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).