A mouse mammary gland organoid protocol to mimic breast morphology in vitro

The protocol is aimed at developing primary mammary gland organoids that have a morphology similar to the one of the in vivo breast, which is organized as a complex network of interconnected branches. The organoids are derived from the mouse mammary gland by mechanical dissociation and enzymatic digestion of the tissue to obtain small mammary tissue fragments that spontaneously organize in sphere-shaped organoids. The sphere-shaped organoids are then transferred in mixed Basement Membrane Extract (BME) and collagen gels, supplemented with growth factors to induce an elongated and branched morphology. By combination of the right ECM stiffness (mixed collagen: Matrigel gels) and growth factor supplementation, we managed to obtain complex organoids, up to 1.2 mm in length and with branches up to the 5th level. This will allow to study how the branching process works in branched organs in our body with reduced use of animal models. Also, mutations can be introduced in the model, which can be used to study tumor initiation and progression.