This study was supported by the National Research Foundation of Korea, Grant NRF-2018R1D1A1A09084044. J. H. Lee was supported by a research grant from Kwangwoon University in 2019. Hyerin Kim was supported by the &#8220;Competency Development Program for Industry Specialists&#8221; of the Korean Ministry of Trade, Industry and Energy (MOTIE), operated by the Korea Institute for Advancement of Technology (KIAT) (No. P0002397, HRD program for Industrial Convergence of Wearable Smart Devices).

1. Device fabrication
<ol>
	<li>Define the region to be printed on paper using printer software (Table of Materials). 
	NOTE: The design has 12 wax-patterned layers in which the diameters of the circular sample areas gradually narrow from 5 mm to 2 mm (Figure 1A).</li>
	<li>Print hydrophobic wax on the designated regions on both sides of the cellulose paper (Table of Materials) using a commercial wax printer (Table of Materials) (Figure 1B).</li>
	<li>Place the wax-printed paper in a laboratory oven for 80 s at 120 &#176;C. 
	NOTE: This step allows the wax to reach the inside of the paper by achieving a thermal reflow of the printed wax. With this incubation protocol, the resolution of the pattern is ~2 mm. Thus, be careful not to print patterns of less than 2 mm. Otherwise the patterns will be blocked by the wax.</li>
	<li>Cut the wax-printed paper with a cutter to make individual devices (Figure 1C).</li>
	<li>Drop 5 &#956;L and 2 &#956;L of permselective membrane (e.g., Nafion; Table of Materials) onto the sample areas in the leftmost and rightmost layers, respectively (Figure 1D).</li>
	<li>Place the layers coated with the permselective membrane on a hot plate at 70 &#176;C for 30 min to evaporate the permselective membrane solvent.</li>
	<li>Seal the outermost surface of the coated layer facing the buffer solution with pressure-sensitive tape, leaving a small hole.</li>
	<li>Fold the printed individual device (i.e., Exo-PAD) back and forth along the white lines. 
	NOTE: By folding the device, all sample areas become convergently connected (Figure 1E). This convergent design focuses the electric field lines when the voltage is applied for ICP, achieving more intensive preconcentration of the microvesicles and exosomes.</li>
</ol>
2. Enrichment and spatial focusing of microvesicles and exosomes by ion concentration polarization
<ol>
	<li>Load 15 &#956;L of the microvesicle and exosome sample (~3 x 1011 particles/mL in 0.1x phosphate buffered saline [PBS] with 0.05% Tween 20) in the convergent sample areas by pipetting and wait a few seconds to ensure complete wetting of all sample areas (Figure 1F).</li>
	<li>Place two acrylic chambers at both ends of the Exo-PAD and clamp the Exo-PAD securely with small binder clips to prevent unfolding (Figure 1G).</li>
	<li>Fill the chambers with 110 &#956;L of 0.1x PBS and insert two Ag/AgCl electrodes (Figure 1H).</li>
	<li>Apply 30 V to the electrodes for 20 min using a current-voltage source measurement system (Table of Materials). 
	NOTE: The applied voltage generates the ICP phenomenon and hence preconcentrates the microvesicles and exosomes on layers 8 and 9 of the Exo-PAD.</li>
</ol>
3. Isolation of the enriched microvesicles and exosomes
<ol>
	<li>Separate the folded Exo-PAD from the acrylic chambers and unfold the device to isolate the enriched microvesicles and exosomes from the other layers (Figure 1I).</li>
	<li>Punch out the sample areas in layers 8 and 9, where the microvesicles and exosomes are enriched, for downstream analysis (Figure 1J).</li>
</ol>
4. Scanning electron microscopy analysis
<ol>
	<li>Fix the enriched microvesicles and exosomes by immersing the punched areas in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer for 1 h and in 1% osmium tetroxide in 0.1 M sodium cacodylate for 1 h. 
	CAUTION: Osmium tetroxide is highly poisonous and hazardous chemical. Because osmium tetroxide can penetrate plastics, it must be stored in glass. Any handling of osmium tetroxide must be performed in a chemical fume hood with double nitrile gloves.</li>
	<li>Dehydrate the fixed microvesicles and exosomes with ascending grades of 200 proof ethanol (i.e., 50%, 70%, 90%, and 100%) for 30 min each.</li>
	<li>Chemically dry the sample by immersing it in hexamethyldisilazane in a desiccator for 30 min. 
	CAUTION: Hexamethyldisilazane is a flammable and moisture-sensitive chemical. It must be stored in a dry and well-ventilated area away from ignition sources.</li>
	<li>Coat the completely dried microvesicles and exosomes with gold/palladium (~20 nm) via sputtering and capture scanning electron microscopy (SEM) images.</li>
</ol>
5. Nanoparticle tracking analysis
<ol>
	<li>Punch out the sample areas in layers 6, 8, and 10 using a biopsy punch after 20 min of device operation.</li>
	<li>Immerse each punched-out area in buffer solution (0.1x PBS with 0.01% Tween 20).</li>
	<li>Vortex for 10 min and centrifuge for 30 s at 6,000 rpm to resuspend the enriched microvesicles and exosomes.</li>
	<li>Remove the punched-out areas from the solution and measure the concentration of microvesicles and exosomes by a nanoparticle tracking analysis (NTA) instrument (Table of Materials).</li>
</ol>

<ol>
	<li>Edgar, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Q+&amp;+A:+What+are+exosomes,+exactly.">Q &amp; A: What are exosomes, exactly.</a> BMC Biology. 14 (1), 1-7 (2016).</li><li>Contreras-Naranjo, J. C., Wu, H. J., Ugaz, V. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidics+for+exosome+isolation+and+analysis:+Enabling+liquid+biopsy+for+personalized+medicine.">Microfluidics for exosome isolation and analysis: Enabling liquid biopsy for personalized medicine.</a> Lab on a Chip. 17 (21), 3558-3577 (2017).</li><li>Simons, M., Raposo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+-+vesicular+carriers+for+intercellular+communication.">Exosomes - vesicular carriers for intercellular communication.</a> Current Opinion in Cell Biology. 21 (4), 575-581 (2009).</li><li>St&#229;hl, A. L., Johansson, K., Mossberg, M., Kahn, R., Karpman, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+and+microvesicles+in+normal+physiology,+pathophysiology,+and+renal+diseases.">Exosomes and microvesicles in normal physiology, pathophysiology, and renal diseases.</a> Pediatric Nephrology. 34 (1), 11-30 (2019).</li><li>Chen, C., Lin, B. R., Hsu, M. Y., Cheng, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paper-based+devices+for+isolation+and+characterization+of+extracellular+vesicles.">Paper-based devices for isolation and characterization of extracellular vesicles.</a> Journal of Visualized Experiments. (98), e52722 (2015).</li><li>Kim, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origami-paper-based+device+for+microvesicle/exosome+preconcentration+and+isolation.">Origami-paper-based device for microvesicle/exosome preconcentration and isolation.</a> Lab on a Chip. 19 (23), 3917-3921 (2019).</li><li>Raposo, G., Stoorvogel, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+vesicles:+Exosomes,+microvesicles,+and+friends.">Extracellular vesicles: Exosomes, microvesicles, and friends.</a> Journal of Cell Biology. 200 (4), 373-383 (2013).</li><li>Lee, Y., El Andaloussi, S., Wood, M. J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exosomes+and+microvesicles:+Extracellular+vesicles+for+genetic+information+transfer+and+gene+therapy.">Exosomes and microvesicles: Extracellular vesicles for genetic information transfer and gene therapy.</a> Human Molecular Genetics. 21, 125-134 (2012).</li><li>Liu, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Field-Free+Isolation+of+Exosomes+from+Extracellular+Vesicles+by+Microfluidic+Viscoelastic+Flows.">Field-Free Isolation of Exosomes from Extracellular Vesicles by Microfluidic Viscoelastic Flows.</a> ACS Nano. 11 (7), 6968-6976 (2017).</li><li>Marczak, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+isolation+and+preconcentration+of+exosomes+by+ion+concentration+polarization.">Simultaneous isolation and preconcentration of exosomes by ion concentration polarization.</a> Electrophoresis. 39 (15), 2029-2038 (2018).</li><li>Livshts, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+exosomes+by+differential+centrifugation:+Theoretical+analysis+of+a+commonly+used+protocol.">Isolation of exosomes by differential centrifugation: Theoretical analysis of a commonly used protocol.</a> Scientific Reports. 5, 1-14 (2015).</li><li>Chiriac&#242;, M. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lab-on-chip+for+exosomes+and+microvesicles+detection+and+characterization.">Lab-on-chip for exosomes and microvesicles detection and characterization.</a> Sensors. 18 (10), 3175 (2018).</li><li>Lobb, R. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+exosome+isolation+protocol+for+cell+culture+supernatant+and+human+plasma.">Optimized exosome isolation protocol for cell culture supernatant and human plasma.</a> Journal of Extracellular Vesicles. 4 (1), 1-11 (2015).</li><li>Taylor, D. D., Shah, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+of+isolating+extracellular+vesicles+impact+down-stream+analyses+of+their+cargoes.">Methods of isolating extracellular vesicles impact down-stream analyses of their cargoes.</a> Methods. 87, 3-10 (2015).</li><li>Han, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrokinetic+size-based+spatial+separation+of+micro/nanospheres+using+paper-based+3d+origami+preconcentrator.">Electrokinetic size-based spatial separation of micro/nanospheres using paper-based 3d origami preconcentrator.</a> Analytical Chemistry. 91 (16), 10744-10749 (2019).</li><li>Yeh, S. H., Chou, K. H., Yang, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sample+pre-concentration+with+high+enrichment+factors+at+a+fixed+location+in+paper-based+microfluidic+devices.">Sample pre-concentration with high enrichment factors at a fixed location in paper-based microfluidic devices.</a> Lab on a Chip. 16 (5), 925-931 (2016).</li></ol>

The authors have nothing to disclose.

Although the Exo-PAD was used successfully for the enrichment and isolation of microvesicles and exosomes, several critical points should be carefully considered: 1) the oven incubation time and temperature during the device preparation, 2) processing time, 3) application of voltage with varying layer numbers and sample area diameters, and 4) applicability to clinical samples.
The incubation time and temperature given in the protocol are optimized conditions to fabricate a reliable device. Longer incubation times or a higher temperature may cause distortion of the convergent design due to the excessive reflow of the printed wax. This will prevent the formation of focused electric field lines and thus increase the migration resistance during the electrokinetic enrichment process.
Processing time is also a factor to consider for maximum enrichment and isolation. A processing time of &#60;20 min results in less preconcentration because there is not sufficient time to allow microvesicle and exosome migration. On the other hand, a processing time of &#62;20 min deteriorates the focusing efficiency and allows dispersion of the enriched microvesicles and exosomes. According to this test, 20 min is the best processing time to achieve maximum enrichment and isolation of microvesicles and exosomes.
The applied voltage is another important factor to consider. In this protocol, considering the number of layers and the sample area diameters, 30 V is used to achieve stable preconcentration and isolation. When an ICP phenomenon is generated in the device, the operation regime can be divided into three distinct regions according to the applied voltage: (1) ohmic, (2) limiting, and (3) overlimiting regions6,15,16. For stable preconcentration and isolation, the device should be operated in the limiting region, where an ion depletion zone is generated and microvesicles and exosomes are stably preconcentrated. In this experimental setting, the operating voltage range (5&#8722;40 V) corresponds to the limiting region for the Exo-PAD. If the applied voltage is &#60;5 V, the device operates in the ohmic region, where the electric current increases linearly, indicating that no ion depletion region is generated for preconcentration. However, if the voltage is &#62;40 V, the ion depletion region is destroyed by strong voltage-driven electroconvection, which destabilizes preconcentration. Considering these factors, it was determined that 30 V was the optimal voltage to use for a device with 12 layers and a sample area diameter of 2&#8722;5 mm. Layer numbers or sample area diameters can be increased to enlarge the sample volume, but these factors, as well as the operating voltage, should be optimized to achieve stable enrichment of microvesicles and exosomes.
The proposed Exo-PAD can be used to isolate microvesicles and exosomes from various clinical samples, including saliva, urine, serum, and plasma. For samples with less protein, such as saliva or urine, microvesicles and exosomes can be easily isolated without major changes, because a small amount of protein in these samples will be filtered out through nonspecific binding to the paper. When operating the device within the limiting region, only the applied voltage needs to be considered, because the ionic strength of samples can be different. For protein-rich samples, such as serum or plasma, both the preconcentration of microvesicles and exosomes and their separation from abundant proteins should be carefully considered. Our recent experiment shows that separation of microvesicles and exosomes from proteins is significantly improved when carbonate buffer is used, because carbonate buffer results in a higher electrical charge difference between microvesicles and exosomes and protein due to the isoelectric point, resulting in better separation efficiency. For this experiment, a substance with abundant proteins coexisting with microvesicles and exosomes was mimicked by mixing purified microvesicles and exosomes labeled with FITC (Ex/Em: 490/520 nm) with BSA labeled with a different fluorophore (Ex/Em: 590/617 nm) in 50 mM carbonate buffer (Table of Materials). As shown in Supplementary Figure 1, most microvesicles and exosomes were preconcentrated on layer 8, as in Figure 2A and they were separated from BSA, which stayed in layers 1&#8211;3. This result suggests that enrichment from a serum or plasma sample requires an additional step adding carbonate buffer to the sample, and clearly demonstrates that the Exo-PAD can purify microvesicles and exosomes from abundant proteins.&#160;

Microvesicles and exosomes are small membrane vesicles measuring 0.2&#8722;1 &#956;m and 30&#8722;200 nm, respectively. They are secreted into the extracellular environment by several different cell types1,2,3,4,5. They contain parental cell information in the form of subsets of DNA, mRNA, miRNA, proteins, and lipids, and circulate throughout the body via various body fluids such as serum, plasma, urine, cerebrospinal fluid, amniotic fluid, and saliva6,7,8,9. Thus, techniques for efficient isolation of microvesicles and exosomes from biological fluids can provide extensive opportunities in the fields of the diagnosis, prognosis, and real-time monitoring of disease, as well as in the development of new therapeutics.
However, the conventional isolation method for microvesicles and exosomes based on ultracentrifugation is extremely time-consuming and causes significant loss and contamination of the sample. This is because it involves several cumbersome pipetting and loading steps and discarding of various reagents with repeated ultracentrifugation5,6,10,11,12. Moreover, the high shear stress induced by ultracentrifugation (~100,000 x g) can cause the physical lysis of microvesicles and exosomes, yielding poor recovery rates (5&#8722;23%)6,13,14. Therefore, a highly efficient, unobtrusive isolation technique for microvesicles and exosomes must be developed to reduce damage and loss, thereby achieving higher recovery rates.
An origami-paper-based device (Exo-PAD) was developed for simpler, gentler, and highly efficient isolation of microvesicles and exosomes6. The design of the Exo-PAD is a multifolded paper with serially connected sample areas that gradually decrease in diameter. The ion concentration polarization (ICP) technique, which is a nano-electrokinetic phenomenon that preconcentrates charged biomolecules, was integrated with this unique design. Using the Exo-PAD resulted in fivefold enrichment of the microvesicles and exosomes in specific layers and their isolation by simply unfolding the device. This article describes the Exo-PAD in detail, from the overall device fabrication and operation to analysis of its use, to illustrate the method and show representative results6.

<table><tbody><tr><td>Ag/AgCl electrodes</td><td>A-M Systems, Inc.</td><td>531500</td><td>0.15&quot; diameter</td></tr><tr><td>Albumin from Bovine Serum (BSA), Alexa Fluor 594 conjugate</td><td>Thermo Fisher Scientific</td><td>A13101</td><td>BSA conjugated with Alexa Fluor 594 (Ex/Em: 590/617 nm)</td></tr><tr><td>Carbonate-Bicarbonate Buffer</td><td>Sigma-Aldrich</td><td>C3041-50CAP</td><td>Carbonate buffer</td></tr><tr><td>CorelDraw software (Coral Co., Canada)</td><td>Corel Corporation</td><td></td><td>Printer software to define wax printing region</td></tr><tr><td>ColorQube 8870</td><td>Xerox Corporation</td><td></td><td>Wax printer</td></tr><tr><td>Chromatography paper grade 1</td><td>Whatman</td><td>3001-861</td><td>Cellulose paper, dimension: 20 * 20 cm</td></tr><tr><td>Fluorescent-labeled exosome standards</td><td>HansaBioMed Life Sciences, Ltd.</td><td>HBM-F-PEP-100</td><td>Exosome labeled with FITC (Ex/Em: 490/520 nm)</td></tr><tr><td>Keithley 2410 current/voltage source-meter</td><td>Keithley Instruments, Inc.</td><td></td><td>Current&amp;ndash;voltage source measurement system</td></tr><tr><td>Nafion perfluorinated resin solution</td><td>Sigma-Aldrich</td><td>31175-20-9</td><td>Permselective membrane, 20 wt.% in the mixture of lower aliphatic alcohols and water; contains 34% water</td></tr><tr><td>NanoSight LM10</td><td>NanoSight Technology</td><td></td><td>Nanoparticle tracking analysis (NTA) machine</td></tr><tr><td>Phosphate-buffered saline (PBS, pH7.4)</td><td>Thermo Fisher Scientific</td><td>10010001</td><td></td></tr></tbody></table>

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.

The operation time must be optimized to achieve the maximum recovery yield of the enriched microvesicles and exosomes. Insufficient time does not allow sufficient migration of the microvesicles and exosomes, which decreases the enrichment, whereas excessive time deteriorates the spatial focusing and hence disperses the microvesicles and exosomes. Thus, through the time optimization step, the maximum preconcentration factor of microvesicles and exosomes and the final location where microvesicles and exosomes are most enriched can be identified. To find the final location of the microvesicles and exosomes, time-lapse migration of fluorescently labeled microvesicles and exosomes was observed with a microscope (Figure 2A) and the fluorescence intensities in all sample areas were quantified using ImageJ software (Figure 2B). Before the enrichment process (Figure 2A, 0 min), microvesicles and exosomes were dispersed in all sample areas with a gradual decrease in the intensity because of the slight filtering action of the paper matrix (Figure 2B, 0 min). After 10 min of processing (Figure 2A, 10 min), microvesicles and exosomes migrated electrokinetically and an instant preconcentration plug appeared on layer 7. Further processing achieved greater preconcentration of microvesicles and exosomes. When the process time reached 20 min, microvesicles and exosomes were strongly focused on layers 8 and 9 (Figure 2A, 20 min) and enriched fivefold, based on the fluorescence intensities (Figure 2B, 20 min).
After completing the preconcentration process, the enriched microvesicles and exosomes were isolated by unfolding the device and the sample area in layer 8 was punched out for further analysis. SEM images were obtained to investigate the integrity of the enriched microvesicles and exosomes. As shown in the SEM images (Figure 3A), significantly more nondamaged microvesicles and exosomes were observed after the enrichment process. Using the obtained SEM images, the size distribution of the enriched microvesicles and exosomes was analyzed (Figure 3B), where the exosomes and microvesicles were distinguished based on a threshold diameter of 200 nm. In other words, the size of exosomes ranged from 95&#8211;195 nm with a mean diameter of 134 nm and that of the microvesicles from 214&#8211;377 nm with a mean diameter of 315 nm.
Subsequently, the actual concentration of the enriched microvesicles and exosomes on the sample areas in layers 6, 8, and 10 was analyzed by NTA. Before enrichment, the concentration was ~2.24 x 1011 particles/mL in layer 8 (Figure 3C, layer 8). After enrichment, the concentration was ~1.25 x 1012 particles/mL in layer 8 (Figure 3C, layer 8), indicating that the microvesicles and exosomes were preconcentrated by a factor of ~5.58. Noticeable preconcentration was not observed on the other layers (Figure 3C, layers 6 and 10). Taken together, these results demonstrate that the proposed paper-based device can enrich microvesicles and exosomes by approximately fivefold.
The isolation efficiency of the device was evaluated as follows: The sample volume that can be accommodated by layer 8 and 9 is ~2.2 &#956;L out of a total 15 &#956;L of sample volume in 10 layers because of the narrowing design of the Exo-PAD. If we assume an ideal case in which all of the microvesicles and exosomes are preconcentrated on layer 8 and 9 and none are lost, then the ideal preconcentration factor should be ~6.8 (15 &#956;L/2.2 &#956;L = ~6.8). However, the experimental preconcentration factor obtained by NTA (Figure 3c) was ~5.58, which means a portion of 1.22 microvesicles and exosomes were lost by lysis or nonspecific binding to the paper matrix. 
<img alt="Equation 1" src="/files/ftp_upload/61292/61292eq1.jpg" /> 
where 2.24 x 1012 particles/mL is the initial concentration of microvesicles and exosomes. From this calculation, the expected loss rate of the Exo-PAD is about 18%; hence, the isolation efficiency is about 82%. This is a significant improvement compared to the poor yields of ultracentrifugation (5%&#8211;23%).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61292/61292fig1.jpg" /> 
Figure 1: Overall Exo-PAD procedures, from assembly to operation. (A) Software design to guide the printing of hydrophobic wax on cellulose paper. (B) The wax-printed paper after incubation in an oven for 80 s at 120 &#176;C. (C) The individual devices are cut out using a cutter. (D) Permselective membrane solution is dropped on the sample areas in the leftmost and rightmost layers and the solvent is evaporated on a hotplate at 70 &#176;C for 30 min. (E) The printed Exo-PAD is folded back and forth along the white lines; all sample areas are thus convergently connected. (F) A microvesicle and exosome sample is loaded in the sample area. (G) Two acrylic chambers are placed at the ends of the device and secured with small binder clips. (H) The acrylic chambers are filled with buffer solution and two Ag/AgCl electrodes are inserted to apply a voltage of 30 V. (I) The device is unfolded to isolate the preconcentrated microvesicles and exosomes. (J) The sample areas in layers 8 and 9 are punched out for analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61292/61292fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61292/61292fig2.jpg" /> 
Figure 2: Observation of fluorescently labeled microvesicles and exosomes during device operation and corresponding fluorescence intensities. (A) Time-lapse observation of microvesicles and exosomes. Scale bar = 5 mm. With 20 min of processing, the microvesicles and exosomes were strongly focused on layers 8 and 9, where (B) they were enriched by approximately fivefold. Error bars represent standard deviation (n = 3). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61292/61292fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61292/61292fig3.jpg" /> 
Figure 3: Analysis of the enriched microvesicles and exosomes. (A) SEM images showing the integrity of the enriched microvesicles and exosomes. After preconcentration, many more microvesicles and exosomes were observed6. Scale bar = 1 &#956;m. (B) Size distribution of the enriched microvesicles and exosomes. The exosome sizes were 95&#8722;195 nm (mean diameter = 134 nm) and microvesicle sizes were 214&#8722;377 nm (mean diameter = 315 nm). (C) The actual concentration of the enriched microvesicles and exosomes evaluated using NTA. After enrichment, the concentration of microvesicles and exosomes increased fivefold, which is consistent with fluorescence results6. Error bars represent standard deviation (n = 3). Panels A and C are reproduced by permission from Kim et al.6. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61292/61292fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Supplementary Figure 1: Microvesicles and exosomes preconcentrated and focused on layer 8 were separated from bovine serum albumin (BSA), which stayed in layers 1&#8211;3. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/61292/61292supfig1large.jpg" target="_blank">Please click here to download this file.</a>

Watch this Scientific Journal Video about Paper-Based Preconcentration and Isolation of Microvesicles and Exosomes at JoVE.com

Paper-Based Preconcentration and Isolation of Microvesicles and Exosomes

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 ...

paper-based-preconcentration-isolation-microvesicles

Nanyang Technological University

Research

JoVE Journal

Biochemistry

6.0K Views.  Kwangwoon University. In dit protocol beschrijven we een eenvoudige methode om een op papier gebaseerd apparaat genaamd de XO Pad te fabriceren, dat is ontworpen voor de effectieve verrijking in isolatie van extracellulaire vikjes. De XO Pad is een eenvoudig apparaat dat extracellulaire blaasjes kan voorconcentraat en isoleren zonder het gebruik van chemicaliën of werkmachines, terwijl ze hogere terugwinningspercentages kunnen vertonen dan conventionele methoden. Begin met het definiëren van de regio die moet wo...

Video: Paper-Based Preconcentration and Isolation of Microvesicles and Exosomes

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

Isolation of Tissue Extracellular Vesicles from the Liver

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in cell-to-cell communication by shuttling bioactive molecules such as RNA or protein from one cell to another. Most studies of EVs have been performed in cell culture models or in EVs isolated from body fluids. There is emerging interest in the isolation of EVs from tissues to study their contribution to physiological processes and how they are altered in disease. The isolation of EVs with sufficient yield from tissues is technically challenging because of the need for tissue dissociation without cellular damage. This method describes a procedure for the isolation of EVs from mouse liver tissue. The method involves a two-step process starting with in situ collagenase digestion followed by differential ultra-centrifugation. Tissue perfusion using collagenase provides an advantage over mechanical cutting or homogenization of liver tissue due to its increased yield of obtained EVs. The use of this two-step process to isolate EVs from the liver will be useful for the study of tissue EVs.

This is a protocol to isolate tissue extracellular vesicles (EVs) from the liver. The protocol describes a two-step process involving collagenase perfusion followed by differential ultracentrifugation to isolate liver tissue EVs.

Extracellular vesicles (EVs) can be released from many different cell types and detected in most, if not all, body fluids. EVs can participate in ...

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.

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 ...

Bioengineering

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 ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Medicine

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

Biology

Purification of High Yield Extracellular Vesicle Preparations Away from Virus

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection setting. The presented method is meant to isolate EVs away from virions (i.e., HIV-1), allowing for a higher efficiency and yield compared to conventional ultracentrifugation methods. Our protocol contains three steps: EV precipitation, density gradient separation, and particle capture. Downstream assays (i.e., Western blot, and PCR) can be run directly following particle capture. This method is advantageous over other isolation methods (i.e., ultracentrifugation) as it allows for the use of minimal starting volumes. Furthermore, it is more user friendly than alternative EV isolation methods requiring multiple ultracentrifugation steps. However, the presented method is limited in its scope of functional EV assays as it is difficult to elute intact EVs from our particles. Furthermore, this method is tailored towards a strictly research-based setting and would not be commercially viable.

This protocol isolates extracellular vesicles (EVs) away from virions with high efficiency and yield by incorporating EV precipitation, density gradient ultracentrifugation, and particle capture to allow for a streamlined workflow and a reduction of starting volume requirements, resulting in reproducible preparations for use in all EV research.

One of the major hurdles in the field of extracellular vesicle (EV) research today is the ability to achieve purified EV preparations in a viral infection ...

Immunology and Infection

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 ...

Paper-Based Preconcentration and Isolation of Microvesicles and Exosomes

Samenvatting

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