Hepatocyte and Stellate cell isolation from mouse liver

From LipidomicsWiki

Hepatocyte and Stellate cell Isolation (SOP)

Procedure according to P.O. Seglen, Meth. Cell Biol. 13 (1976) 29-83 adapted by L. Riccalton-Banks et al. Mol. Cell. Biochemistry 248 (2003), 97-102

1. Preliminaries

• Prepare perfusion set-up (Masterflex pump controller)

• Prepare Krebs-Henseleit, perfusion buffer, collagenase buffer and incubation medium
i. Measure 25 ml 10x concentrated Krebs-Henseleit and add to a 250 ml graduated cylinder containing 0,525 g NaHCO3. Make up to 250 ml with dist. Water. Bubble with carbogen for 15 min (check if the pH is 7.35 and 7.45).
ii. Add 150 ml of this buffer to a 250 ml Erlenmeyer flask containing 0.54 g glucose. Place in water-bath at 37 oC. Keep gassing with carbogen until used.
iii. Add 100 ml of this buffer to a 200 ml Erlenmeyer containing 0.36 g glucose and 1 gram fatty acid free bovine serum albumin (BSA).
iv. Prepare medium for the hepatocyte and stellate cell incubations (DMEM)
v. Take from freezer (-200C):
1. one vial concentrated calcium-free perfusion buffer; add the thawed content to a 250 ml graduated cylinder containing 0.45 g glucose. Make up to 125 ml with dist. water. Put in a water-bath of 37 oC.
2. one vial concentrated collagenase buffer; add the thawed content to a 50 ml graduated cylinder. Containing 0.18 g glucose. Make up to 50 ml with dist. water. Place in water-bath at 37 oC.

• Take from the refrigerator the bottle with heparinized physiological saline (1 ml of heparine of 5000 U per 500 ml 0.9% NaCl) and place in water-bath at 37 oC.

2. Actual cell isolation

• Wear gloves and rinse them with 70% ethanol
• Anaesthetize a mouse (male minimum 15 week of age BUT preferably 20 weeks) with Isoflurane using a vaporizer system.
• Rinse the tubing with perfusion buffer and remove any possible air bubbles
• Fix the animal upside down on a cork-covered board that is placed in a stainless steel tray.
• Sterilize the belly using 70% ethanol
• Cut the skin on both sides from the tail onward all the way to the armpits of the front legs.
• Cut along the same lines the abdominal muscle layers up to the diaphragm.
• Pipet some warm heparinized saline on the exposed intestines.
• Expose the portal vein by placing the intestines on the right hand-side outside the body. Drench again some heparinized saline.
• Insert two ligatures loosely tied with a single knot around the portal. The first one close to the liver, the second one as far away from the liver as possible.
• Insert a flattened very small tooth pick under the portal in the opening of the second ligature or something else which gives you the option to lift the portal vein.
• Carefully free the portal vein from adhering fat and connective tissue (optional).
• Perfuse the liver through the portal vein:
- Make a slit (perpendicular) in the ventral aspect of the portal 0.2 cm from the match, using a very small surgical scissor.
- Insert in the slit a canula with perfusion fluid slowly pumped (dripping) through it (make sure that no air bubbles are present in the tubing). Once the canula is in immediately severe both the aorta and the vena cava inferior by making a deep cut in the caudal muscles all the way to the backbone (avoid blowing up the liver!).
- Secure the canula by tying the two ligatures as tight as possible.
- Open the chest. Cut eosophagus and aorta.
• Perfuse the liver for 10 minutes with the calcium-free buffer at a flow rate of 10 ml/min.
• Stop the pump and switch to the bottle containing the collagenase solution. Perfuse the liver for 10 min at a flow-rate of 5 ml/minute. Carefully excise the liver and allow some drops of collagenase buffer (the more the better) to ooze from the liver.
• Place the liver in an evaporating basin and add about 75 ml warm Krebs-Henseleit + glucose (from the 250ml Erlenmeyer flask with the 200 ml). Peel the liver capsule off on one side of all lobes using forceps with very fine tips. Gently combing (fine dog comb) and shaking the liver vigorously using the canula as handle. Comb some more and shake again. Repeat till only connective tissue is left. This crude cell suspension is used to collect hepatocytes and stellate cells (3).

3. Procedure for Hepatocyte and Stellate cell purification

• Pass the crude cell suspension over a sieve (250 μm) into a 250 ml beaker. Transfer the suspension to a 500 ml Erlenmeyer flask. Incubate this flask at 370C in a shaking water-bath (adjust shaking rate such that the cells don’t settle or that foam formation is avoided; some where around 30 – 60 oscillations/min).
• After 15 min, pass the cell suspension over a double layer of sieves (top screen 250 μm, bottom screen 100 μm) into a 250 ml beaker. Divide the suspension over 4 plastic centrifuge tubes and spin 2 min at 50 x g
• Collect the supernatant. This contains the stellate cells which will be further processed as shown in the flow diagram below. The cell pellets are resuspended thoroughly in a few ml of warm Krebs-Henseleit + glucose/BSA. Combine 4 tubes in two. Make up with the same buffer to halfway the tubes and spin again exactly 2 min at 50 x g as before.
• Collect the supernatant (combine with previous collected supernatant for stellate cell isolation!!) and resuspend the cell pellets thoroughly in a few ml of warm Krebs-Henseleit + glucose/BSA. Combine 2 tubes in one. Make up with the same buffer to halfway the tube and spin again (using a balancing tube filled with water). This time 3 min at 120 x g.
• Aspirate carefully the supernatant. Determine the wet weight of the cell pellet. Resuspend the pellet thoroughly in a few ml of warm incubation medium and make up to 10x the wet weight with incubation medium (e.g. wet weight 5.5 g: final volume 55 ml). This will give a cellular protein concentration of approximately 12 mg/ml.
• Pass the final cell suspension over a sieve (60 μm) into a 250 ml beaker. Hepatocytes ready to plate or to be used in suspension cultures.

Stellate cell isolation from the 50 x g spins:

• The combined supernatants from the 50 x g spins are processed as shown in the flow diagram:

IMAGE MISSING

Stock Solutions

Krebs-Henseleit buffer (10x concentrated)

NaCl 34.48 g
KCl 1.76 g
KH2PO4 0.81 g
MgSO4 1.48 g
CaCl2.2 aq. 1.84 g or CaCl2 anh. 1.39 g

Make up to 500 ml with dist. water, keep stored in refrigerator

Before use take 25 ml plus 0.525 g NaHCO3; make up to 250 ml.
Gas 15 min with carbogen.
Subsequently, adjust pH to 7.35-7.45 at room temperature.

Perfusion buffer (25x concentrated)

NaCl 20.75 g
KCl 1.25 g
Hepes 6 g

Dissolve in about 75 ml dist. water, adjust pH to 7.6 at 200C or 7.4 at 37 oC using 1 M NaOH, make up to 100 ml).
Divide in portions of 5 ml and store at –200C

Before use take 1 portion and make up to 125 ml
Include 0.45 g glucose (= 20 mM)

Collagenase buffer (10x concentrated)

NaCl 2.0 g
KCl 0.25 g
Hepes 12.0 g
CaCl2.2 aq. 0.35 g or CaCl2 anh. 0.265 g

Dissolve in about 40 ml water, adjust pH to 7.8 at 200C or 7.6 at 37 oC using 10 M NaOH, make up to 50 ml
Add 0.2 g type I collagenase from Sigma (this is the only brand and the only quality suitable for the purpose!!!), dissolve. Centrifuge 30 min at 3,000 x g.
Divide in portions of 5 ml and store at –200C

Before use take 1 portion and make up to 50 ml.
Include 0.18 g glucose (= 20 mM)

N.B. Use A.R. quality throughout preparation of reagents!