Three-Dimensional Co-Culture Method for the Study of Indirect Interactions Between Different Cell Types Across a...

Three-dimensional (3D) co-culture is a rapidly evolving technique for researchers looking to accurately study cell-cell interactions using in vitro experiments. The limitations of monolayer cell culture, including limited interactions between the cellular and extracellular environment and disturbed cell morphology, are addressed by simulating the in vivo cellular environment. Using a scaffold to provide structural support and including biologically active extracellular matrix components, 3D cultures display behaviours and morphologies more congruent with tissue. Incorporating multiple cell types into this kind of 3D environment allows for the study of cell-cell interactions inside a biomimetic model system. A wide range of 3D co-culture technologies has emerged, each with its own advantages and challenges. Often these technologies require specialized equipment, a complex setup, or specific technical knowledge. As well, there exist a few standardized methods for studying indirect cell-cell interactions between two cell types separated by a reconstituted basement membrane. Here, we describe a 3D co-culture method that requires only fundamental technical skills and uses more widely applicable materials to successfully recapitulate indirect cell-cell interactions across a basement membrane. A monolayer of cells is covered in a layer of extracellular matrix, in the form of Matrigel, and a second cell type is seeded on top. The resultant co-culture is maintained for five days, at which point cells are analyzed for morphological changes by immunofluorescence or extracted from the co-culture for more detailed genomic, transcriptomic, or proteomic analyses. This protocol is ideal for studying the impact of cell-cell communication on cell behaviour when physical contact is prohibited by a basement membrane. As researchers continue to opt for more in vivo-relevant cell culture methods, a streamlined approach is necessary to avoid high barriers to entry.