Intestinal Organoids in Colitis Research: Focusing on Variability and Cryopreservation

In recent years, stem cell-derived organoids have become a cell culture standard that is widely used for studying various scientific issues that were previously investigated through animal experiments and using common tumor cell lines. After their initial hype, concerns regarding their standardization have been raised. Here, we aim to provide some insights into our experience in standardizing murine colonic epithelial organoids, which we use as a replacement method for research on inflammatory bowel disease. Considering good scientific practice, we examined various factors that might challenge the design and outcome of experiments using these organoids. First, to analyze the impact of antibiotics/antimycotics, we performed kinetic experiments using ZellShield® and measured the gene expression levels of the tight junction markers Ocln, Zo-1, and Cldn4, the proliferation marker Ki67, and the proinflammatory cytokine Tnfα. Because we found no differences between cultivations with and without ZellShield®, we then performed infection experiments using the probiotic Escherichia coli Nissle 1917 as an already established model setup to analyze the impact of technical, interexperimental, and biologic replicates. We demonstrate that interexperimental differences pose the greatest challenge for reproducibility and explain our strategies for addressing these differences. Additionally, we conducted infection experiments using freshly isolated and cryopreserved/thawed organoids and found that cryopreservation influenced the experimental outcome during early passages. Formerly cryopreserved colonoids exhibited a premature appearance and a higher proinflammatory response to bacterial stimulation. Therefore, we recommend analyzing the growth characteristics and reliability of cryopreserved organoids before to their use in experiments together with conducting several independent experiments under standardized conditions. Taken together, our findings demonstrate that organoid culture, if standardized, constitutes a good tool for reducing the need for animal experiments and might further improve our understanding of, for example, the role of epithelial cells in inflammatory bowel disease development

Meteorin-like promotes heart repair through endothelial KIT receptor tyrosine kinase

Effective tissue repair after myocardial infarction entails a vigorous angiogenic response, guided by incompletely defined immune cell-endothelial cell interactions. We identify the monocyte- and macrophage-derived cytokine METRNL (meteorin-like) as a driver of postinfarction angiogenesis and high-affinity ligand for the stem cell factor receptor KIT (KIT receptor tyrosine kinase). METRNL mediated angiogenic effects in cultured human endothelial cells through KIT-dependent signaling pathways. In a mouse model of myocardial infarction, METRNL promoted infarct repair by selectively expanding the KIT-expressing endothelial cell population in the infarct border zone. Metrnl-deficient mice failed to mount this KIT-dependent angiogenic response and developed severe postinfarction heart failure. Our data establish METRNL as a KIT receptor ligand in the context of ischemic tissue repair