108 Chapter 4 middle part of the organoids. For each cell the number of peroxisomes per square micron is calculated from three biological replicates. Electron microscopy Hepatic organoids were incubated in DMEM on ice to dissolve the Matrigel. Organoids were washed in 0.1M sodium cacodylate (103256; Millipore) pH 7.2 and fixed for 75 min with 2% glutaraldehyde (G7661; Sigma-Aldrich) in sodium cacodylate. Organoids were post fixed with 1% osmium tetroxide (19134; Electron Microscopy Sciences) and 1% ferrocyanide in sodium cacodylate for 60 min. Samples were en bloc stained O/N with 0.5% uranyl acetate, dehydrated in series of ethanol and embedded in epon. 80 nm thin sections were collected on carbon evaporated formvar copper grids and analyzed with a CM12 transmission electron microscope (Philips) running at 100 kV. Therapeutic interventions with different PPAR-α agonists Mature hepatic organoids were cultured in complete DM until day 12. The organoids were then placed in amino acid free DM supplemented with different PPAR-alpha agonists at different concentrations. WY-14643 (Axon Medchem) was tested at two different concentrations 100 and 200 µM based on literature reports60–62. Docosahexaenoic acid (DHA) (Sigma Aldrich) was tested at two different concentrations 50 and 100 µM63,64. Linoleic Acid (Sigma Aldrich) was tested at two different concentrations 50 and 150 µM65,66. The vehicles DMSO and fatty acid-free BSA were tested in the same volume as that of the higher concentration of the compounds used. Transfection of Hek293T cells for production of γ-retroviral particles. Hek293T packaging cells were seeded in a 10 cm standard tissue culture dish to reach a confluency of 80%. Transfection of cells was performed using 7.0 μg of Retroviral transfer plasmid pMRX-IP/GFP-LC3-RFP-LC3ΔG (kindly provided by Noboru Mizushima, Department of Biochemistry and Molecular Biology, University of Tokyo, Tokyo, Japan; Addgene plasmid #84572; (http://n2t.net/ addgene:84572; RRID:Addgene_84572)36, 7.5 μg pGag/Pol, 2.5 μg pVSV-G packaging vectors and 1 μg pAdvantage expression enhancer. The plasmid mix was added to PEI (Polysciences Inc. Cat N: 23966-2) transfection reagent prepared in Opti-MEMTM Reduced Serum Medium (Gibco, Cat N: 31985062) at 1:5 ratio (plasmid DNA Mix:PEI). The final mix was added to Hek293T cells dropwise. Media from viral particles producing-cells (10mL) was collected
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