Isolation and Culture of Mouse Kupffer Cells

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

1. Perfusion and Cell Collection (Figure 1)

1. Prepare freshly all reagents described in the material table.
2. Warm the EGTA (Ethylene Glycol Tetra-acetic Acid)/HBSS (Hank's Balanced Salt Solution) Solution (50 ml per mouse) and the Collagenase Solution (100 ml per mouse) for 30 min at 40 °C.
3. Rinse the pump flexible tubing first with 70% ethanol. Pour 40 ml of EGTA/HBSS Solution into a centrifuge tube immersed in the water bath and rinse the pump flexible tubing with pre-warmed EGTA/HBSS Solution.
4. Perform terminal anesthesia using a barbiturate to reliably produce unconsciousness before respiratory depression and death. Inject phenobarbitone at 1 mg/kg, i.p. into a female or male CD1 mouse (35-45 g). Confirm the anesthesia by toe pinching.
5. Shave abdominal hairs and sterilize the abdominal surface using a 70% ethanol solution.
6. Cut through the abdominal cavity and expose the portal vein and inferior vena cava by moving the intestine laterally to the left of the abdomen.
7. Start the pump at a speed of 1-3 ml/min with EGTA/HBSS Solution and cannulate the portal vein using a 23 g butterfly needle (wings cut). Clamp the section of the portal vein cannulated with the 23 G needle and then flip the serrefine forceps at the surface of the opened mouse abdomen. The liver should quickly pale within the first 30 sec of EGTA/HBSS Solution perfusion.
8. Rapidly incise the lower part of the inferior vena cava to avoid excess pressure building in the liver, and then increase the flow rate gradually to 7 ml/min, over the first minute of perfusion. The animal dies due to bleeding secondary to vena caval venipuncture.
9. When less than 5 ml of EGTA/HBSS Solution is remaining in the centrifuge tube, fill it up with Collagenase Solution (40-50 ml). Increase the flow rate gradually to 10 ml/min, over 30 sec.
10. Make the liver swell by applying pressure to the inferior vena cava, using tweezers, for 5-10 sec intervals. This can be done periodically (5-10 times during digestion). This step will improve the liver cell dissociation and reduce the perfusion time with collagenase.
11. When less than 10 ml of Collagenase solution remains within the centrifuge tube, pour 40-50 ml of pre-warmed Collagenase solution into the centrifuge tube.
12. After 10-15 min of perfusion and about 70-80 ml of Collagenase Solution perfused, apply a small pressure on the surface of the liver with a forceps. A print of the pressure indicates that liver cells are dissociated.
13. Remove the liver from the abdominal cavity in one piece and place it in a centrifuge tube containing 20-30 ml of Kupffer Cells Isolation Medium. Keep liver cells on ice or at 4 °C for a maximum period of 3 hr to avoid affecting liver cell viability.
14. Repeat the perfusion procedure on several animals if needed, while keeping the perfused liver on ice. Pool one to three livers for purification and proceed with stage 3.

2. Preparation of the Density Gradient for Centrifugation  (Figure 2)

1. Proceed with the following steps (sections 2, 3 & 5) under sterile conditions and keep cells on ice or at 4 °C. Prepare the SIP (Solution of Isotonic coated silica Particles) by mixing 15.3 ml of coated silica particle solution with 1.7 ml of 10 x PBS.
2. To make 20 ml of 25% SIP solution, mix 5 ml of SIP with 15 ml of PBS. To make 20 ml of 50% SIP solution, mix 10 ml of SIP with 10 ml of PBS.
3. Fill one centrifuge tube with 20 ml of the 50% SIP solution. Tilt the centrifuge tube at an angle close to 90° and slowly add the 25% SIP solution (20 ml) on the side wall of the tube using a 25 ml serological pipette, without touching the surface of the 50% SIP layer. When adding the 25% SIP solution, progressively reduce the angle of the tube. Keep the density gradient on ice until use.

3. Kupffer Cell Purification (Figure 3)

1. Put one perfused liver in a Petri dish with 15 ml of Kupffer Cell Isolation Medium. Rupture the Glisson's capsule (membrane of the liver) using scissors and release all liver cells into the Kupffer cell isolation medium. Filter the solution through a 100 µm cell strainer to be collected in a centrifuge tube. Repeat the perfusion procedure on additional perfused livers and pool the cells in the same centrifuge tube (15 ml from one mouse, and up to 45 ml from three mice).
2. Centrifuge the cell suspension at 50 x g for 2 min at 4 °C. Parenchymal cells (hepatocytes) will be in the pellet and non-parenchymal cells (including Kupffer cells) will be in the supernatant.
3. Collect the supernatant in a clean centrifuge tube and centrifuge at 50 x g for 2 min each. Repeat this step three more times.
4. Centrifuge at 1,350 x g for 15 min to pellet non-parenchymal cells. Discard the supernatant and resuspend the pellet in 10 ml of Kupffer Cell Isolation Medium.
5. Add the non-parenchymal cell solution on the discontinuous isotonic gradient 25/50% as described in 2.3. Centrifuge at 850 x g for 15 min without acceleration or break.
6. Localize the enriched Kupffer cell fraction appearing turbid within the 25% SIP fraction close to the 25/50% SIP interface (Figure 3). Using a 10 ml serological pipette, aspirate about 12 ml of the enriched Kupffer cell fraction. Transfer cells into a centrifuge tube containing 35-40 ml of Kupffer Cell Isolation Medium. Mix gently and centrifuge at 1,350 x g for 15 min at 4 °C to pellet cells.
7. Discard the supernatant and resuspend cells in 5-10 ml of pre-warmed Kupffer Cell Culture Medium. Count cells (hemocytometer) and measure the viability using trypan blue staining.
8. Plate the purified non-parenchymal cells in 24-well plates at a density of 5 x 10⁵ cells/well. Incubate the cells at 37 °C and 5% CO₂ (Figure 3). After 30 min, gently remove the medium, wash with pre-warmed Hank's Balanced Salt Solution (HBSS) once then replace it with 500 µl of fresh and pre-warmed Kupffer Cell Culture Medium (per well).