The use of adult-stem-cell-derived organoids makes the protocols ideally suited for applications where faithful recapitulation of wild-type epithelial cell types is of importance and where other microenvironmental influences need to be excluded. They are suitable for the investigation of single microbial species effects but could also be amended for combinations of different microorganisms. ![]() The protocols outlined below have broad applications in the study of microbe–epithelium interactions in cancer, infectious diseases and homeostasis. ![]() Perform read-outs in analogy to 3D coculture. For MOI calculations, a representative well can be harvested and cells counted as described above. Upon reaching confluence, add microbes to the apical or basal side in a medium suitable for exposure. Seed 100,000 cells in 100 µL per well of a tissue-culture-treated 96-well plate or a Transwell insert of 6.5 mm diameter.Īfter 3 d or when the layer is close to confluence, switch to any differentiation medium if needed. Resuspend in expansion medium with 10 µm RhoKi at a density of 1,000,000 cells per 1 mL. Spin for 5 min at 300 g, and discard supernatant.ĭissociate the organoids to single cells as described previously 16 (basic protocol 5). Harvest organoids from the culture in 1 mL of cold DMEM, and transfer to a 15 mL Falcon tube. Transwell inserts grant apical and basal access to the cell layer, whereas standard cell culture plates allow for the greatest scalability.Īfter the coating process, remove the supernatant and allow the BME coating to dry in the incubator for another 15–30 min. Below, we provide a protocol to grow and differentiate organoids in monolayers for cocultures.Ĭoat the bottom of a 96-well plate or a 24-well plate Transwell insert with 5% (vol/vol) BME in DMEM for 30 min at 37 ☌. Injection of intestinal organoids allows coculture under the most physiological conditions, but standardization and scalability of this protocol is difficult to achieve. We also describe deviations from this coculture protocol that can be performed without the need for microinjections, such as viral exposure 8, 15 or 2D cocultures in Transwell plates (Box 1). Based on the development of microinjection procedures for various organoid systems and pathogens in our group, we here provide a comprehensive protocol for the preparation and microbe coculture of human intestinal organoids (Fig. ![]() This necessitates microinjection of gut microbiota into the lumen of organoids for a faithful representation of epithelium–microbe orientation. The polarization of human small intestinal and colonic organoids results in a cystic shape with the inside corresponding to the gut lumen. The protocols for microbe–epithelium cocultures presented here are based on human adult-stem-cell-derived organoids, which have been described previously 14. While these protocols mostly entail bacterial cocultures, dedicated protocols for viral 11 and parasitic 12, 13 organoid exposure have recently been published and can be directly coupled to the methods described here. Furthermore, we present protocols for 3D and 2D apical and basal exposure to microbes for medium-throughput studies and for pathogens with a nonapical infection route. In this protocol, we lay out a broadly applicable procedure for the injection of microbes into the organoid lumen, as employed in recent studies 5, 10. This underlines the importance of sophisticated in vitro model systems to gain mechanistic insights into microbial effects on the epithelium and enable the development of therapeutics targeted at microbial–epithelial interaction processes. ![]() Nevertheless, many links of microbes to diseases remain correlative owing to the difficulty of modeling host–microbe relationships in a reductionist yet meaningful way. The important role of the intestinal microbiota in healthy organ function and diseased states is increasingly recognized. 8, 9) and genotoxic Escherichia coli 10 infections. Amongst others 2, 3, 4, adult-stem-cell-derived organoids have been harnessed to study Cryptosporidium parvum 5, Helicobacter pylori 6, norovirus 7, severe acute respiratory syndrome coronavirus 2 (refs. In recent years, these near-physiological models have led to substantial advances in stem cell biology, disease modeling and the study of host–microbe interactions. 1), adult-stem-cell-derived organoids have vastly expanded the possibilities to grow healthy epithelial cell types. Since they were first reported in 2009 (ref.
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