Anisotropic substrate topographies affect floor‐plate patterning of NE organoids. A,B) NE organoids growing on PDMS chip with linear grooves of different dimensions (Chip 5 µm, Chip 10 µm, Chip 20 µm) and control PDMS chip with flat surface. Bright‐field images (Scale bars: 100 µm) and immunofluorescence staining (Scale bars: 50 µm) of F‐actin (phalloidin) and FOXA2 in NE organoids on A) day 14 and B) day 18, showing representative examples of successful floor‐plate patterning in NE organoids. C–E) Quantification of area, lumen‐to‐organoid size ratio, number density of NE organoids and percentage of floor‐plate patterned NE organoids on (C) day 14, (D) day 16, and (E) day 18, n ≥ 3. F–H) Quantification of F‐actin (phalloidin) intensity in NE organoids on (F) day 14, (G) day 16, and (H) day 18, n ≥ 3. Total mean fluorescence intensity (MFI) of F‐actin (phalloidin) were shown as dot plots (left) and the corresponding MFI of each slide along the Z‐stack were indicated as curve graphs (right). Error bars in (C–H) represent S.E.M. P‐values of statistical significance were represented as: * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, using one‐way analysis of variance (ANOVA) followed by Dunnett multiple comparisons test.