Inclusion of macromolecular crowders in three-dimensional human gingival fibroblast cultures

Abstract Fibroblast-derived extracellular matrix (ECM) niche regulates cell phenotype and behavior relevant to development, tissue homeostasis, wound healing, and pathological conditions. Improved understanding of cell-ECM interactions requires culture models that mimic the extracellular microenvironment found in vivo. Unlike in vivo, cultured cells reside in aqueous media containing a low concentration of macromolecules. Inclusion of inert macromolecular crowders (MMCs) allows to better mimic in vivo-like conditions in vitro in a phenomenon known as macromolecular crowding (MMC). We present here a standardized protocol to utilize MMCs (Ficoll 70/400) to generate human gingival fibroblast cultures that can be propagated at least for 21 days. The resulting cultures are composed of cells embedded in their own three-dimensional ECM and display gene expression and ECM composition that are distinct from that observed under non-crowded conditions.