In Vivo Murine Liver Perfusion: A Method to Infuse the Rodent Liver with Solution via the Portal Vein

Published: April 30, 2023

Abstract

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.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board. 1. Preparation Preparation of solutions and culture media Prepare all culture media and buffer according to the Table of Materials. Preparation of instruments Set up a water bath (>10 L) at 42 °C, a peristaltic pump with variable speed, and clam…

Representative Results

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 close…

Materials

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 Comments
Collagenase Type IV Sigma Aldrich C5138
Isoflurane  Abcam ab144581

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In Vivo Murine Liver Perfusion: A Method to Infuse the Rodent Liver with Solution via the Portal Vein. J. Vis. Exp. (Pending Publication), e20084, doi: (2023).

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