106 Chapter 4 In a broader perspective it will be interesting to test co-supplementation with vitamin B3 (nicotinamide or nicotinamide ribose), which was previously found to rescue mitochondrial function in the liver of LPD-treated mice2. A combination of nutritional supplements might synergistically improve mitochondrial and peroxisomal function as well as organ function. Conclusions In conclusion, we found that docosahexaenoic acid prevents the loss of some peroxisomal and mitochondrial proteins in a hepatic organoid model of malnutrition. Our results warrant testing of DHA as a potential dietary supplement in a more physiologically relevant animal model of severe malnutrition. METHODS Isolation of biliary ducts and organoid culture Hepatic organoids lines were established using mouse biliary ducts isolated from the liver of male C57BL/6J mice between 3 and 5 weeks of age (Jackson Laboratory, Bar Harbor, ME, USA) following the previously reported protocol59. Ductal fragments were placed in isolation medium (IM) consisting of Advanced DMEM/F12 (Gibco), supplemented with 10 mM HEPES, 1x GlutaMax, 1% Penicillin-Streptomycin (all Gibco), 1x N2-Supplement (Invitrogen), 1X B27Supplement without vitamin A (Invitrogen), 10 mM Nicotinamide (Sigma Aldrich), 1.25 mM N-AcetylCysteine (Sigma Aldrich), 10% Rspondin-1 condition medium (kindly provided by Calvin J. Kuo), 30% Wnt3a conditioned medium (kindly provided by Hans Clevers), 100 ng/ml Noggin, 50 ng/ml HGF, 100 ng/ml FGF-10, 50 ng/ml EGF (all Peprotech), 10 nM Leu-gastrin (Sigma Aldrich) and 10 µM Y 27632 dihydrochloride (Axon Medchem). Three days after isolation, Wnt3a CM and Noggin were removed from the medium. Y 27632 dihydrochloride was removed 4 days after isolation. Medium without Wnt3a-CM and Noggin was used to keep the organoids in culture and referred to as expansion medium (EM). Medium was changed every 2-3 days and they were passaged every 7-9 days in a split ratio 1:6 – 1:9 on average. In order to differentiate the organoids into more mature hepatocyte-like organoids, after passage they were kept in EM medium for 3 days and then
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