Organotypic culture of untransformed and tumorigenic primary mammary epithelial cells.

Three-dimensional (3D) culture systems represent an important means to study untransformed and neoplastic cells. These cultures can recapitulate organotypic growth by developing a polarized phenotype, forming specialized cell-cell contacts, and attaching to an underlying basement membrane. All of these features are necessary for the proper control of single-cell behavior within a growing structure. By employing 3D cultures, specific aspects of single cells, such as their capacity to proliferate, survive, and differentiate, can be followed in real time. This protocol describes how to generate 3D cultures of primary mammary epithelial cells.