Name: isolation of tissue extracellular vesicles from the liver
Uploaded: 2019-08-21T12:30:00.000+00:00
Duration: 5 s
Description: Watch this Scientific Journal Video about Isolation of Tissue Extracellular Vesicles from the Liver at JoVE.com

This study was supported by funding from National Cancer Institute Grant CA-217833.

All studies involving animals were performed in accordance with a protocol that was approved by the Mayo Clinic Institutional Animal Care and Use Committee.
1. Bench Preparation
<ol>
	<li>Prepare a water bath and set it to 37 &#176;C. The other necessary equipment and set-up are shown in Figure 1.</li>
	<li>Measure 100 mg of collagenase type IV and add it to a 125 mL flask containing 100 mL of HBSS in a water bath (40 &#176;C). Ensure that the collagenase has been dissolved by swirling the liquid in the flask, and also make sure that the flask is submerged completely in the water bath. For greater efficiency, allow the collagenase to dissolve for at least 30 min even though it may appear to dissolve instantly.</li>
	<li>Submerge a 125 mL flask containing 50 mL of HBSS in a water bath at 40 &#176;C. This will be used for initial flushing.</li>
	<li>Spray down the surface of all instruments such as the scissors and forceps with 70% ethanol. Prepare a few clean cotton swabs.</li>
	<li>Rinse out the tubing of the pump by running 70% ethanol through the pump and remove any residual ethanol by rinsing it twice with running water.</li>
	<li>Lay an absorbent bench pad on the lab bench and place a hard box container on top. This will be needed to contain any excess liquids during the mouse perfusion. Wrap a polystyrene foam pad with aluminum foil and place it inside the container.</li>
	<li>Place a sterile 10 cm culture dish close to the bench. This will be used to hold the digested liver after the perfusion is completed.</li>
	<li>Pour 10 mL of collagenase medium (step 1.2) into a sterile culture dish in advance.</li>
	<li>Using a winged blood collection set, connect the 23-gauge to the free end of the pump tubing (cutting the wings off the butterfly cannula can lead to better handling). Pass until the tip of the cannula of blood collection is filled.</li>
</ol>
2. Animal Preparation
<ol>
	<li>Before starting anesthesia using isoflurane, make sure that an adequate amount of supply gas is available for the duration of the procedure. Turn on the oxygen (O2) supply to the induction chamber at 1-2 L, then turn on isoflurane between 2-4% using a flowmeter.</li>
	<li>Put the mouse into the induction chamber and close the top door. Monitor the mouse until it is recumbent. 
	NOTE: The gases in the chamber will keep the mice anesthetized for several minutes.</li>
	<li>Place the mouse on a polystyrene foam pad wrapped with aluminum foil. Switch the flow from the induction chamber to a nosecone. Ensure that anesthesia is adequate. If the mouse has started responding, gently restrain it in a nose cone until the it is fully anesthetized.</li>
	<li>Ensure anesthesia by monitoring respiration and response to stimulation during the procedure. Adjust the rate flowmeter as needed to ensure adequate anesthesia. The paws must be nonresponsive to the pinch test under anesthesia. Additional details may be obtained from the laboratory guide for the care of animals.</li>
	<li>Tape or pin down all four limbs of the mouse.</li>
	<li>Clean the skin over the abdomen by spraying with 70% ethanol and wiping it off with gauze and an alcohol pad. This step is critical to avoid contamination from the mouse fur.</li>
	<li>Make a wide-open cut through the skin from the anterior to the pelvis bone using a set of sterile scissors. Be careful not to cut any internal organs . Displace the intestines to the animal&#39;s left side&#160;by using a cotton tipped applicator to gently to expose the portal vein (PV) and inferior vena cava (IVC).</li>
</ol>
3. Cannulation and Perfusion (0.5 h)
<ol>
	<li>Using curved forceps, place a thread underneath the portal vein and tie a knot loosely to prepare cinching tightly after cannulation.</li>
	<li>Insert the cannula (23G from the blood collection set) into the portal vein 5-10 mm below the ligature. Do not insert the cannula past the first portal branch, otherwise the right anterior lobe can be inadequately perfused.The cannula can be fixed or fastened with thread using a stopper knot.</li>
	<li>Start the pump to infuse in HBSS at a low flow rate (1-2 mL/min). Once the cannulation is confirmed to be successful, the liver will begin to blanch.</li>
	<li>Cut the IVC to relieve pressure and allow excessive fluid within the liver to drain. This is best performed using the operators other hand so the cannula is not moved.</li>
	<li>Slowly increase the flow rate to 8 mL/min and complete the perfusion using the entire 50 mL volume of HBSS through the liver, over the next 5 min.</li>
	<li>Change the collagenase containing medium (step 1.2) into the beaker just before the HBSS is starting to run out. Ensure that air bubbles are not present and do not flow into the liver when changing the medium.</li>
	<li>Apply transient pressure to the IVC at 5 s intervals by clamping with forceps. This will cause the liver to swell and help with tissue digestion and dissociation (7-8 min). As digestion progresses, the liver will swell and become white. The liver can swell uniformly to approximately twice its original size.</li>
	<li>Turn off the pump and remove the cannula once digestion is completed. The completion of liver digestion will depend on the size of the mouse and condition of the liver. A dent in the liver can be observed if a cotton-tipped applicator is used to gently probe the liver.</li>
	<li>Remove the gallbladder from the liver, being careful not to tear it. Using a washed pair of scissors and forceps, extract the liver from the mouse into a sterile 10 cm culture dish containing phosphate-buffered saline (PBS) for surface washing. Transfer the liver carefully into the sterile 10 cm culture dish containing collagenase medium (from step 1.8). This is a critical step for avoiding contamination from the mouse blood and bile.</li>
	<li>Grab and tear apart the liver with two clean forceps while gently shaking the cells from the liver. As this happens, the medium will become clouded. All hepatocytes can be shaken away, leaving behind the connective tissue and vascular tissue.</li>
	<li>Triturate the cell solution many times using a 3 mL syringe until the undigested parts of the liver are shaken off. Pour it off into a 50 mL conical tube with 70 &#181;m nylon cell strainers to filter any undigested connective tissue. Wash the dish with HBSS to collect the remaining cells and fill up the 50 mL conical tube.</li>
	<li>Centrifuge softly the 50 mL conical tube in a swinging bucket rotor at 50 x g for 10&#160;min at 4 &#176;C.</li>
	<li>Transfer the supernatant to a new 50 mL conical tube.</li>
</ol>
4. Isolation of EVs (5 h)
<ol>
	<li>Centrifuge the supernatant at 300 x g for 10 min at 4 &#176;C. Transfer the supernatant into a new 50 mL conical tube.</li>
	<li>Centrifuge the supernatant at 2000 x g for 20 min at 4 &#176;C to remove cell debris and aggregates.</li>
	<li>Transfer the supernatant to a round-bottom tube and centrifuge the supernatant at 10,000 x g for 70 min at 4&#176;C.</li>
	<li>Collect the supernatant and place into a polycarbonate ultracentrifuge tube and centrifuge at 100,000 x g for 70 min at 4 &#176;C.</li>
	<li>Collect the pellet in an ultracentrifuge tube which is then washed by re-suspending in PBS. Centrifuge the supernatant further at 100,000 x g for 70 min at 4 &#176;C.</li>
	<li>The final pellet comprising of cellular nanovesicles can be directly used for experiments or re-suspended with 1000 &#181;L of PBS and stored at -80 &#176;C.</li>
</ol>
5. Assessment of Quality and Yield of Isolations
<ol>
	<li>Assess the size distribution and concentration using nanoparticle tracking analysis or tunable resistive pulse sensing following the instrument manufacturer&#39;s protocols.</li>
	<li>Perform further isolation and purification of specific vesicle populations by various approaches such as the addition of a sucrose gradient or cushion, immunoaffinity techniques, or size exclusion chromatography, based on specific experimental needs.</li>
</ol>

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The authors have nothing to disclose.

This protocol describes an optimal and reproducible method for the isolation of hepatic tissue EV using a two-step perfusion process via the portal vein followed by differential ultracentrifugation. Important steps of the procedure include cannula placement, collagenase concentration and digestion time, flow speed of the medium, handling of the tissue after digestion, and classical differential ultracentrifugation.
Cell separation is achieved by separation from connective tissue components after digestion using collagenase type IV.The concentration of collagenase used for perfusion can range from 0.1 to 5 mg/mL. There can be considerable batch-to-batch variation in the efficacy of collagenase for tissue digestion. Concentrations of collagenase from 0.5 to 5 mg/mL were tested, but the concentration used did not have a major impact on the yield of EVs obtained. Using a higher concentration of collagenase will result in a more rapid swelling and whitening of the liver. The goal is to obtain satisfactory cell dissociation without excessive contamination or damage. An optimal concentration of collagenase used in these isolations is 1-2 mg/mL perfused for 7-8 min at a flow rate of 8 mL/min. A perfusion procedure that is too long will increase the risk of destroying the thin connective tissue within the liver as well as increase technical risks such as needle dislodgment from the portal vein or air trapping with the vein.
The most challenging aspect of this protocol is the cannulation of the portal vein. This can be challenging to perform, particularly in mice in the 18- to 25-g size range. The techniques for collagenase perfusion were originally developed for use in rats and subsequently adopted for use in mice after numerous modifications and adjustments. Cannulation using a 23G blood collection set is easier than placement of a catheter in blood vessels of small luminal diameter. Fixing the cannula using thread with a stopper knot is recommended to avoid dislodgement and the knot also serves as a marker of portal vein location in case the cannula comes off the vessel.
For downstream analysis, it is extremely important to have minimal contamination from cells. There are several important considerations in the handling of tissue after digestion. First, the forceps and scissors are changed when the liver is extracted to avoid blood contamination. Second, it is critical that the gallbladder be carefully removed from the liver to avoid tearing and unwanted contamination from bile. Third, once the liver has been removed from the mouse, the liver is washed very gently using PBS to remove any blood. Minimizing contamination with cells should be given a higher priority than reduction in the yield of EVs obtained.
Ultracentrifugation is the most commonly used method for the isolation and purification of EVs8,9,10,11. This approach will remove most parenchymal cells such as hepatocytes or cholangiocytes and non-parenchymal cells such as Kupffer cells, sinusoidal endothelial cells, and stellate cells, In addition, cell debris, cell aggregations, and dead cells will also be removed by differential centrifugation. Further purification and isolation of specific populations can be performed by size exclusion chromatography to remove any non-vesicular protein aggregates or lipoproteins.
A limitation of this protocol is that it may not capture all tissue vesicles, given the possibility that some vesicles may be removed in the perfusate. If a global assessment is needed, collection of perfusate and isolation of vesicles within perfusate should be considered. A further limitation is the potential for cell damage. To monitor the potential impact of excessive cell death, cell viability can be monitored and incorporated within quality parameters for tissue EV isolations. In conclusion, this procedure describes an optimized workflow using a two-step perfusion technique via the portal vein followed by differential ultracentrifugation for obtaining liver tissue EVs from mouse livers at a high yield. These tissue EVs are suitable for downstream analyses such as characterization of biomolecular composition and other studies that aim to characterize their physiological or pathophysiological roles or potential applications as disease markers.

Extracellular vesicles (EVs) are membrane-bound vesicles that are released from many different types of cells in the body. EVs contain a cargo of molecules that include RNA, DNA, and protein. Transfer of this cargo by EVs from one cell to another is postulated as one mechanism by which cells within tissues communicate with each other1. The majority of information regarding the cargo or roles of EVs in normal health and diseases has been derived from studies on EVs obtained from cells in culture or collected from the circulation or other body fluids2. In order to understand their physiological roles in vivo, a robust method is necessary for the isolation of tissue EVs that captures all populations of EVs and avoids cellular damage or contamination3. The overall goal of the method described herein is to isolate tissue EVs from mouse livers.
Most cell types in the liver have been shown to produce EVs, and the study of EV-based signaling is advancing basic knowledge and understanding of hepatic diseases. However, the combined impact of EVs from different cell types within tissues is only partially understood. Isolation of EVs from liver tissues is necessary in order to understand the in situ contributions of EVs within the tissue milieu. The approach described herein is based on a two-step perfusion to enhance tissue dissociation and minimize cell damage. Subsequently, EVs are isolated from the dissociated liver tissue. Approaches using two-step perfusion for isolation of hepatocytes have been used since the early 1950s4. These methods for hepatocyte isolation have been modified and continuously improved and are now standard approaches for isolation of hepatocytes in cultures, in cell suspensions, and from tissues5,6,7. In the first step, the liver is subjected to a non-recirculating perfusion with calcium-free buffer,Hank&#39;s balanced salt solution (HBSS). In the second step, the liver is perfused with collagenase to dissolve the extracellular matrix for further separation of desmosomal cell-to-cell junctions. An optimal treatment time for collagenase dissolution is 7 to 10 minutes. A shorter duration of treatment will cause incomplete dissolution and retain cell contacts in the liver, whereas a longer duration may cause liver damage or portal vein disruption. EVs are then isolated using differential centrifugation to remove cells and cellular debris. This results in EV collection in high yields that can be used for further downstream analyses or studies.

<table><tbody><tr><td>125 mL Erlenmeyer flask</td><td>Fisher scientific</td><td>FB500125</td><td></td></tr><tr><td>125 mL Erlenmeyer flask</td><td>Fisher scientific</td><td>FB500125</td><td></td></tr><tr><td>Curved non-serrated scissors</td><td>Fine Science Tools</td><td>14069-12</td><td></td></tr><tr><td>Curved forceps</td><td>Fine Science Tools</td><td>13009-12</td><td></td></tr><tr><td>Masking tape</td><td>Home supply store</td><td></td><td></td></tr><tr><td>Aluminum foil</td><td>Home supply store</td><td></td><td></td></tr><tr><td>Styrofoam pad</td><td>Home supply store</td><td></td><td></td></tr><tr><td>Absorbent Bench Underpad</td><td>Scientific inc.</td><td>B1623</td><td></td></tr><tr><td>Masterflex L/S Digital Miniflex Pump</td><td>Cole-parmer</td><td>ZX-07525-20</td><td></td></tr><tr><td>Water bath</td><td>Thermo Electron Precision</td><td>2837</td><td></td></tr><tr><td>Blood collection sets</td><td>Becton Dickinson</td><td>367292</td><td></td></tr><tr><td>Petri dish, clear lid 100x15</td><td>Fisher Scientific</td><td>FB0875712</td><td></td></tr><tr><td>Falcon 70mm Nylon Cell Strainers</td><td>Fisher scientific</td><td>352350</td><td></td></tr><tr><td>50 mL conical tubes</td><td>Fisher Scientific</td><td>12-565-270</td><td></td></tr><tr><td>Cotton Tipped Applicators</td><td>Moore medical</td><td>69622</td><td></td></tr><tr><td>25mL Serological Pipet</td><td>Falcon</td><td>357525</td><td></td></tr><tr><td>Ohmeda Isotec 4 Isoflurane Vaporiser</td><td>BioSurplus</td><td>203-2751</td><td></td></tr><tr><td>O2 gas</td><td></td><td></td><td></td></tr><tr><td>Isoflurane</td><td></td><td></td><td></td></tr><tr><td>&amp;nbsp;Levo Plus Motorized Pipette Filler</td><td>Scilogex</td><td>74020002</td><td></td></tr><tr><td>Centrifuge 5804R</td><td>Sigma-aldrich</td><td>22628048</td><td></td></tr><tr><td>Beckman Coulter Optima L-100 XP</td><td>Beckman</td><td>969347</td><td></td></tr><tr><td>Beckman Coulter Avanti JXN-26</td><td>Beckman</td><td>B34182</td><td></td></tr><tr><td>70 Ti Fixed-Angle Rotor</td><td>Beckman</td><td>337922</td><td></td></tr><tr><td>JA-25.50Fixed-Angle Rotor</td><td>Beckman</td><td>363055</td><td></td></tr><tr><td>Nalgene Round-bottom tube</td><td>Thermo Scientific</td><td>3118-0028</td><td></td></tr><tr><td>Polycarbonate ultracentrifuge tubes with cap assembly</td><td>Beckman</td><td>355618</td><td></td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>HyClone Hank&#039;s Balanced Salt Solution (HBSS), Ca/Mg free</td><td>Fisher scientific</td><td>SH30588.01</td><td></td></tr><tr><td>Collagenase, Type IV, powder</td><td>Fisher scientific</td><td>17104019</td><td></td></tr><tr><td>Phosphate Buffered Saline (PBS)</td><td>Fisher scientific</td><td>SH30256.01</td><td></td></tr></tbody></table>

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.

The apparatus needed for these isolations comprises of standard laboratory equipment, making this a relatively simple and cost-effective approach. Isolations have been performed from twelve- to thirty-week-old male and female Balb/c or FVB mice. The tray holding the mouse is lined with aluminum foil inside a hard-walled container that collects excess fluids during the perfusion. The flasks containing HBSS or collagenase-containing medium are submerged in a water bath (40 &#176;C) ready to be used. Two sterile 10 cm culture dishes are used. One is needed for surface washing with PBS, and the other for hepatocyte separation from the connective tissue components.
In this method, the liver is perfused in a non-continuous manner via the portal vein in preference to cannulation from the inferior vena cava. An alternative and commonly used perfusion approach is to perform retrograde perfusion by cannulating the inferior vena cava and cutting the portal vein for drainage. However, portal vein cannulation is easy to access and involves a short distance to the liver, as the portal vein feeds directly into the liver8. The selection of an insertion point for cannulation is crucial for optimal success (Figure 2). The cannula is placed past the branches of the stomach and pancreatic veins but not beyond the first portal branch (right and left hepatic portal veins).Once the optimal insertion location in the portal vein is identified, curved forceps are used to place a thread underneath portal vein and tie a loose knot. The needle of cannula is fixed with thread using a stopper knot to stop the needle from falling out.
Figure 3&#160;outlines the overall processing scheme for the differential centrifugation for liver tissue EVs isolation. Ultracentrifugation removes cells, debris and other impurities. The first four centrifugation steps (50 x g, 300 x g, 2,000 x g, 10,000 x g) are designed to remove hepatocytes, intact other cells, dead cells, or cell debris respectively. (Figures 4A and 4B). After these steps, ultracentrifugation is again performed at 100,000 x g to collect the pellet (Figure 4C). The pellet is washed by re-suspending in PBS and subjected to a final ultracentrifugation at 100,000 x g. The pellet after ultracentrifugation is absolutely visible and viscid in this procedure compared to EVs from conditioned culture media. Pipetting many times is required until any brown aggregates are out of sight and completely dissolved. The final pellet is re-suspended with 1000 &#181;L of PBS (Figure 4D). Ultracentrifugation removes impurities and other soluble contaminants from the plasma, which can affect functional experimental outcomes. The centrifugation is be carried out at 4 &#730;C.
From mouse liver, this method yields a tissue EV concentration that ranges from 1.74 to 4.00 x 1012 with a mean of 3.46 x 1012 particles per mL as determined by nanoparticle tracking analysis (NTA) (Figure 5). The mean size of the isolated liver tissue EVs was 157.7 nm, with a mode size of 144.5 nm and EV sizes ranging from 100-600 nm by NTA. The yield of EV will depend on factors such as the liver weight and losses within the perfusate or ultracentrifugation steps.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58649/58649fig1.jpg" /> 
Figure 1: Bench preparation. The materials and their locations are: (A) pump, (B) warmed 125 mL flask containing HBSS and water suction port, (C) nose cone connected to an Isoflurane vaporiser, (D) water exhaust port of the pump connecting with needle, (E) tray lined with aluminum foil inside a hard walled container, and (F) 10 cm culture dish in which collagenase medium is poured in advance. <a href="https://www.jove.com/files/ftp_upload/58649/58649fig1large.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/58649/58649fig2.jpg" /> 
Figure 2: Cannulation site. The anatomy of the mouse abdomen is shown. Using curved forceps, a thread is placed underneath the portal vein (PV) and a loose knot is tied. The insertion location is near the liver, 5-10 mm below the ligature, but not beyond the first portal branch (left and right hepatic portal veins). The cannula is fixed or fastened using thread with a stopper knot. This knot serves as a marker of the PV location if the cannula is dislodged. <a href="https://www.jove.com/files/ftp_upload/58649/58649fig2large.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/58649/58649fig3.jpg" /> 
Figure 3: Schematic of centrifugation steps. The goal is to remove unwanted cells and other components and isolate EVs. The first four centrifugation steps are designed to remove hepatocytes and other cells, dead cells, or cell debris using differential centrifugation. After these steps, ultracentrifugation is performed at 100,000 x g to collect the pellet of EVs. The pellet is washed by re-suspending in PBS and subjected to a final ultracentrifugation at 100,000 x g. All the centrifugation steps are carried out at 4 &#730;C. <a href="https://www.jove.com/files/ftp_upload/58649/58649fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58649/58649fig4.jpg" /> 
Figure 4: Differential centrifugation. (A) After centrifugation at 50 x g for 10 min, a pellet containing hepatocytes is observed. (B) A round-bottom tube is used for centrifugation at 10,000 x g for 70 min to remove cell debris. (C) A polycarbonate ultracentrifuge tube is used for centrifugation at 100,000 x g for 70 min. The pellet is collected in one tube and washed by re-suspending with PBS. (D) The final pellet is re-suspended in 1000 &#181;L of PBS. <a href="https://www.jove.com/files/ftp_upload/58649/58649fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/58649/58649fig5.jpg" /> 
Figure 5: Representative result. The size and concentration of liver tissue EVs can be determined by nanoparticle tracking analysis (NTA). <a href="https://www.jove.com/files/ftp_upload/58649/58649fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Isolation of Tissue Extracellular Vesicles from the Liver at JoVE.com

Isolation of Tissue Extracellular Vesicles from the Liver

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 ...

isolation-of-tissue-extracellular-vesicles-from-the-liver

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11.4K Views.  Mayo Clinic. 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.

Video: Isolation of Tissue Extracellular Vesicles from the Liver

Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for culturing or for obtaining cell lysates. In this protocol, the portal vein is used as the site for catheterization, rather than the vena cava, as this limits contamination of other possible cell types in the final liver preparation. No special instrumentation is required throughout the procedure. A water bath is used as a source of heat to maintain the temperature of all the buffers and solutions. A standard peristaltic pump is used to drive the fluid, and a refrigerated table-top centrifuge is required for the centrifugation procedures. The only limitation of this technique is the placement of the catheter within the portal vein, which is challenging on some of the mice in the 18 - 25 g size range. An advantage of this technique is that only one vein is utilized for the perfusion and the access to the vein is quick, which minimizes ischemia and reperfusion of the liver that reduces hepatic cell viability. Another advantage to this protocol is that it is easy to distinguish live from dead hepatocytes by eyesight due to the difference in cellular density during the centrifugation steps. Cells from this protocol may be used in cell culture for any downstream application as well as processed for any biochemical assessment.

The goal of this protocol is to obtain high viability and high yield of hepatocytes and sinusoidal endothelial cells from liver. This is accomplished by perfusing the liver with a type IV collagenase solution via the portal vein, followed by differential centrifugation to obtain hepatocytes and sinusoidal endothelial cells.

This protocol demonstrates a method for obtaining high yield and viability for mouse hepatocytes and sinusoidal endothelial cells (SECs) suitable for ...

Extraction of Extracellular Vesicles from Whole Tissue

Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or prognostic biomarker assays, and likely serve as important players in the progression of a vast spectrum of diseases. Current research is focused on the characterization of vesicles secreted from multiple cell and tissue types in order to better understand the role of EVs in the pathogenesis of conditions including neurodegeneration, inflammation, and cancer. However, globally consistent and reproducible techniques to isolate and purify vesicles remain in progress. Moreover, methods for extraction of EVs from solid tissue ex vivo are scarcely described. Here, we provide a detailed protocol for extracting small EVs of interest from whole fresh or frozen tissues, including brain and tumor specimens, for further characterization. We demonstrate the adaptability of this method for multiple downstream analyses, including electron microscopy and immunophenotypic characterization of vesicles, as well as quantitative mass spectrometry of EV proteins.

Here, we provide a detailed protocol to isolate small extracellular vesicles (EVs) from whole tissues, including brain and tumor specimens. This method offers a reproducible technique to extract EVs from solid tissue for further downstream analyses.

Circulating and interstitial small membrane-bound extracellular vesicles (EVs) represent promising targets for the development of novel diagnostic or ...

Cancer Research

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

An Enrichment Method for Small Extracellular Vesicles Derived from Liver Cancer Tissue

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's interstitial space. The efficient enrichment of these sEVs is an important prerequisite to the study of their biological function and a key to the development of clinical detection techniques and therapeutic carrier technology. It is difficult to isolate sEVs from tissue because they are usually heavily contaminated. This study provides a method for the rapid enrichment of high-quality sEVs from liver cancer tissue. The method involves a four-step process: the incubation of digestive enzymes (collagenase D and DNase &#921;) with tissue, filtration through a 70 &#181;m cell strainer, differential ultracentrifugation, and filtration through a 0.22 &#181;m membrane filter. Owing to the optimization of the differential ultracentrifugation step and the addition of a filtration step, the purity of the sEVs obtained by this method is higher than that achieved by classic differential ultracentrifugation. It provides an important methodology and supporting data for the study of tissue-derived sEVs.

Here we describe the enrichment of small extracellular vesicles derived from liver cancer tissue through an optimized differential ultracentrifugation method.

Small extracellular vesicles (sEVs) derived from tissue can reflect the functional status of the source cells and the characteristics of the tissue's ...

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

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 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 ...

Biology

Isolation and Characterization of Mouse Primary Liver Sinusoidal Endothelial Cells

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. LSECs have a distinct morphology characterized by the presence of fenestrae and the absence of basement membrane. LSECs play essential roles in many pathological disorders in the liver, including metabolic dysregulation, inflammation, fibrosis, angiogenesis, and carcinogenesis. However, little has been published about the isolation and characterization of the LSECs. Here, this protocol discusses the isolation of LSEC from both healthy and nonalcoholic fatty liver disease (NAFLD) mice. The protocol is based on collagenase perfusion of the mouse liver and magnetic beads positive selection of nonparenchymal cells to purify LSECs. This study characterizes LSECs using specific markers by flow cytometry and identifies the characteristic phenotypic features by scanning electron microscopy. LSECs isolated following this protocol can be used for functional studies, including adhesion and permeability assays, as well as downstream studies for a particular pathway of interest. In addition, these LSECs can be pooled or used individually, allowing multi-omics data generation including RNA-seq bulk or single cell, proteomic or phospho-proteomics, and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq), among others. This protocol will be useful for investigators studying LSECs&#39; communication with other liver cells in health and disease and allow an in-depth understanding of the role of LSECs in the pathogenic mechanisms of acute and chronic liver injury.

Here we outline and demonstrate a protocol for primary mouse liver sinusoidal endothelial cell (LSEC) isolation. The protocol is based on liver collagenase perfusion, nonparenchymal cell purification by low-speed centrifugation, and CD146 magnetic bead selection. We also phenotype and characterize these isolated LSECs using flow cytometry and scanning electron microscopy.

Liver sinusoidal endothelial cells (LSECs) are specialized endothelial cells located at the interface between the circulation and the liver parenchyma. ...

Refining Techniques for Murine Spleen-Derived Exosome Isolation

Isolation and Characterization of Exosomes Derived from Mouse Spleen Tissues

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed that Exo derived from humoral or cell-supernatant are promising targets for novel diagnostic or prognostic biomarkers, underscoring their significant role in disease pathogenesis. Tissue-derived Exo (Ti-Exo) have attracted increasing attention due to its ability to accurately reflect tissue specificity and the microenvironment. Ti-Exo, present in interstitial space, play crucial roles in intercellular communication and cross-organ signaling. Despite their recognized value in elucidating disease mechanisms, isolating Ti-Exo remains challenging due to the complexity of tissue matrices and variability in extraction methods. In this study, we developed a practical protocol for isolating exosomes from mice spleen tissue, providing a reproducible technique for subsequent identification analysis and functional studies. We used Type I collagenase digestion combined with differential ultracentrifugation to isolate spleen-derived Exo. The characteristics of isolated Exo were determined through electron microscopy, the nano-flow cytometer, and the western blot. The isolated spleen-derived Exo displayed the typical morphology of lipid bilayer vesicles, with particle sizes ranging from 30 nm to 150 nm. In addition, the expression profile of exosome markers confirmed the presence and purity of exosomes. Taken together, we successfully established a practical protocol for isolating spleen-derived Exo in mice.

Here, we present a protocol to isolate mouse spleen-derived exosomes using a combination of digestion with collagenase type I and ultracentrifugation.

Exosomes (Exo) are lipid-bilayer structures secreted by various cells, including those of animals, plants, and prokaryotes. Previous studies have revealed ...

Medicine

Isolation and Analysis of Traceable and Functionalized Extracellular Vesicles from the Plasma and Solid Tissues

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important players in the maintenance of organismal homeostasis and the progression of a wide spectrum of diseases. While the current research focuses on the characterization of endogenous exosomes with the endosomal origin, microvesicles blebbing from the plasma membrane have gained increasing attention in health and sickness, which are featured by an abundance of surface molecules recapitulating the membrane signature of parent cells. Here, a reproducible procedure is presented based on differential centrifugation for extracting and characterizing EVs from the plasma and solid tissues, such as the bone. The protocol further describes subsequent profiling of surface antigens and protein cargos of EVs, which are thus traceable for their derivations and identified with components related to potential function. This method will be useful for correlative, functional, and mechanistic analysis of EVs in biological, physiological, and pathological studies.

The present protocol describes a method to extract extracellular vesicles from the peripheral blood and solid tissues with subsequent profiling of surface antigens and protein cargos.

Circulating and tissue-resident extracellular vesicles (EVs) represent promising targets as novel theranostic biomarkers, and they emerge as important ...