36 Chapter 2 of invasive sampling (e.g. biopsies) in this vulnerable patient population living in low-resource settings. To overcome these limitations, animal models have been developed and used to start unravelling the role of organelle dysfunction induced by severe malnutrition. Data from low-protein fed rodents supports the existence of a link between loss of peroxisomes, impaired mitochondrial function and the development of hepatic steatosis in severe malnutrition29,30. While a role of peroxisomes and mitochondria is also suspected in malnutritioninduced intestinal dysfunction, this has not yet been described. In vivo work is extremely valuable but does not offer the high-throughput capacity of in vitro models to analyse in-depth organelle dynamics and screen for potential interventions. In cultured cells, amino acid deprivation can lead to peroxisomal degradation29 and altered mitochondrial respiration31,32. However, these twodimensional cell cultures are less suitable for extrapolating to organ function. Organoids have the advantages of cell culture – ease of sampling and control of nutrient concentrations – while maintaining broader functionality of the modelled organ33,34. The aim of this study was to develop and characterize translational organoid models of severe malnutrition of the liver and the intestine. Amino-acid deprivation in organoids compromised both hepatic function and intestinal structure and function in a similar way as found in vivo with a low-protein diet. Functional changes were accompanied by reduced mitochondrial and peroxisomal proteins, which could mostly be restored by the resupplementation of amino acids or pharmacological interventions with rapamycin or fenofibrate. Restoration of protein levels aligned with signs of improved fatty acid oxidation in hepatic organoids and increased tight junction protein claudin-3 in intestinal organoids. We conclude that organoid models are suitable to elucidate pathophysiological processes involved in severe malnutrition and to test novel therapeutic interventions. RESULTS Hepatic and intestinal organoid lines were established from mouse biliary duct fragments and intestinal crypts, respectively. To mimic the effect of a low-protein diet, the organoids were cultured in a medium without any amino acids. Nevertheless, most amino acids were still detected at low levels in the medium, presumably originating from Matrigel degradation as well as from growth factors. (Supplementary Table 1).
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