution in PBS and wait 1 min.</li>
	<li>Rinse the samples with 30 ml PBS shaken at 100 rpm for 30 sec.</li>
	<li>Add a 5 &#956;l colorimetric substrate containing 1:1 volume ratio of hydrogen peroxide and 3,3&#8217;,5,5&#8217;-tetramethylbenzidine (TMB) and scan using a desktop scanner.</li>
</ol>
6. Downstream Assay Example 3: RNA Isolation
<ol>
	<li>Immerse the samples in 35 &#956;l of polyvinylpyrrolidone-based RNA isolation aid and 265 &#956;l of lysis/binding buffer to lyse EVs captured. Vortex vigorously.</li>
	<li>Add 30 &#956;l homogenate provided in the isolation kit to the lysate. Vortex vigorously and put on ice for 10 min.</li>
	<li>Extract the RNA using acid phenol-chloroform separation described in the following steps.
	<ol>
		<li>Take 330 &#956;l of phenol-chloroform from the bottom layer of the bottle and add to the homogenate/lysate. Vortex vigorously.</li>
		<li>Centrifuge at 10,000 x g for 5 min. Transfer the aqueous (upper) phase to a clean tube and note the volume removed.</li>
	</ol>
	</li>
	<li>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 &#176;C.</li>
	<li>Concentrate and further purify the RNA using an RNA cleanup kit according to the manufacture&#8217;s protocol.</li>
</ol>

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The authors declare that they have no competing financial interests.

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 fluids. We chose chromatography cellulose paper grade 1 mainly for its proper pore size and low protein adsorption. Compared to another commonly used porous materials, nitrocellulose, cellulose paper has much lower nonspecific protein adsorption34. We also find little nonspecific capture of EVs in this study. In addition, its particle retention level, 11 &#956;m, is larger than the size of EVs35, allowing for specific capture in contrast to physical trapping of EVs. Here, we focus on EVs of a smaller size by passing serum samples through a 0.8 &#956;m filter, which step may be modified when larger EVs are to be studied.
Chemical surface modification can be a key to achieve a stable and high coverage of capture molecules on the surface as a means of improving the efficiency of EV isolation. Surface modifications of cellulose have been reviewed36,37. There are high numbers of hydroxyl (-OH) groups on the surface of cellulose and they can react with silane to give stable chemical Si-O-C bonds after being activated with oxygen plasma or other condensation process. The silane used in this study is (3-mercaptopropyl) trimethoxysilane, (MeO)3Si-(CH2)3-SH. The thiol (-SH) group can react with GMBS to provide a short space arm (0.73 nm) and a crosslinking group that couples with primary amines. Capture molecules with amine groups can be conjugated to GMBS directly. In this study, however, we conjugated NeutrAvidin instead to provide a general scheme to conjugate various capture molecules that are biotin-labeled. However, consensus on EV subtype-specific markers has yet to be reached17. NeutrAvidin has a very strong affinity to biotin and a near-neutral isoelectric point (pH 6.3), minimizing nonspecific interactions with negatively charged objects, e.g. the surface of EVs. In contrast, avidin with an isoelectric point of 10.5 carries positive charges at neutral pH. A higher concentration of anti-CD63 antibody is used than that in Chen et al.20, mainly due to the small volume applied and the larger surface-to-volume ratio of paper device. However, the amount and compositions of proteins adsorbed onto the paper fibers have not been characterized, which may be sample-dependent. Hence, care must be taken when interpreting the results of protein analysis.
Collected biological samples should be handled gently and processed rapidly to prevent artifactually increased EV levels. It is recommended to remove cells and platelets, if any, before storage at -80 &#176;C. A current standardization of sample handling in EV research can be found in this review17.
Proper laboratory hygiene practice should be employed. Always wear gloves and change gloves frequently to minimize the introduction of dust particles and ribonucleases. Isolation of RNA from EVs in cell culture-conditioned media can yield more than 70 times greater concentrations using paper devices as compared to that extracted from EVs concentrated using the ultracentrifuge method (unpublished).
The protocol described herein uses cellulose paper devices modified chemically with capture molecules to isolate different subgroups of EVs. The method is simple, efficient, and especially valuable when the sample volume is limited.

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 routes1-9. 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 precipitation17-19, and microfluidic techniques20-22. 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 nonspecific14,23,24. 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 polymers25-27, 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.

<table><tbody><tr><td>Chromatography Paper</td><td>GE Healthcare Life Sciences</td><td>3001-861</td><td>Whatman&amp;reg; Grade 1 cellulose paper</td></tr><tr><td>(3-Mercaptopropyl) trimethoxysilane</td><td>Sigma Aldrich</td><td>175617</td><td>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.</td></tr><tr><td>Ethanol</td><td>Fisher Scientific</td><td>BP2818</td><td>Absolute, 200 Proof, molecular biology grade</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>BioShop Canada Inc.</td><td>ALB001</td><td>Often referred to as Cohn fraction V.</td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-&amp;#947;-maleimidobutyryloxy succinimide ester (GMBS)</td><td>Pierce Biotechnology</td><td>22309</td><td>GMBS is an amine-to-sulfhydryl crosslinker. GMBS is moisture-sensitive.</td></tr><tr><td>Avidin</td><td>Pierce Biotechnology</td><td>31000</td><td>NeutrAvidin has 4 binding sites for biotin and its pI value is 6.3, which is more neutral than native avidin</td></tr><tr><td>Biotinylated mouse anti-human anti-CD63</td><td>Ancell</td><td>215-030</td><td>clone AHN16.1/46-4-5</td></tr><tr><td>biotinylated annexin V</td><td>BD Biosciences</td><td>556418</td><td>Annxin V has a high affinity for phosphatidylserine (PS)</td></tr><tr><td>Primary anti-CD9 and secondary antibody</td><td>System Biosciences</td><td>EXOAB-CD9A-1</td><td>The secondary antibody is horseradish peroxidise-conjugated</td></tr><tr><td>Serum separation tubes</td><td>BD Biosciences</td><td>367991</td><td>Clot activator and gel for serum separation</td></tr><tr><td>Annexin V binding buffer</td><td>BD Biosciences</td><td>556454</td><td>10x; dilute to 1x prior to use.</td></tr><tr><td>TMB substrate reagent set</td><td>BD Biosciences</td><td>555214</td><td>The set contains hydrogen peroxide and 3,3&amp;rsquo;,5,5&amp;rsquo;-tetramethylbenzidine (TMB)</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>RNA isolation kit</td><td>Life Technologies</td><td>AM1560</td><td>MirVana RNA isolation kit</td></tr><tr><td>Polyvinylpyrrolidone-based RNA isolation aid</td><td>Life Technologies</td><td>AM9690</td><td>Plant RNA isolation aid contains polyvinylpyrrolidone (PVP) that binds to polysaccharides.</td></tr><tr><td>RNA cleanup kit</td><td>Qiagen Inc.</td><td>74004</td><td>MinElute RNA cleanup kit is designed for purification of up to 45&amp;nbsp;&amp;mu;g RNA.</td></tr><tr><td>Plasma chamber</td><td>March Instruments</td><td>PX-250</td><td></td></tr><tr><td>Scanning electron microscope</td><td>Hitachi Ltd.</td><td>S-4300</td><td></td></tr><tr><td>Desktop scanner</td><td>Hewlett-Packard Company</td><td>Photosmart B110</td><td>8-bit color images were captured. Cameras and smart phones may be also used.</td></tr><tr><td>Image-record system</td><td>J&amp;amp;H Technology Co</td><td>GeneSys G:BOX Chemi-XX8</td><td>16-bit fluroscence images were captured. Fluroscence microscopes may be also used.</td></tr></tbody></table>

paper-based devices for isolation and characterization of extracellular vesicles

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 &#956;l. 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.

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 elsewhere28-30. 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 with 20 &#956;g/ml fluorophore R-phycoerythrin-conjugated biotin molecules (PE-biotin) in step 1.3. The paper test zones are then rinsed with PBS to remove unbound PE-biotin molecules. In contrast, 10 &#956;l 20 &#956;g/ml PE-biotin in PBS containing 1% (w/v) BSA and 0.09% (w/v) sodium azide is directly spotted on the paper test zone for 3 &#215; 10 min, followed by PBS rinse for conducting the physisorption of PE-biotin dye molecules. Chemically modified paper devices (&#34;paper+crosslinker+dye&#34;) along with unprocessed paper devices (&#34;paper&#34;), paper devices modified with the same protocol without the application of PE-biotin molecules (&#34;paper+crosslinker&#34;) and paper devices with physisorbed PE-biotin molecules (&#34;physisorbed-dye&#34;) are scanned using an image-record system set to 540-560 nm excitation, 575 nm-long-pass emission, and the data is saved in the form of 16-bit files and analyzed using ImageJ software. As shown in Figure 2A, fluorescence intensity of test zones prepared by the chemical conjugation method is substantially higher, as compared to physically coated paper test zones (p-value &#60; 0.0005, n = 5, Student&#8217;s t-test). The capturing of EVs is also specific as exemplified in Figure 2B, C, where few EVs are retained on the paper device when the capture molecules are replaced by PBS solution.
EVs are heterogeneous in sizes, specific contents, and biogenesis routes. To bind them, paper devices coated with two different EV capture molecules, anti-CD63 antibodies and annexin V, were prepared. CD63, a member of the tetraspanin family (which includes CD9, CD63, CD81 and CD82 molecules) is enriched on EV membrane31, and annexin V has a strong affinity for phosphatidylserine (PS)32,33. It is found that EVs are released constitutively during early apoptosis or under stress conditions, in which process normal cell phospholipid asymmetry is lost, leading to increased exposure of PS on the outer leaflet of the EV membrane. Analyses of SEM images show that the size distributions of CD63+ EVs and PS+ EVs are different, with mean diameters of 80 and 43 nm, respectively (Figure 3).The two-tailed Student&#8217;s t-test is used to determine the statistical p-value (p-value &#60; 0.0001). Surprisingly, CD63+ EVs appear rounder and larger than PS+ EVs on average, which biological significance remains to be elucidated.
RNA from EVs captured on the paper device can be extracted. Analyses with a bioanalyzer showed similar overall profiles for total RNAs extracted from CD63+ and PS+ EVs (Figure 4A). Compared to the amounts of RNA extracted using the microfluidic and ultracentrifugation techniques, respectively about twice and 39 times more RNAs per unit volume of serum sample were extracted utilizing the paper device, albeit using a smaller sample volume (Table 1). P-ELISA can be carried out for the detection of EVs. Anti-CD9 primary antibody and HRP-conjugated secondary antibody are used in this study. Figure 4B displays grey value changes produced by the enzymatic reaction of HRP for paper devices applied with 10 &#956;l PBS, 50% serum (diluted with PBS), or 100% serum, respectively. The grey value increases linearly within the range of serum samples tested.
Paper devices are especially advantageous when the sample amount is limited such as in the case of aqueous humor, when only &#60; 200 &#956;l/eye can be collected. Multiple samples are often needed and pooled together if other isolation methods, e.g., ultracentrifugation, are to be used. EVs from patient aqueous humor samples isolated with paper devices coated with anti-CD63 antibodies can be seen in SEM images as showed in Figure 5.
<img alt="Figure 1" src="/files/ftp_upload/52722/52722fig1.jpg" /> 
Figure 1. The fabrication and operation procedure of paper-based assays aimed at isolation and characterization of extracellular vesicles.&#160;(A) Filter paper is cut into the desired shape and laminated. (B) The surface of the paper is modified with a brief treatment of oxygen plasma and reacted with 3-mercaptopropyl trimethoxysilane, N-&#947;-maleimidobutyryloxy succinimide ester, and NeutrAvidin, in that order. Biotinylated capture molecules, e.g., antibodies, can then be immobilized via the strong affinity between biotin and NeutrAvidin molecules. (C) Biosamples can be pipette onto the functionalized paper device. Extracellular vesicles can be isolated. (D) Downstream assays, such as SEM, transcriptome, and ELISA (shown), can be performed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52722/52722fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52722/52722fig2.jpg" /> 
Figure 2. Extracellular vesicles (EVs) can be captured on functionalized paper devices with good specificity and sensitivity.&#160;(A) Fluorescently labeled biotin molecules are highly immobilized onto the paper device via chemical conjugation process (&#34;paper+crosslinker+dye&#34;) in contrast to physical adsorption (&#34;physisorbed-dye&#34;). Unprocessed paper (&#34;paper&#34;) and conjugation molecules (&#34;paper+crosslinker&#34;) contribute little to the fluorescence intensity. (B) A representative SEM image showing few EVs are captured on the paper device non-specifically when the capture molecules is replaced by PBS containing 1% (w/v) BSA. (C) Many EVs are retained on paper devices coated with anti-CD63 antibodies. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52722/52722fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3-mercaptopropyl" src="/files/ftp_upload/52722/52722fig3.jpg" /> 
Figure 3. Subgroups of Extracellular vesicles (EVs) can have different size distributions.&#160;(A) and (B) are representative SEM images of EVs captured on paper devices coated with anti-CD63 antibodies and annexin V molecules, respectively. (C) CD63+ EVs appear larger than PS+ EVs under experimental conditions used in this study (p-value &#60; 0.0001, n = 124 and 203, Student&#8217;s t-test). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52722/52722fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52722/52722fig4.jpg" /> 
Figure 4. Downstream transcriptome and ELISA assays. (A) Bioanalyzer data showing the intensities (y-axis, in arbitrary units) and size (x-axis, in nucleotides (nt)) distributions of fluorescently-labeled total RNA extracted from EVs captured on anti-CD63 antibody- or annexin V-coated filter papers. The migrating peak at ~25 nt represents an internal standard. (B) Grey value changes produced by the enzymatic reaction of horseradish peroxidase (HRP) in the P-ELISA assay for paper devices applied with 10 &#956;l samples of PBS, 50% serum (diluted with PBS) and 100% serum samples. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52722/52722fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52722/52722fig5.jpg" /> 
Figure 5. Extracellular vesicles (EVs) in aqueous humor samples can be captured on functionalized filter paper without sample pooling. SEM images of isolated CD63+ EVs in aqueous humor samples from patients with (A) age-related macular degeneration, (B) diabetic macular edema, and (C) polypoidal choroidal vasculopathy, respectively. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52722/52722fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>isolation method</td>
			<td>capture molecule</td>
			<td>serum vol. (&#181;l)</td>
			<td>average RNA extracted (ng)</td>
			<td>average extracted RNA/serum 
			(pg/&#181;l)</td>
			<td>ratio of average RNA/serum</td>
			<td>ref</td>
		</tr>
		<tr>
			<td>microfluidic device</td>
			<td>anti-human CD63</td>
			<td>400</td>
			<td>4.14</td>
			<td>10.4</td>
			<td>20</td>
			<td>20</td>
		</tr>
		<tr>
			<td>paper device</td>
			<td>anti-human CD63</td>
			<td>72</td>
			<td>1.49 &#177; 0.08</td>
			<td>20.7</td>
			<td>39</td>
			<td>22</td>
		</tr>
		<tr>
			<td>paper device</td>
			<td>annexin V</td>
			<td>72</td>
			<td>0.99 &#177; 0.26</td>
			<td>13.8</td>
			<td>26</td>
			<td>22</td>
		</tr>
		<tr>
			<td>ultracentrifugation</td>
			<td>-</td>
			<td>2,000</td>
			<td>1.06 &#177; 0.64</td>
			<td>0.53</td>
			<td>1</td>
			<td></td>
		</tr>
		<tr>
			<td>serum</td>
			<td>-</td>
			<td>72</td>
			<td>1.23 &#177; 0.58</td>
			<td>17</td>
			<td>32</td>
			<td>22</td>
		</tr>
	</tbody>
</table>
Table 1. Total RNA extracted from serum samples from healthy volunteers analyzed with a bioanalyzer (n = 4 for all conditions).

Watch this Scientific Journal Video about Paper-based Devices for Isolation and Characterization of Extracellular Vesicles at JoVE.com

Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

This protocol details a method to isolate extracellular vesicles (EVs), small membranous particles released from cells, from as little as 10 &#956;l 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.

Extracellular vesicles (EVs), membranous particles released from various types of cells, hold a great potential for clinical applications. They contain ...

paper-based-devices-for-isolation-characterization-extracellular

Nanyang Technological University

Research

JoVE Journal

Bioengineering

11.4K Views.  National Tsing Hua University.  Dit protocol Gegevens methode extracellulaire blaasjes (EV), kleine membraneuze deeltjes vrijgemaakt uit cellen te isoleren, van slechts 10 pl serummonsters. Deze benadering omzeilt de noodzaak van ultracentrifugatie, vereist slechts enkele minuten testtijd en maakt de isolatie van EV uit monsters van beperkte omvang. 

Video: Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

Rapid Fluorescence-based Characterization of Single Extracellular Vesicles in Human Blood with Nanoparticle-tracking Analysis

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released from many cell types and play a pivotal role in regulating cell-cell communication and a diverse range of biological processes. Many different methods for the characterization of EVs have been described. However, most of these methods have the disadvantage that the preparation and characterization of the samples are very time-consuming, or it is extremely difficult to analyze specific markers of interest due to their small size and due to the lack of discrete populations. While methods for analysis of EVs have been considerably improved over the last decade, there is still no standardized method for characterization of single EVs. Here, we demonstrate a semi-automated method for characterization of single EVs by fluorescence-based nanoparticle-tracking analysis. The protocol that is presented addresses the common problem of many researchers in this field and provides the complete workflow for rapid isolation of EVs and characterization with PKH67, a general cell membrane linker, as well as with specific surface markers such as CD63, CD9, vimentin, and lysosomal-associated membrane protein 1 (LAMP-1). The presented results show a high level of reproducibility, as confirmed by other methods, such as Western blotting. In the conducted experiments, we exclusively used EVs isolated from human serum samples, but this method is also suitable for plasma or other body fluids and can be adjusted for characterization of EVs from cell culture supernatants. Irrespective of the future progress of research on EV biology, the protocol that is presented here provides a rapid and reliable method for rapid characterization of single EVs with specific markers.

In this protocol, we describe the complete workflow for rapid isolation of extracellular vesicles from human whole blood and characterization of specific markers by fluorescence-based nanoparticle-tracking analysis. The presented results show a high level of reproducibility and can be adjusted to cell culture supernatants.

Extracellular vesicles (EVs), including exosomes, are specialized membranous nano-sized vesicles found in bodily fluids that are constitutively released ...

Biochemistry

Paper-Based Preconcentration and Isolation of Microvesicles and Exosomes

Microvesicles and exosomes are small membranous vesicles released to the extracellular environment and circulated throughout the body. Because they contain various parental cell-derived biomolecules such as DNA, mRNA, miRNA, proteins, and lipids, their enrichment and isolation are critical steps for their exploitation as potential biomarkers for clinical applications. However, conventional isolation methods (e.g., ultracentrifugation) cause significant loss and damage to microvesicles and exosomes. These methods also require multiple repetitive steps &#160;of ultracentrifugation, loading, and wasting of reagents. This article describes a detailed method to fabricate an origami-paper-based device (Exo-PAD) designed for the effective enrichment and isolation of microvesicles and exosomes in a simple manner. The unique design of the Exo-PAD, consisting of accordion-like multifolded layers with convergent sample areas, is integrated with the ion concentration polarization technique, thereby enabling fivefold enrichment of the microvesicles and exosomes on specific layers. In addition, the enriched microvesicles and exosomes are isolated by simply unfolding the Exo-PAD.

Presented is a protocol to fabricate a paper-based device for the effective enrichment and isolation of microvesicles and exosomes.

Microvesicles and exosomes are small membranous vesicles released to the extracellular environment and circulated throughout the body. Because they ...

Optimization of Flow Cytometric Sorting Parameters for High-Throughput Isolation and Purification of Small Extracellular Vesicles

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the physiological and pathological state of a cell. However, there is no standard method for sEV isolation, which prevents downstream biomarker identification and drug intervention studies. In this article, we provide a detailed protocol for the isolation and purification of 50-200 nm sEV by a flow cell sorter. For this, a 50 &#181;m nozzle and 80 psi sheath fluid pressure were selected to obtain a good sorting rate and stable side stream. Standard sized polystyrene microspheres were used to locate populations of 100, 200, and 300 nm particles. With additional optimization of the voltage, gain, and forward scatter (FSC) triggering threshold, the sEV signal could be separated from the background noise. These optimizations provide a panel of critical sort settings that enables one to obtain a representative population of sEV using FSC vs. side scatter (SSC) only. The flow cytometry-based isolation method not only allows for high-throughput analysis but also allows for synchronous classification or proteome analysis of sEV based on the biomarker expression, opening numerous downstream research applications.

This protocol provides a rapid and size-specific isolation method for small extracellular vesicles by optimizing the size of the air spray nozzle, sheath fluid pressure, sample flow pressure, voltage, gain, and triggering threshold parameters.

Small extracellular vesicles (sEV) can be released from all cell types and carry protein, DNA, and RNA. Signaling molecules serve as indicators of the ...

Microfluidic Device for Continuous Extracellular Vesicle Isolation from Human Urine

Single Step Isolation of Extracellular Vesicles from Large-Volume Samples with a Bifurcated A4F Microfluidic Device

Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. Nevertheless, the current methodologies for isolating EVs present significant challenges, such as complexity, time consumption, and the need for bulky equipment, which hinders their clinical translation. To address these limitations, we aimed to develop an innovative microfluidic system based on cyclic olefin copolymer-off-stoichiometry thiol-ene (COC-OSTE) for the efficient isolation of EVs from large-volume samples in a continuous manner. By utilizing size and buoyancy-based separation, the technology used in this study achieved a significantly narrower size distribution compared to existing approaches from urine and cell media samples, enabling the targeting of specific EV size fractions in future applications. Our innovative COC-OSTE microfluidic device design, utilizing bifurcated asymmetric flow field-flow fractionation technology, offers a straightforward and continuous EV isolation approach for large-volume samples. Furthermore, the potential for mass manufacturing of this microfluidic device offers scalability and consistency, making it feasible to integrate EV isolation into routine clinical diagnostics and industrial processes, where high consistency and throughput are essential requirements.

Author Spotlight: Asymmetric Field Flow Fractionation for Bioreactor Integration

Extracellular vesicles hold immense promise for biomedical applications, but current isolation methods are time-consuming and impractical for clinical use. In this study, we present a microfluidic device that enables the direct isolation of extracellular vesicles from large volumes of biofluids in a continuous manner with minimal steps.

Extracellular vesicles (EVs) hold immense potential for various biomedical applications, including diagnostics, drug delivery, and regenerative medicine. ...

Size Exclusion Chromatography for Separating Extracellular Vesicles from Conditioned Cell Culture Media

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain proteins, nucleic acids, and lipids that are reflective of the parent cell from which they are derived. Proper separation of EVs from other components in a sample allows for characterization of their associated cargo and lends insight into their potential as intercellular communicators and non-invasive biomarkers for numerous diseases. In the current study, oligodendrocyte derived EVs were isolated from cell culture media using a combination of state-of-the-art techniques, including ultrafiltration and size exclusion chromatography (SEC) to separate EVs from other extracellular proteins and protein complexes. Using commercially available SEC columns, EVs were separated from extracellular proteins released from human oligodendroglioma cells under both control and endoplasmic reticulum (ER) stress conditions. The canonical EV markers CD9, CD63, and CD81 were observed in fractions 1-4, but not in fractions 5-8. GM130, a protein of the Golgi apparatus, and calnexin, an integral protein of the ER, were used as negative EV markers, and were not observed in any fraction. Further, when pooling and concentrating fractions 1-4 as the EV fraction, and fractions 5-8 as the protein fraction, expression of CD63, CD81, and CD9 in the EV fraction was observed. The expression of GM130 or calnexin was not observed in either of the fraction types. The pooled fractions from both control and ER stress conditions were visualized with transmission electron microscopy and vesicles were observed in the EV fractions, but not in the protein fractions. Particles in the EV and protein fractions from both conditions were also quantified with nanoparticle tracking analysis. Together, these data demonstrate that SEC is an effective method for separating EVs from conditioned cell culture media.

The protocol here demonstrates that extracellular vesicles can be adequately separated from conditioned cell culture media using size exclusion chromatography.

Extracellular vesicles (EVs) are nano-sized lipid-membrane bound structures that are released from all cells, are present in all biofluids, and contain ...

Neuroscience

Isolation and Characterization of Microvesicles from Peripheral Blood

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived microvesicles (MVs, diameter &#62; 100 nm) is a fundamental cellular process that occurs in all living cells. These vesicles transport proteins, lipids and nucleic acids specific for their cell of origin and in vitro studies have highlighted their importance as mediators of intercellular communication. EVs have been successfully isolated from various body fluids and especially EVs in blood have been identified as promising biomarkers for cancer or infectious diseases. In order to allow the study of MV subpopulations in blood, we present a protocol for the standardized isolation and characterization of MVs from peripheral blood samples. MVs are pelleted from EDTA-anticoagulated plasma samples by differential centrifugation and typically possess a diameter of 100 - 600 nm. Due to their larger size, they can easily be studied by flow cytometry, a technique that is routinely used in clinical diagnostics and available in most laboratories. Several examples for quality control assays of the isolated MVs will be given and markers that can be used for the discrimination of different MV subpopulations in blood will be presented.

Extracellular vesicles present in blood have been suggested as novel biomarkers for various diseases. Here, we present a protocol for the isolation of large plasma membrane-derived microvesicles from peripheral blood samples and their subsequent analysis by conventional flow cytometry and Western Blotting.

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived ...

Biology

Isolation and Characterization of Human Adipocyte-Derived Extracellular Vesicles using Filtration and Ultracentrifugation

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can modulate the cellular status of other cells locally in tissue microenvironments or through liberation into peripheral blood. Adipocyte-derived EVs are elevated in the peripheral blood and show alterations in their cargo (RNA and protein) during metabolic disturbances, including obesity and diabetes. Adipocyte-derived EVs can regulate the cellular status of neighboring vascular cells, such as endothelial cells and adipose tissue resident macrophages to promote adipose tissue inflammation. Investigating alterations in adipocyte-derived EVs in vivo is complex because EVs derived from peripheral blood are highly heterogenous and contain EVs from other sources, namely platelets, endothelial cells, erythrocytes and muscle. Therefore, the culture of human adipocytes provides a model system for the study of adipocyte derived EVs. Here, we provide a detailed protocol for the extraction of total small EVs from cell culture media of human gluteal and abdominal adipocytes using filtration and ultracentrifugation. We further demonstrate the use of Nanoparticle Tracking Analysis (NTA) for quantification of EV size and concentration and show the presence of EV-protein tumor susceptibility gene 101 (TSG101) in the gluteal and abdominal adipocyte derived-EVs. Isolated EVs from this protocol can be used for downstream analysis, including transmission electron microscopy, proteomics, metabolomics, small RNA-sequencing,&#160;microarrays and can be&#160;utilized in functional in vitro/in vivo studies.

We describe the isolation of human adipocyte-derived extracellular vesicles (EVs) from gluteal and abdominal adipose tissue using filtration and ultracentrifugation. We characterize the isolated adipocyte-derived EVs by determining their size and concentration by Nanoparticle Tracking Analysis and by western blotting for the presence of EV-protein tumor susceptibility gene 101 (TSG101).

Extracellular vesicles (EVs) are lipid enclosed envelopes that carry biologically active material such as proteins, RNA, metabolites and lipids. EVs can ...

Single-Particle Interferometric Reflectance Imaging Characterization of Individual Extracellular Vesicles and Population Dynamics

Extracellular vesicles (EVs) are nanometer-sized vesicles with a lipid bilayer that are secreted by most cells. EVs carry a multitude of different biological molecules, including protein, lipid, DNA, and RNA, and are postulated to facilitate cell-to-cell communication in diverse tissues and organs. Recently, EVs have attracted significant attention as biomarkers for diagnostics and therapeutic agents for various diseases. Many methods have been developed for EV characterization. However, current methods for EV analysis all have different limitations. Thus, developing efficient and effective methods for EV isolation and characterization remains one of the crucial steps for this cutting-edge research field as it matures. Here, we provide a detailed protocol outlining a single-particle interferometric reflectance imaging sensor (SP-IRIS), as a method that is capable of detecting and characterizing EVs from unpurified biological sources and purified EVs by other methodologies. This advanced technique can be used for multi-level and comprehensive measurements for the analysis of EV size, EV count, EV phenotype, and biomarker colocalization.

This protocol presents single-particle interferometric reflectance imaging that is designed for the multi-level and comprehensive measurements of extracellular vesicles (EV) size, EV count, EV phenotype, and EV biomarker colocalization.

Extracellular vesicles (EVs) are nanometer-sized vesicles with a lipid bilayer that are secreted by most cells. EVs carry a multitude of different ...

Isolation and Characterization of Cyanobacterial Extracellular Vesicles

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential biotechnology models. Recent work has demonstrated that both marine and freshwater cyanobacteria produce extracellular vesicles -&#160;small membrane-bound structures released from the outer surface of the microbes. While vesicles likely contribute to diverse biological processes, their specific functional roles in cyanobacterial biology remain largely unknown. To encourage and advance research in this area, a detailed protocol is presented for isolating, concentrating, and purifying cyanobacterial extracellular vesicles. The current work discusses methodologies that have successfully isolated vesicles from large cultures of Prochlorococcus, Synechococcus, and Synechocystis. Methods for quantifying and characterizing vesicle samples from these strains are presented. Approaches for isolating vesicles from aquatic field samples are also described. Finally, typical challenges encountered with cyanobacterial vesicle purification, methodological considerations for different downstream applications, and the trade-offs between approaches are also discussed.

The present protocol providesdetailed descriptions for isolation, concentration,and characterization of extracellular vesicles from cyanobacterial cultures. Approaches for purifying vesicles from cultures at different scales, trade-offs among methodologies, and considerations for working with field samples are also discussed.

Cyanobacteria are a diverse group of photosynthetic, Gram-negative bacteria that play critical roles in global ecosystems and serve as essential ...

Single Extracellular Vesicle Transmembrane Protein Characterization by Nano-Flow Cytometry

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and first-generation particle analysis to comprehensive multi-parameter measurements such as nano-flow cytometry (nFCM). nFCM is a form of flow cytometry that utilizes instrumentation specifically designed for nano-particle analysis, allowing for thousands of EVs to be characterized per minute both with and without the use of staining techniques. High resolution side scatter (SS) detection allows for size and concentration to be determined for all biological particles larger than 45 nm, while simultaneous fluorescence (FL) detection identifies the presence of labeled markers and targets of interest. Labeled subpopulations can then be described in quantitative units of particles/mL or as a percentage of the total particles identified by side scatter.
Here, EVs derived from conditioned cell culture media (CCM) are labeled with both a lipid dye, to identify particles with a membrane, and antibodies specific for CD9, CD63, and CD81 as common EV markers. Measurements of comparison material, a concentration standard and a size standard of silica nanospheres, as well as labeled sample material are analyzed in a 1-minute analysis. The software is then used to measure the concentration and size distribution profile of all particles, independent of labeling, before determining the particles that are positive for each of the labels.
Simultaneous SS and FL detection can be utilized flexibly with many different EV sources and labeling targets, both external and internal, describing EV samples in a comprehensive and quantitative manner.

The latest generation of EV characterization tools are capable of single EV analysis across multiple parameters simultaneously. Nano-flow cytometry measures all biological particles larger than 45 nm without labeling and identifies specific characteristics of subpopulations by a variety of fluorescent labeling techniques.

Single particle characterization has become increasingly relevant for research into extracellular vesicles, progressing from bulk analysis techniques and ...

Paper-based Devices for Isolation and Characterization of Extracellular Vesicles

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