RESUMO
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.
RESUMO
Cd14 and Alpk1 both encode pathogen recognition receptors and are known candidate genes for affecting severity in inflammatory bowel diseases. CD14 acts as a coreceptor for bacterial lipopolysaccharide (LPS), while ALPK1 senses ADP-D-glycero-beta-D-manno-heptose, a metabolic intermediate of LPS biosynthesis. Intestinal barrier integrity can be influenced by CD14, whereas to date, the role of ALPK1 in maintaining barrier function remains unknown. We used colon-derived 3D organoids, first characterised for growth, proliferation, stem cell markers, and expression of tight junction (TJ) components using qPCR and immunohistochemistry. They showed characteristic crypt stem cells, apical shedding of dead cells, and TJ formation. Afterwards, organoids of different genotypes (WT, Il10 -/-, Cd14 -/-, and Alpk1 -/-) were then stimulated with either LPS or Escherichia coli Nissle 1917 (EcN). Gene expression and protein levels of cytokines and TJ components were analysed. WT organoids increased expression of Tnfα and tight junction components. Cd14 -/- organoids expressed significantly less Tnfα and Ocln after LPS stimulation than WT organoids but reacted similarly to WT organoids after EcN stimulation. In contrast, compared to WT, Alpk1 -/- organoids showed decreased expression of different TJ and cytokine genes in response to EcN but not LPS. However, Western blotting revealed an effect of ALPK1 on TJ protein levels. These findings demonstrate that Cd14, but not Alpk1, alters the response to LPS stimulation in colonic epithelial cells, whereas Alpk1 is involved in the response upon bacterial challenge.
