Preparation of Extracellular Matrices and Cell/Matrix Functional Units Produced by Cultured and Primary Fibroblasts

Fibroblast-derived matrices (FDMs) provide a biologically relevant in vitro alternative to conventional two-dimensional (2D) substrates by enabling fibroblasts to assemble and organize their own extracellular matrix (ECM) environment, closely mimicking the physiological stromal microenvironment. This unit describes methods for generating interstitial matrices in which fibroblasts assemble their own multilayered extracellular scaffold, termed a fibroblast/ECM functional unit, that yields a cell-free FDM anchored to the culture surface, following decellularization. The resulting FDM is a polymerized, native-like scaffold that can be repopulated with cells, compressed, or solubilized for biomechanical and biochemical analysis. Unlike synthetic hydrogels or basement membrane extracts such as Matrigel, FDMs preserve native topological and mechanical features of the interstitial ECM. The Basic Protocol covers FDM production from cultured (NIH-3T3) and primary fibroblasts, including poly-D-lysine/gelatin surface preparation, L-ascorbic acid-driven ECM deposition, and detergent-based decellularization. We have 12 Support Protocols (SPs) extending this workflow to cover the following: phenotypic evaluation by immunofluorescence and fiber orientation analysis (SP1); chemical rigidification (SP2); cell attachment (SP3) and morphology (SP4) assays; α-SMA phenotype evaluation (SP5); lysis of fibroblast/ECM units and Western blot validation of FAK phosphorylation and α-SMA levels (SP6); mechanical matrix compression (SP7); matrix solubilization (SP8); primary fibroblast isolation by nonenzymatic (SP9) and enzymatic (SP10) methods; hTERT lentiviral immortalization (SP11); and biochemical fractionation by sequential detergent extraction (SP12). These protocols enable acquisition of physiologically relevant data on cell-matrix interactions, signaling, and matrix composition that differ fundamentally from results obtained using conventional 2D or synthetic substrates. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Preparation of extracellular matrices produced by cultured or primary fibroblasts Support Protocol 1: Phenotypic evaluation of unextracted multilayered fibroblast/ECM units Support Protocol 2: Fixing extracted matrices to limit their viscoelastic properties Support Protocol 3: Cell attachment assay Support Protocol 4: Determination of cell shape Support Protocol 5: Evaluation of α-SMA phenotype - functional analysis of FDMs Support Protocol 6: Lysis of fibroblast/ECM units or FDM-replated fibroblasts for Western blot analyses Support Protocol 7: Mechanical compression of the fibroblast-derived matrix Support Protocol 8: Solubilization of FDM-derived matrix Support Protocol 9: Isolation of primary fibroblasts by a nonenzymatic procedure Support Protocol 10: Isolation of primary fibroblasts by enzymatic tissue digestion Support Protocol 11: Generation of hTERT lentiviruses for primary fibroblast immortalization Support Protocol 12: Biochemical fractionation of multilayered fibroblast/ECM units.