A simple and effective method for mature kidney organoid containing a collecting duct using retinoic acid and bone morphogenetic protein 7
During kidney development, an intermediate mesoderm (IM) give rise to distinct structures: the ureteric bud (UB) and the metanephric mesenchyme (MM). These structures differentiate further into the collecting duct and nephron, forming a mature kidney. Generating functional kidney organoids has been challenging due to incomplete development of the UB. Researchers have overcome this limitation by differentiating UB and MM separately and co-culturing them to generate kidney organoids. However, this study developed a co-culture-free method using retinoic acid (RA), plays important role in the anterior IM differentiation and BMP7 secreted by UB in vivo development. This protocol provided not only simplifies the complexity of kidney organoid generation but also advances our understanding of the crucial signaling pathways involved in kidney development. Overall design: Two-dimensional (2D)-cultured IM cells were collected, dissociated using accutase, seeded in a 96-well ultra-low attachment plate (Corning; 1 × 105 cells/well), and gently centrifuged thereafter. The cell aggregates were cultured with 100 ng/mL FGF9, 1 µg/mL heparin, and 5 µM Y27632 (Tocris) in basal differentiation medium for 1 day. After incubation at 37 °C (5% CO2), 5–6 cell aggregates were transferred to a collagen-coated Transwell (#3491, Corning) and treated with 5 µM CHIR99021 in basal differentiation medium for 1 h. The medium was replaced by basal differentiation medium containing 100 ng/mL FGF9 and 100 ng/mL BMP7 (Peprotech), and the cells were cultured for 10–14 days. The medium was replaced daily with care taken so that the cultured medium did not overflow over the membrane.