“Preparation and incubation of precision-cut liver and intestinal slices for application in drug metabolism and toxicity studies.” Nature protocols 5, no. 9 (September 2010): 1540-51.

Materials

• Krumdieck slicer

• Incubator: 37^oC – 80% O₂ – 5% CO₂

• Surgery material (knife, scissor, forceps)

• 12 wells plate

• 1ml safelock tubes

• Liquid N₂

Solutions

• Culture medium: William E + Glutamax + Glucose (1.375gr/500mL William E medium) + Gentamycine (500μL/500mL)

• Krebs solution: for 3L ( 300mL 10x Krebs + 6.30g NaHCO₃ + 13.50g Glucose + 7.14g HEPES + 2.7L cold ultra pure water)

• University of Wisconsin organ preservation solution (UW)

Protocol

1. Assemble the Krumdieck slicer. Turn on the cooler, connect the cooler tube with the slicer. Turn on the flow cabinet. Set the incubator to 37^oC – 80% O₂ – 5% CO₂

2. Prepare Krebs: Carbogenate Krebs for 20 minutes and adjust to pH 7.4 by NaOH 5M

3. Preparation culture medium: 1.3 ml medium (WilliamE+Glucose+Gentamycine) per well.

   Note: Medium must be filled into plates, oxygenated in incubator for 30min-1h before incubate with slice.

4.  For each condition: 3 slices for ATP, 3 slices for RNA and 3 slices for histology.

5. Pick up the liver and store in ice-cold UW-solution.

6. Preparation 5 mm liver cores:

   Rat: Transfer the liver to a Petri dish that has a silicone insert. Keep the surface of the liver wet by pouring UW on it. Turn on the drill at speed setting 2. Secure the tissue by holding it down loosely by hand. Prepare cores by rapidly pressing the hollow rotating tissue coring tool perpendicularly into the tissue until it touches the silicone patch at the bottom of the petri dish. Cores should be cylindrical, with similar diameters at the two ends.

   Mouse: Make cores from the liver with 6mm biopsy punch. Store cores in ice-cold UW.

7. Put the liver core into the core holder of Krumdieck slicer, and start making slices. The thickness of the liver slice is measured by wet weight (3-5 mg).

   Collect slices and put 1 slice/well for incubation. Use the thermal pad to keep the plate warm.

8. Collecting sample (harvesting slices) 

               for ATP (1 slice/1ml SONOP/ tube),

               for RNA (3 slices/ empty safelock tube)

               for histology (3 slices/4%Formalin well)

      ATP and RNA samples must be snap-frozen in liquid Nitrogen.

      3x1mL medium for each experiment condition will be collected into micronic-well.

• Clean the slicer: Disassmble the slicer, put all parts in soap, wash carefully and transfer to water box. Rinse with Demiwater and 70% ethanol and allow them to dry.

Remarks: