In order to successfully replicate native cell and tissue function in the laboratory, the artificial growth environment needs to be physiologically relevant. Traditionally, cells have been grown on plastic substrates such as a Petri dish that often forces cells to flatten out into a two-dimensional (2D) morphology. This approach is highly unrealistic and fails to replicate the complex 3D architecture and behaviour found in vivo. This chapter therefore describes some of the materials now available that offer cells a 3D interface for growth in vitro. A brief review of the advantages and disadvantages of each technology is discussed, along with some of the current methodologies available to ‘talk’ and ‘listen’ to cells in this new 3D environment. These methodologies are exemplified in this chapter with a practical case study using the Alvetex®Scaffold. Finally, a mention of the future developments of 3D substrates is also included, such as those 3D models that include various mechanical and biochemical properties representative of the native environment.