hPSC differentiation and generation of kidney micro-organoids: Initially, hPSC were differentiated into IM using a modified Takasato's protocol (Takasato et al., 2016, 2015). Briefly, hPSC were dissociated into single cells using TrypLE Select and seeded on to Matrigel-coated plates at a density of 15,000 cells/cm2 using MEF conditional media or E8 media with Revitacell (Thermo Fisher Scientific), and left to adhere overnight at 37°C in a standard cell culture incubator. Differentiation into kidney organoids involves two steps. Step 1: Primitive streak was induced by treating 2D monolayer cultures of hPSCs with 7 µM CHIR99021 (Tocris Bioscience) for 4 days in TeSR-E6 media (Stemcell Technologies). PS cells were driven into IM from day 5 to day 7 by treating with 200 ng/ml FGF9, 1 µg/ml heparin and 1 µM CHIR99021. This resulted in the induction of a mixture of IM cells. Step 2: To form micro-organoids, IM cells were washed with 1 ml of 0.1 M PBS on day 7 and then cells were dissociated using either 1.5 ml of EDTA or TrypLE Select for 3 min at 37°C. Dissociated cells were washed with plain DMEM and centrifuged at 0.4 g. The cell pellet was resuspended in 2 ml of Stage 1 media [base media containing 200 ng/ml FGF9, 1 µg/ml heparin, 1 µM CHIR99021, 0.1% PVA, 0.1% MC, 10 µM Rho kinase inhibitor (Tocris Bioscience)]. The cell suspension was transferred to 6 cm2 low adhesion plates (Greiner Bio-One). Organoids spontaneously formed after placing the culture dishes on an orbital shaker (Ratek) rotating at 60 rpm in a standard cell culture incubator at 37°C and 5% CO2. After 24 h. Stage 1 media was replaced with Stage 2 media (base media containing 200 ng/ml FGF9, 1 µg/ml heparin, 1 µM CHIR99021, 0.1% PVA, 0.1% MC) for another 4 days. From day 7+5 onwards all organoids were refreshed with Stage 3 media (base media containing 0.1% PVA, 0.1% MC) and continued until day 7+18 or the desired end point by refreshing on alternative days. Full paper
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