Source: Cabral, F., et al. Purification of Hepatocytes and Sinusoidal Endothelial Cells from Mouse Liver Perfusion. J. Vis. Exp. (2018).
This video describes a procedure for mouse liver perfusion through the portal vein using collagenase solution prior to isolating liver cells.
All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
1. Preparation
2. Animal Procedure
3. Liver Perfusion
Figure 1: Schematic representation of the perfusion suite. The flasks are held in place by clamps (not shown); the tubing containing fluid is swapped from the 1L flask to the 125 mL flask during the procedure is represented by the red dotted line. Note, that the oxygen feed does not bubble directly into the solutions in the flasks. The corks should also be notched to allow a closed-circuit fluid flow with the tubing inserted within the notch. This is critical during warm-up of the tubing and for ejection of all air within the tubing. The air bleed is composed of the T-connector which is connected to Tygon tubing and pinch clamps for quick open and closure of the system.
Figure 2: Image of the vasculature of the mouse abdomen. Insertion of the catheter should be just below the gastric and pancreatic vein junctions coming off the portal vein (green dot) and the tip should be placed near the left and right hepatic portal veins (yellow dot) which forms a fork into the main lobes of the liver. Once correct placement is achieved, the catheter should be stabilized with thread using a simple overhand knot. K = kidney, L = liver, SI = small intestine.
Figure 3: Catheter placement in the portal vein. (A) The forceps are used to ensure blood engorgement of the portal vein and to straighten out the vein for catheter placement. (B) The catheter is lined up in parallel with the portal vein with the bevel up. (C) The bevel of the needle within the catheter is inserted into the vein, not through the vein. (D) The needle of the catheter is retracted, and blood will backflow through the catheter as indicated by the tip of the forceps.
Figure 4: Proper liver perfusion. (A) After catheter placement, the female Luer end of the catheter is connecting to the male Luer end (cut from a 1 mL syringe) of the pump tubing. (B) After cutting one of the major descending blood vessels, blood and PBS are drained from the abdomen by slicing the side of mouse with scissors. (C) The liver should blanch while the blood is flushed out and (D) will swell when under pressure by squeezing shut the cut blood vessel with forceps.
1 L Erlenmeyer flask | Fisher Scientific | S63274 | |
250 mL Erlenmeyer flask | Fisher Scientific | S63271 | |
Silicone tubing | Cole-Parmer | 96400-14 | This tubing runs from the flasks through the pump to the T connector and then to the 1.0 mL syringe that is connected to the catheter. |
Tygon tubing | Fisher Scientific | R3603 | Used as an adaptor between 96400-14 and pipettes and T connector. This may also be used for the oxygen tubing. |
T-connectors | Cole-Parmer | EW-06294-82 | |
Quick dissconnects | Fisher Scientific | 6150-0010 | |
Pinch Clamps | Fisher Scientific | 6165-0002 | |
Masterflex L/S Variable speed Pump; model 7553-70 | Cole-Parmer | EW-07559-00 | Periplastic pump with variable speed |
Pump head; model 7014-20 | Cole-Parmer | EW-07014-20 | |
Glass graduated 1.0 mL pipettes | Fisher Scientific | 13-678 | |
Curved non-serrated scissors | Fine Science Tools | 14069-12 | |
Dumont forceps | Fine Science Tools | 11252-20 | |
Curved forceps | Fine Science Tools | 13009-12 | |
10 mL syringe | Fisher Scientific | 03-377-23 | Only barrel of syringe will be needed |
Sterlized spoon | Home supply store | ||
Cotton ball(s) | Home supply store | ||
Polyester sewing thread | Home supply store | ||
Masking tape | Home supply store | ||
Thumb tacks | Home supply store | ||
Styrofoam pad | 50 mL conical rack | ||
Cookie/baking sheet | Home supply store | ||
Absorbant underpads | Fisher Scientific | 14-206-64 | |
19 L water bath | Fisher Scientific | TSCOL19 | |
BD Insyte Autogaurd Shielded IV Catheter 24 guage | Becton Dickinson | 381412 | Plastic cathetar with retractable needle |
Crystallizing dishes 100×50 | VWR | 89000-290 | |
Polystyrene petri dishes | Sigma Aldrich | P5481-500EA | |
50 mL conical tubes | Fisher Scientific | 12-565-270 | |
Graduated pipettes (5 mL) | Fisher Scientific | 170355 | |
Graduated pipettes (25 mL) | Fisher Scientific | 170357 | |
EasyStrainer 100 µM | Greiner bio-one | 542000 | 100 µm filter |
EasyStrainer 40 µM | Greiner bio-one | 542040 | 40 µm filter |
REAGENTS | |||
Name | Compound | Grams (g/L) | Millimolar (mM) |
Buffer 1; pH 7.4 | NaCl | 8.3 | 142 |
KCl | 0.5 | 6.7 | |
HEPES | 2.4 | 10 | |
BSA | 15 | 0.226 | |
Buffer 2; pH 7.4 | NaCl | 3.9 | 66.74 |
KCl | 0.5 | 6.71 | |
CaCl2 | 0.7 | 6.31 | |
HEPES | 24 | 100 | |
BSA | 15 | 0.226 | |
Phenol Red | 0.01 | 0.03 | |
PBS; pH 7.4 | NaCl | 8 | 137 |
KCl | 0.2 | 2.7 | |
Na2HPO4-7H2O | 1.15 | 4.3 | |
KH2PO4 | 0.2 | 1.4 | |
OTHER REAGENTS | |||
Name | Company | Catalog Number | コメント |
Collagenase Type IV | Sigma Aldrich | C5138 | |
Isoflurane | Abcam | ab144581 |