52 Chapter 2 dysfunction in severe malnutrition. These models are also suitable to explore therapeutic interventions. METHODS Isolation of intestinal crypts and biliary duct fragments and organoid culture The use of crypts from the small intestine and ductal fragments from the liver of male C57BL/6J mice between 3 to 5 weeks of age (Jackson Laboratory, Bar Harbor, ME, USA) was approved by the Central Authority for Scientific Procedures on Animals (CCD) of the Netherlands and from the University of Groningen Ethical Committee for Animal Experiments (Animal Use Protocol Number: 171504-01-001/3). The small intestine was dissected and divided in three sections: from proximal to distal, the duodenum, jejunum and ileum. Jejunum was used to generate organoids according to a previously described protocol1. Intestinal organoids were kept in complete culture medium consisting of AdvDMEM/F12 plus 10mM HEPES, 1X GlutaMax and 1% Penicillin-Streptomycin (all Gibco), 1X N-2 Supplement (Invitrogen, CA, USA), 1X B-27 Supplement (Invitrogen), 1.25 mM N-Acetylcysteine (Sigma Aldrich, MO, USA), 10% Rspondin Conditioned Medium (provided by Calvin J. Kuo), 50 ng/ml EGF (Peprotech, NJ, USA) and 100 ng/mL Noggin (Peprotech). Medium was changed every 2-3 days. Intestinal organoids were passaged every 5-7 days. Ductal fragments from livers were isolated following the protocol by Broutier et al35. Biliary duct fragments were kept in culture expansion medium consisting of Advanced DMEM/F12 supplemented with 10mM HEPES, 1X GlutaMax, 1% Penicillin-Streptomycin, 1X N-2 Supplement, 1X B-27 Supplement, 10mM Nicotinamide (Sigma Aldrich), 1.25mM N-Acetylcysteine, 10% RSpondin Conditioned Medium, 30% Wnt3a CM (provided by Hans Clevers), 100 ng/ml Noggin, 50 ng/ml HGF (Peprotech), 100 ng/ml FGF-10 (Peprotech), 50 ng/ml EGF, 10nM Leu-gastrin (Sigma Aldrich). Three days after the isolation Noggin and Wnt3a CM were removed from the medium. The medium was changed every 2-3 days. Liver organoids were passaged every 6-7 days. Expansion medium promotes the proliferation of liver stem cells as well as cholangiocytes. After three days in expansion medium, organoids were transferred to hepatocytedifferentiation medium which consisted of Advanced DMEM/F12 supplemented with 10mM HEPES, 1X GlutaMax, 1% Penicillin-Streptomycin, 1X N-2 Supplement,
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