Slicing and incubation rat intestinal slices

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: 37oC – 80% O2 – 5% CO2

  • Surgery material (knife, scissor, forceps)

  • 24 wells plate

  • 1ml safelock tubes

  • Liquid N2

Solutions

  • Culture medium: William E + Glutamax + Glucose (1.375gr/500mL William E medium) + Gentamycine (500μL/500mL) + Fungizon (500μL/50mL)

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

  • 3% Low melting agarose (in 0.9% NaCl)

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 cabinet to 37oC – 80% O2 – 5% CO2

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

  3. Prepare medium: 0.5mL medium (WilliamE+Glucose+Gentamycine+Fungizon) per well.

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

  1. For each experiment condition: 3 slices for ATP, 6 slices for RNA, and 3 slices for histology

  2. Pick up the intestine, store in ice-cold Krebs solution

  3. Preparing Intestinal core:

  • Choose the part of intestine: duodenum first ?cm, jejunum is next ?cm, following is ileum till the ceacum, after the ceacum is the colon.

  • Cut the part into 5-6 pieces, each 2-3cm long. Use hockey stick gently press to push out the feces, flush with Krebs by Pasteur pipet several times to clean the inside.

  • Use surgical thread to close one end of the piece. Pump agarose 3% into the piece (agarose must be at 37oC-38oC). Quickly close by the forceps and put into cold Krebs solution. Hold on for a moment.

  • Take the core out and cut it in half. Embed the core into agarose in the core holder. Wait few minutes for agarose to solidify.

  1. Slicing

  • Fill the Krumdieck slicer with Krebs

  • Put the intestinal core into the holder place of Krumdieck slicer

  • Turn on, and start slicing, adjust the thickness of the slice

  • The thickness of the intestine slice is measured by weight (without agarose, wet mass is around 1-2mg~400um thickness)

  1. Put 1 slice/well for incubation. Using thermal pad to keep the plate warm during handling.

  2. Plate with slices is incubated in incubator while shaking at 90rpm.

  3. Collecting sample (harvesting slice)

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

               for RNA (6 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.

  1. 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:

 Always keep tissue material cold and wet