53 2 Organoids as a model to study intestinal and liver dysfunction in severe malnutrition 1X B-27 Supplement, 1 mM N-Acetylcysteine, 100 ng/ml FGF-10, 50 ng/ml EGF, 10nM Leu-gastrin, 50nM A-83-01 (Tocris, Bristol, UK) and 10uM DAPT (Sigma Aldrich). After day 13, 3μM dexamethasone (Sigma Aldrich) was added into the medium. Organoids were kept in differentiation medium until day 16. Malnutrition in organoids using amino acid starvation and amino acid re-supplementation studies To mimic malnutrition in organoids, a custom-made amino acid free Advanced DMEM/F12 was used (Invitrogen). Apart from this amino acid free DMEM, the formulation of the culture media was identical as described above for the respective organs. Intestinal organoids were grown in complete culture medium for three days after passage before being placed in low amino acid culture medium for 48 hours. For the amino acid re-supplementation studies, organoids were placed in complete medium for further 48 hours after the starvation. All organoids conditions were collected at the end of day 7. Liver progenitor organoids were grown in complete expansion medium for 3 days after passage before being placed in differentiation medium until day 12. From day 12 to day 14 they were grown in amino acid-free medium. At the end of day 14 the organoids were placed back in complete differentiation medium until the end of day 16. For simple amino acid starvation, the organoids were starved from day 14 to day 16. All the organoids conditions were collected at the end of day 16. Therapeutic interventions Intestinal organoids were grown in complete culture medium until three days after the passage, then placed in amino acid-free medium for 48 hours that was supplemented with rapamycin (Sigma Aldrich, MO, USA) at a final concentration of 2nM. Hepatic organoids were grown in complete differentiation medium until day 14. From day 14 to day 16 they were grown in amino acid-free medium supplemented with fenofibrate (Sigma Aldrich) at a final concentration of either 10 or 100μM, as indicated.
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