Isolation of primary hepatocytes from mouse liver

From LipidomicsWiki

Solutions

Krebs-Henseleit buffer 1 (KHB1): NaCl 115 mM; NaHCO3 25 mM; KCl 5.9 mM; MgCl2 1.18 mM; NaH2PO4 1.23 mM; Na2SO4 1,2 mM; CaCl2 1.25 mM, glucose 6 mM.

Krebs-Henseleit buffer 2 (KHB2): NaCl 115 mM; NaHCO3 25 mM; KCl 5.9 mM; MgCl2 1.18 mM; NaH2PO4 1.23 mM; glucose 6 mM (= KHB1 without CaCl2 and Na2SO4).

Collagenase solution: 20 mg of collagenase type CLS II (200U/mg, Worthington) in 100 ml KHB2 having 3% bovine serum albumin ( fraction V, Sigma-Aldrich) and 100 µM CaCl2 .

DMEM (Dulbecco's Modified Eagle Medium), ice cold
Iodixanol (40 %, Axis Shield), ice cold

KHB1, KHB2 and collagenase solution each are filter-sterilized. KHB1 and KHB2 are placed into CO2 incubator for saturation with CO2.

Mouse perfusion

Before perfusion KHB2 and collagenase solution are brought to 37 °C, tubings are washed with ethanol and flushed with KHB2.

1. The mouse is anesthized subcutaneously with Narkodorm (60 μl/100g weight, CP-Pharma) and perfused through the Vena porta hepatis with KHB2 and then with collagenase solution at 37°C for 10 min each.

2. Liver is carefully removed and transferred into a Petri dish, filled with 4-5 ml collagenase solution. In this solution the liver is cut into small pieces, pressed through a household sieve and flushed with ice-cold KHB1.

3. The cell suspension obtained is filtrated through a cell strainer (70 µm nylon, BD Falcon) into a 50 ml Greiner-tube.

4. To filtrated cell suspension approximately 10 ml ice cold DMEM is added and subsequently centrifuged at 50 g (~ 500 rpm, Beckman CS-6R rotor) for 2 min at 4 °C.

5. Supernatant is discarded and hepatocyte pellet is suspended in approximately 10 ml ice
cold DMEM.

6. Hepatocytes from 3 mice are collected by above mentioned protocol and pooled.

7. Pooled cell suspension is mixed with 40 % Iodixanol solution to final iodixanol concentration of 17 % and 2 ml ice cold KHB1 are overlayed.

8. Centrifugation is carried out at 400g (~ 1400 rpm, Beckman CS-6R rotor) for 20 min at 4 °C. Allow the rotor to decelerate freely (no brake) for protection of gradient.

9. Discard supernatant and retrieve hepatocyte cell pellet.