This research was supported by internal funds from CSU (to KMD), ATCC contract #2016-0550-0002 (a subcontract of NIAID HHSN272201600013C), and The Bill and Melinda Gates Foundation (OPP1039688) (to KMD/NKG). We thank the NSF Research Experience for Undergraduates (REU) summer program at Colorado State University for additional support.

This work was determined not to be human subject research upon initial review from Colorado State University&#39;s Institutional Review Board (IRB), as all human samples were obtained as de-identified samples from the Bioreclamation IVT biorepository and were collected under IRB approved protocols.
1. Preparation of Crude Sample (either Plasma or Serum) by Pelleting Larger Vesicles and Cellular Debris
Note: Safety consideration: plasma, serum, and oth...

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An optimized method to increase the purity and yield of exosomes from blood will increase the ability to accurately mine extracellular vesicles as a source of biomarkers for several diseases. The standard methods currently used to isolate exosomes, specifically, ultracentrifugation and precipitation methods, have several disadvantages including exosome aggregation and pelleting of soluble proteins7,17,18. Alternatively, the use ...

Exosomes are nanovesicles (ranging in size from 30 nm to 150 nm) released by almost all the cells in the human body to facilitate cell-to-cell communication processes1,2. Interestingly, the composition of exosomes changes depending on the cells of origin as well as the health status of the individual3,4,5. Additionally, exosomes can be retrieved from several biological fluids such as: saliva, urine, and blood2. Because of these features, exosomes are considered to be a good source of disease biomarkers. Unfortunately, there is no standard method for the isolation of exosomes. Some laboratories consider multistep centrifugation, with a final high-speed step at 100,000 x g using density gradients as the gold standard method for exosome isolation. However, recent studies have shown that ultracentrifugation induces aggregation of exosomes with soluble proteins and other exosomes, in addition to affecting exosome integrity, both of which may hamper downstream applications6,7. Other common methods of exosome isolation include, but are not limited to: precipitation by commercial polyethylene glycol (PEG) based reagents, centrifugal ultrafiltration, and size-exclusion chromatography (SEC). The commercial reagents using polyethylene glycol (PEG) polymers enrich exosomes by causing them to precipitate and form a pellet. Limitations using this polymer are contamination with residual PEG polymer and an abundance of soluble non-exosomal proteins in the final product. Ultrafiltration utilizes centrifugation to purify and concentrate vesicles using a cellulose membrane; exosomes are retained above the filter, while smaller impurities and other proteins pass through the membrane7,8. Just like other methods, centrifugal ultrafiltration has a limited capacity to purify exosomes due to the retention of high levels of non-exosomal proteins, including protein complexes and aggregates. Finally, SEC purification uses porous resin to separate molecules by size. SEC has shown promising results, overcoming most of the problems experienced with other methods by capturing the majority of contaminant proteins and preserving exosomal integrity, since isolation is based on gravity or low-pressure systems7,9. However, the co-isolation of larger protein aggregates and lipoproteins10 during SEC affects the purity of the final exosome preparation. While some methods have been tested for exosome purification from cell culture supernatants and plasma7, or only plasma9,11, there is no information about the performance of methods directly comparing blood plasma and serum from the same individual.
Here, we focus on the purification of blood exosomes by comparing a variety of workflows to determine if vesicle enrichment techniques are translatable between plasma and serum. Nanoparticle tracking analysis and western blot were used to quantify the differences in exosome concentration, purity, and protein composition in the end products. The final method, detailed in this protocol, increases vesicle numbers and reduces non-exosomal protein levels. Importantly, a drastic reduction of common co-precipitating proteins including albumin and apolipoproteins is demonstrated. The high abundance of these two proteins in blood and the frequency at which they co-purify with exosomes causes inconsistencies between &#34;purified&#34; samples, skewing the downstream analyses. This protocol includes the use of a commercially available SEC resin; the resin is composed of porous beads in which proteins smaller than 700 kDa can enter the beads. Once inside, the proteins are retained by an octylamine ligand. The eluate is composed of exosomes and molecules larger than 700 kDa12. Additionally, in order to reduce the protein aggregates and apolipoproteins which evade bead trapping, we include a protease digestion step in the workflow. Currently, there is no single technique capable of maximizing exosome yield while reducing co-purifying non-exosomal proteins. This study shows that a purification protocol that combines a protease digestion pretreatment with multiple recovery and purification methods can be used to increase exosome yield and purity from blood serum and plasma.

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			<td height="21" style="height:21px;width:281px;">Nanosight</td>
			<td style="width:149px;">Malvern</td>
			<td style="width:123px;">NS300</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Benchtop microcentrifuge&#160;</td>
			<td style="width:149px;">Thermo</td>
			<td style="width:123px;">Legend Mico21</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Benchtop centrifuge&#160;</td>
			<td style="width:149px;">Beckman Coulter</td>
			<td style="width:123px;">Allegra 6R</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Waterbath&#160;</td>
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			<td style="width:124px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Microplate reader</td>
			<td style="width:149px;">BIOTEK</td>
			<td style="width:123px;">Epoch</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Pierce BCA Protein Assay Kit</td>
			<td style="width:149px;">THERMO</td>
			<td style="width:123px;">23227</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Micro BCA Protein Assay Kit</td>
			<td style="width:149px;">THERMO</td>
			<td style="width:123px;">23235</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Phosphate buffered saline</td>
			<td style="width:149px;">Corning</td>
			<td style="width:123px;">21-040-CV</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">96 well plate</td>
			<td style="width:149px;">Corning</td>
			<td style="width:123px;">15705-066</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">Amicon Ultra-0.5 Centrifugal Filter Unit with Ultracel-100 membrane</td>
			<td style="width:149px;">EMD-Millipore</td>
			<td style="width:123px;">UFC510096</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Amicon Ultra-4 Centrifugal Filter Units</td>
			<td style="width:149px;">EMD-Millipore</td>
			<td style="width:123px;">UFC800324</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Capto Core 700</td>
			<td style="width:149px;">GE Healthcare</td>
			<td style="width:123px;">17548103</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Poly-Prep&#160;Chromatography Columns</td>
			<td style="width:149px;">BIORAD</td>
			<td style="width:123px;">7311550</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">NuPAGE 4-12% Bis-Tris Protein Gels</td>
			<td style="width:149px;">THERMO</td>
			<td style="width:123px;">NP0323BOX</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Nitrocellulose Membrane, Roll, 0.2 &#181;m</td>
			<td style="width:149px;">BIORAD</td>
			<td style="width:123px;">1620112</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Proteinase K,&#160;Tritirachium album</td>
			<td style="width:149px;">EMD-Millipore</td>
			<td style="width:123px;">539480</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">SimplyBlue SafeStain</td>
			<td style="width:149px;">THERMO</td>
			<td style="width:123px;">LC6065</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">SIGMAFAST&#160;BCIP/NBT</td>
			<td style="width:149px;">Millipore-SIGMA</td>
			<td style="width:123px;">B5655</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">4-Chloro-1-naphthol</td>
			<td style="width:149px;">Millipore-SIGMA</td>
			<td style="width:123px;">C6788</td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:281px;">Anti-CD63 Antibody (rabbit anti-human) with goat anti-rabbit HRP secondary antibody</td>
			<td style="width:149px;">SYSTEM BIOSCIENCES</td>
			<td style="width:123px;">EXOAB-CD63A-1</td>
			<td style="width:124px;">Primary and secondary antibodies</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:281px;">ALB Antibody (F-10)</td>
			<td style="width:149px;">SANTA CRUZ BIOTECHNOLOGY, INC.</td>
			<td style="width:123px;">sc-271605</td>
			<td style="width:124px;">Primary antibody</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:43px;width:281px;">apoB Antibody (C1.4)</td>
			<td style="width:149px;">SANTA CRUZ BIOTECHNOLOGY, INC.</td>
			<td style="width:123px;">sc-13538</td>
			<td style="width:124px;">Primary antibody</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:42px;width:281px;">apoA-I Antibody (B-10)</td>
			<td style="width:149px;">SANTA CRUZ BIOTECHNOLOGY, INC.</td>
			<td style="width:123px;">sc-376818</td>
			<td style="width:124px;">Primary antibody</td>
		</tr>
	</tbody>
</table>

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.

The data presented below were obtained using paired plasma and serum samples from five healthy blood donors. To determine the effects of each processing step, five variations of the presented protocol were performed, and exosome recovery and non-exosome protein removal were compared. The methods trialed included: 1) SEC 700 kDa + Ultrafiltration 3 kDa; 2) Ultrafiltration 100 kDa; 3) Proteinase K + Ultrafiltration 100 kDa; 4) SEC 700 kDa + Ultrafiltration 100 kDa, and 5) Proteinase K + Ult...

Watch this Scientific Journal Video about Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum at JoVE.com

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

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 ...

The overall goal of this procedure is to purify vesicles from blood while reducing co-purifying proteins and complexes which can interfere with downstream analysis including nanoparticle tracking and proteomics. This method can help answer key questions such as what are the proteomic changes in serum or plasma vesicles during the disease state as compared to a healthy state. The main advantage of this technique is the increased accuracy in vesicle enumeration.In removing abundant non-vesicle contaminants, we increase the identification of low abundance proteins and the potential biomarkers of disease. The implication of this method extend to the discovery of novel diagnostic biomarkers. It enables more accurate determination of the proteomic composition of extracellular vesicles in complex matrices, like blood.Generally, individuals new to vesicle purification will struggle because there are many published techniques. The reproducability of downstream proteomic analysis is affected by the co-purification of abundant serum proteins. Equilibrate a serum or plasma sample by placing the tube under non-shaking conditions in a four degree Celsius refrigerator overnight.The next day, aliquot 250 microliters of the sample into a microcentrifuge tube using a 1, 000 microliter pipette. Centrifuge the sample at 18, 000 times G for 30 minutes at four degrees Celsius. Using a 200 microliter pipette, remove 200 microliters of the cleared supernatant and add it to a new microcentrifuge tube.Avoid any pelleted material while transferring the supernatant. Quantify the protein content of the sample by BCA assay using the manufacturer's protocol. To perform the assay, dilute the sample one to 50 in one X phosphate buffered saline.Test each sample in duplicate. Prepare a proteinase K solution at a concentration of 50 micrograms per milliliter in PBS by first adding the corresponding amount of proteinase K to a conical tube. Then add the buffer and vortex for 30 seconds three times at room temperature.Next, add 50 microliters of proteinase K solution to the 10 milligram sample aliquot and mix gently by pipetting up and down using a 200 microliter pipette. Incubate the sample at 37 degrees Celsius in a water bath for 30 minutes, placing the tube in a float tube rack. To prevent potential leakage, avoid complete immersion of the tube in the water bath.Following incubation, transfer the sample and rack to a 60 degree Celsius water bath for 10 minutes to inactivate the proteinase K.Next, rinse an ultrafiltration device containing a 100 kilodalton molecular weight cutoff filter with PBS prior to use. Use a 1, 000 microliter pipette to add 500 microliters of PBS to the filter. After spinning the device at 3, 700 times G for five minutes, discard any remaining retentate as well as the eluate.Increase the volume of the sample to 500 microliters by adding PBS. Then pipette the sample into the pre-rinsed ultrafiltration device. Centrifuge the ultrafiltration device in a fixed angle benchtop centrifuge at 3, 700 times G and four degrees Celsius until the sample has reduced to a volume of 50 microliters.Next, add 500 microliters of PBS directly onto the filter membrane. And pipette up and down five times before centrifuging again as before. Transfer the final 50 microliter retentate to a new microcentrifuge tube.Rinse the filter membrane with 200 microliters of PBS, pipetting up and down 10 times. And transfer the wash to the tube. Then, place the filter holder in the inverse position in a new tube.Run a reverse spin recovery for five minutes at 2, 000 times G to retrieve the remaining sample from the membrane. Place the columns in a size-appropriate rack. Add 850 microliters of SEC slurry with beads having a molecular weight cutoff of 700 kilodaltons using a 1, 000 microliter pipette.Uncap the bottom of the column and let the liquid drain into a waste container for five minutes. Once drained, add 10 microliters of PBS to wash the resin. Allow 20 minutes for the wash to drain from the column.Place the concentrated proteinase K treated sample into a 15 milliliter conical tube. And increase the sample volume to five milliliters by adding PBS with a serological pipette. Mix the tube gently by rocking for one minute.Then, slowly apply the sample to the prepared column with a serological pipette. Collect the flow-through in a new 15 milliliter conical tube. Pipette the collected material over the resin again to remove any remaining material below 700 kilodaltons, collecting the flow-through in a new 15 milliliter conical tube.Wash the resin twice by adding one milliliter of PBS each time. Collect the flow-through in a tube. The total final volume will be roughly seven milliliters.Rinse an ultrafiltration device with a three kilodalton molecular weight filter as before. Centrifuge in a swinging bucket rotor at 3, 700 times G and four degrees Celsius. Buffer will pass through the filter and can be discarded.Concentrated exosomes will be retained above the filter. Centrifuge the sample until the volume above the filter is reduced to 200 microliters. Quantify the protein content of the sample by BCA assay using the manufacturer's protocol.Add five micrograms of purified exosomes to one milliliter of PBS. And vortex for 15 seconds on low medium speed. Place the sample in a one milliliter disposable syringe.If available, set the syringe in an automatic syringe pump set to inject the diluted exosome sample at a rate of 30 microliters per minute. Define the script for analysis to run three technical replicates for a minimum of 30 seconds using a constant flow for every replicate. For the analysis, set the capture threshold at five.Finally, determine the soluble protein reduction as described in the text protocol. Representative results of nanoparticle tracking analysis of purified serum vesicles are shown. The vesicle size is roughly 100 nanometers, as expected.Shown here is a Coomassie-stained protein gel comparing the crude serum, pre and post SEC fractions showing the reduction in albumen and other non-vesicle contaminants. A Western blot displays the retention of CD63, a hallmark exosome protein after proteinase K treatment and purification. The proteinase K step is designed to digest soluble proteins.CD63 is a transmembrane protein and less susceptible to proteinase K at the determined concentration and incubation length. Here, a Western blot shows the depletion of albumen, the most abundant protein in blood and a common co-purifying protein after purification. Once mastered, this technique can be done in six hours.After watching this video, you should have a good understanding of how to use centrifuge ultrafiltration, protease digestion, and size exclusion chromatography to obtain vesicles from biological fluids. It's important to perform protein quantitation by BCA or equivalent technique prior to a chromatography and nanoparticle tracking. Results will vary if protein concentrations exceed those noted in this protocol.Don't forget that working with biological fluids can be extremely hazardous and precautions, such as wearing personal protective equipment including gloves, lab coat, and safety glasses should always be taken while performing this procedure. Following this procedure, other methods like liquid chromatography mass spec can be performed in order to determine the exact proteins contained in the vesicles purified. Though this method can provide insight into the composition of blood vesicles, it can also be applied to other sample types, such as urine, CSF, or cell culture supernatants.

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Research

JoVE Journal

Biochemistry

16.2K Views.  Colorado State University. The overall goal of this procedure is to purify vesicles from blood while reducing co-purifying proteins and complexes which can interfere with downstream analysis including nanoparticle tracking and proteomics. This method can help answer key questions such as what are the proteomic changes in serum or plasma vesicles during the disease state as compared to a healthy state. The main advantage of this technique is the increased accuracy in vesicle enumeration.In removing abundant non-v...