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

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Preparation

1. Preparation of solutions and culture media
   1. Prepare all culture media and buffer according to the Table of Materials.
2. Preparation of instruments
   1. Set up a water bath (>10 L) at 42 °C, a peristaltic pump with variable speed, and clamps for holding flasks. Prepare curved scissors and forceps (Figure 1).
   2. On or adjacent to the water bath, set up a baking s....

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

Name	Company	Catalog Number	Comments
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 100x50	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