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.
125 mL Erlenmeyer flask | Fisher scientific | FB500125 | |
125 mL Erlenmeyer flask | Fisher scientific | FB500125 | |
Curved non-serrated scissors | Fine Science Tools | 14069-12 | |
Curved forceps | Fine Science Tools | 13009-12 | |
Masking tape | Home supply store | ||
Aluminum foil | Home supply store | ||
Styrofoam pad | Home supply store | ||
Absorbent Bench Underpad | Scientific inc. | B1623 | |
Masterflex L/S Digital Miniflex Pump | Cole-parmer | ZX-07525-20 | |
Water bath | Thermo Electron Precision | 2837 | |
Blood collection sets | Becton Dickinson | 367292 | |
Petri dish, clear lid 100×15 | Fisher Scientific | FB0875712 | |
Falcon 70mm Nylon Cell Strainers | Fisher scientific | 352350 | |
50 mL conical tubes | Fisher Scientific | 12-565-270 | |
Cotton Tipped Applicators | Moore medical | 69622 | |
25mL Serological Pipet | Falcon | 357525 | |
Ohmeda Isotec 4 Isoflurane Vaporiser | BioSurplus | 203-2751 | |
O2 gas | |||
Isoflurane | |||
Levo Plus Motorized Pipette Filler | Scilogex | 74020002 | |
Centrifuge 5804R | Sigma-aldrich | 22628048 | |
Beckman Coulter Optima L-100 XP | Beckman | 969347 | |
Beckman Coulter Avanti JXN-26 | Beckman | B34182 | |
70 Ti Fixed-Angle Rotor | Beckman | 337922 | |
JA-25.50Fixed-Angle Rotor | Beckman | 363055 | |
Nalgene Round-bottom tube | Thermo Scientific | 3118-0028 | |
Polycarbonate ultracentrifuge tubes with cap assembly | Beckman | 355618 | |
Reagents | |||
HyClone Hank's Balanced Salt Solution (HBSS), Ca/Mg free | Fisher scientific | SH30588.01 | |
Collagenase, Type IV, powder | Fisher scientific | 17104019 | |
Phosphate Buffered Saline (PBS) | Fisher scientific | SH30256.01 |
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) 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) 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.