In vitro cell culture is a vital research tool for cell biology, pharmacology, toxicology, protein production, systems biology and drug discovery. Traditional culturing methods on plastic surfaces do not accurately represent the in vivo environment, and a paradigm shift from two-dimensional to three-dimensional (3-D) experimental techniques is underway. To enable this change, a variety of natural, synthetic and semi-synthetic extracellular matrix (ECM) equivalents have been developed to provide an appropriate cellular microenvironment. We describe herein an investigation of the properties of four commercially available ECM equivalents on the growth and proliferation of primary human tracheal scar fibroblast behavior, both in 3-D and pseudo-3-D conditions. We also compare subcutaneous tissue growth of 3-D encapsulated fibroblasts in vivo in two of these materials, Matrigel and Extracel. The latter shows increased cell proliferation and remodeling of the ECM equivalent. The results provide researchers with a rational basis for selection of a given ECM equivalent based on its biological performance in vitro and in vivo, as well as the practicality of the experimental protocols. Biomaterials that use a customizable glycosaminoglycan-based hydrogel appear to offer the most convenient and flexible system for conducting in vitro research that accurately translates to in vivo physiology needed for tissue engineering.