Published: August 21st, 2019
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 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.
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'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.
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
2. Animal Preparation
3. Cannulation and Perfusion (0.5 h)
4. Isolation of EVs (5 h)
5. Assessment of Quality and Yield of Isolations
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 °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 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 µL 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 ˚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.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 ˚C. Please click here to view a larger version of this figure.
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 µL of PBS. Please click here to view a larger version of this figure.
Figure 5: Representative result. The size and concentration of liver tissue EVs can be determined by nanoparticle tracking analysis (NTA). Please click here to view a larger version of this figure.
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.
The authors have nothing to disclose.
This study was supported by funding from National Cancer Institute Grant CA-217833.
|125 mL Erlenmeyer flask
|125 mL Erlenmeyer flask
|Curved non-serrated scissors
|Fine Science Tools
|Fine Science Tools
|Home supply store
|Home supply store
|Home supply store
|Absorbent Bench Underpad
|Masterflex L/S Digital Miniflex Pump
|Thermo Electron Precision
|Blood collection sets
|Petri dish, clear lid 100x15
|Falcon 70mm Nylon Cell Strainers
|50 mL conical tubes
|Cotton Tipped Applicators
|25mL Serological Pipet
|Ohmeda Isotec 4 Isoflurane Vaporiser
|Levo Plus Motorized Pipette Filler
|Beckman Coulter Optima L-100 XP
|Beckman Coulter Avanti JXN-26
|70 Ti Fixed-Angle Rotor
|Nalgene Round-bottom tube
|Polycarbonate ultracentrifuge tubes with cap assembly
|HyClone Hank's Balanced Salt Solution (HBSS), Ca/Mg free
|Collagenase, Type IV, powder
|Phosphate Buffered Saline (PBS)
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