57 2 Organoids as a model to study intestinal and liver dysfunction in severe malnutrition Branched chain fatty acid and very long chain fatty acid measurements Mature hepatic organoids were incubated with 25µM phytol (Sigma Aldrich) for 48 hours prior to quenching in ice-cold methanol. Methanol was evaporated at 37°C under a steady stream of N2 for 40 minutes. Next, the pellet was reconstituted in PBS. Samples were hydrolysed in two steps. First, an acid hydrolysis step was performed to release the fatty acids from the different moieties they are bound to without affecting phytanic acid . This was followed by a basic hydrolysis to convert esterified fatty acids into free fatty acids. Pentafluorobenzyl bromide (PFB-Br) was added for the derivatization. The PFB derivatives were analysed on the GC/MS (Agilent 7890/5975 inert XL GCMS System, Agilent Technologies, Amstelveen, The Netherlands) in negative chemical ionization mode (NCI). Results were normalized for protein content. Acylcarnitines measurements Mature hepatic organoids were incubated with 1mM L-carnitine (Sigma Aldrich) for 24 hours prior to collection in TBS. Intracellular acylcarnitines were measured following the previously published protocol70 Imaging of the liver and intestinal organoids and processing of the images Organoid cultures were followed over time with an AxioObserver Z1 compound microscope (Carl Zeiss), 2.5x and 5x objectives and an AxioCam MRm3 CCD camera (Carl Zeiss). Raw images were processed using the ZEN 3.1 blue edition software. The number and size of organoids were assessed in the whole well. A set of 25 organoids were manually counted in each well, tracked over time and measured throughout the different time points. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. FUNDING This work was supported by the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No
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