As the 1990s progressed, other researchers started testing 3D organoid cultures of different origin. Mammary gland is not confined only to acini. To dissect the mechanisms underlying branching morphogenesis of the mammary gland we used small pieces of the gland of virgin mice that we referred to as organoids, within collagen gels. We showed that morphogenesis depends on the interplay of growth factors, morphogens, and matrix metalloproteinases (MMP; Simian et al., 2001). Previously, Montesano and collaborators had described the roles of hepatocyte growth factor and TGF-β on branching morphogenesis of clonally derived mammary epithelial cells in collagen I gels (Soriano et al., 1995, 1996). Both epimorphin (Hirai et al., 1992) and MMPs were required for morphogenesis, but neither was required for proliferation. These results provided the first direct evidence for a crucial role of MMPs in branching in mammary epithelium. A study in 2006 showed that tissue geometry determines the site of branching morphogenesis (Nelson et al., 2006). Using a micropatterning approach to control the initial 3D structure of mouse mammary epithelial tubules, it was determined that tubules dictate the
