A step-by-step protocol for generating human fibroblast cell-derived completely biological extracellular matrix scaffolds.

Native extracellular matrix (ECM) based scaffolds are far more superior in structural and compositional complexity than other engineered scaffolding materials such as hydrogels, electrospun fibers, and three-dimensional (3D) printed substrates. Due to the presence of native structural proteins and other macromolecules, native ECM can better restore the crucial cell-ECM crosstalk and provide a highly biomimetic microenvironment to cells. Allogenic or xenogeneic tissues have been derived by decellularization to obtain native ECM scaffolds. However, their applicability is limited by batch to batch variation, risk of pathogen transfer, undesirable immune response and scarcity of donors. Human dermal fibroblasts (hDFs) can be prescreened and maintained in a pathogen-free condition. Herein, we have described a step-by-step protocol to generate a completely biological ECM scaffold by decellularization of hDF cell sheets. Decellularization was achieved by using an anionic surfactant sodium dodecyl sulfate (SDS) and ethylene diamine tetraacetate (EDTA). The resulting ECM sheet was organized into a nanofibrous scaffold, containing major ECM structural proteins as well as other macromolecules including collagens, fibronectin, laminin and elastin. This cell-derived nanofibrous ECM is a promising scaffold material for constructing highly biomimetic functional tissues.