A broad range of technologies have been developed to enable three dimensional (3D) cell culture. Few if any however are adaptable for routine everyday use in a straightforward and cost effective manner. Alvetex(®) is a rigid highly porous polystyrene scaffold designed specifically to enable routine 3D cell culture. The scaffold is engineered into thin membranes that fit into conventional cell culture plasticware. The material is inert and offers a polystyrene substrate familiar to cell biologists worldwide. The 3D geometry of the scaffold provides the environment in which cells grow, differentiate, and function to form close relationships with adjacent cells thus creating the equivalent of a thin tissue layer in vitro. This chapter introduces the features required by a technology that enables routine 3D cell culture. Using Alvetex(®) as a product that satisfies these requirements, its application is demonstrated for the growth of a recognised cell line. Procedures detailing the use of Alvetex(®) for 3D cell culture are provided. This is followed by a series of detailed methods describing ways to analyse such cultures including histological techniques, immunocytochemistry, and scanning electron microscopy. Examples of data generated from these methods are shown in the corresponding figures. Additional notes are also included where further information about certain procedures is required. The use of Alvetex(®) in combination with these methods will enable investigators to routinely produce complex 3D cultures to research the growth, differentiation, and function of cells in new ways.