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Improvements in the physiological relevance of cell-based assays have been enabled by the development of various interdisciplinary methods. However, due to their complexity, in vivo structures such as basement membranes (BMs), which regulate the phenotype of adherent cells, are still difficult to mimic in vitro. The reconstruction of a physiologically relevant BM is crucially important to develop cell-based assays with the capacity for drug screening and disease modelling. Here, we review the biophysical and biochemical properties of BMs in vivo and their interactions with neighbouring cells. We discuss the current methods used to mimic BM functions in cell-based assays according to the type of targeted applications. In doing so, we examine the advantages and limitations of each method as well as exploring approaches to improve the physiological relevance of engineered or cell-derived BMs in vitro.