Figure 6.

Limitations and troubleshooting of the mammary organoid branching protocol. (A) Overview of common pitfalls during the mammary branching organoid protocol. (B) Example of flattened organoid (3D rendering) on the bottom of the cell culture dish, top view. White arrows indicate 2D sheets of cells; colors indicate depth coding. (C) Confocal image of ductal organoid structures showing 3D growth, side view. (D,E) Confocal images (3D rendering) of a hyperbranched organoid with lack of branch elongation (D) and an elongated organoid after careful removal of all supernatant from the organoid pellet before adding the 7B3C mixture (E). Cdh1-CFP signal in cyan. (F) Brightfield image showing local and uni-directional branching of an organoid with a large core area due to inhomogeneous collagen I distribution within the gel. (G) Brightfield image of a branched and elongated organoid obtained after thorough mixing of the 7B3C matrix prior to plating. Scale bars represent 100 μm.