Paper-Based Preconcentration and Isolation of Microvesicles and Exosomes

Podsumowanie

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

Streszczenie

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  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.

Wprowadzenie

Microvesicles and exosomes are small membrane vesicles measuring 0.2−1 μm and 30−200 nm, respectively. They are secreted into the extracellular environment by several different cell types^1,2,3,4,5. 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 saliva^6,7,8,9. 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 ultracentrifugation^5,6,10,11,12. 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 (5−23%)^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.

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Protokół

1. Device fabrication

1. 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).
2. 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).
3. Place the wax-printed paper in a laboratory oven for 80 s at 120 °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.
4. Cut the wax-printed paper with a cutter to make individual devices (Figure 1C).
5. Drop 5 μL and 2 μL of permselective membrane (e.g., Nafion; Table of Materials) onto the sample areas in the leftmost and rightmost layers, respectively (Figure 1D).
6. Place the layers coated with the permselective membrane on a hot plate at 70 °C for 30 min to evaporate the permselective membrane solvent.
7. Seal the outermost surface of the coated layer facing the buffer solution with pressure-sensitive tape, leaving a small hole.
8. 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.

2. Enrichment and spatial focusing of microvesicles and exosomes by ion concentration polarization

1. Load 15 μL 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).
2. 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).
3. Fill the chambers with 110 μL of 0.1x PBS and insert two Ag/AgCl electrodes (Figure 1H).
4. 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.

3. Isolation of the enriched microvesicles and exosomes

1. 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).
2. Punch out the sample areas in layers 8 and 9, where the microvesicles and exosomes are enriched, for downstream analysis (Figure 1J).

4. Scanning electron microscopy analysis

1. 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.
2. Dehydrate the fixed microvesicles and exosomes with ascending grades of 200 proof ethanol (i.e., 50%, 70%, 90%, and 100%) for 30 min each.
3. 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.
4. Coat the completely dried microvesicles and exosomes with gold/palladium (~20 nm) via sputtering and capture scanning electron microscopy (SEM) images.

5. Nanoparticle tracking analysis

1. Punch out the sample areas in layers 6, 8, and 10 using a biopsy punch after 20 min of device operation.
2. Immerse each punched-out area in buffer solution (0.1x PBS with 0.01% Tween 20).
3. Vortex for 10 min and centrifuge for 30 s at 6,000 rpm to resuspend the enriched microvesicles and exosomes.
4. 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).

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Wyniki

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 enri...

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Dyskusje

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. Lon...

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Materiały

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