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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\u2010free FDM anchored to the culture surface, following decellularization. The resulting FDM is a polymerized, native\u2010like 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.\n                  <\/jats:p>\n                  <jats:p>The Basic Protocol covers FDM production from cultured (NIH\u20103T3) and primary fibroblasts, including poly\u2010D\u2010lysine\/gelatin surface preparation, L\u2010ascorbic acid\u2013driven ECM deposition, and detergent\u2010based 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; \u03b1\u2010SMA phenotype evaluation (SP5); lysis of fibroblast\/ECM units and Western blot validation of FAK phosphorylation and \u03b1\u2010SMA 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).<\/jats:p>\n                  <jats:p>\n                    These protocols enable acquisition of physiologically relevant data on cell\u2013matrix interactions, signaling, and matrix composition that differ fundamentally from results obtained using conventional 2D or synthetic substrates. \u00a9 2026 The Author(s).\n                    <jats:italic>Current Protocols<\/jats:italic>\n                    published by Wiley Periodicals LLC.\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Basic Protocol<\/jats:bold>\n                    : Preparation of extracellular matrices produced by cultured or primary fibroblasts\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Support Protocol 1<\/jats:bold>\n                    : Phenotypic evaluation of unextracted multilayered fibroblast\/ECM units\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Support Protocol 2<\/jats:bold>\n                    : Fixing extracted matrices to limit their viscoelastic properties\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Support Protocol 3<\/jats:bold>\n                    : Cell attachment assay\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Support Protocol 4<\/jats:bold>\n                    : Determination of cell shape\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Support Protocol 5<\/jats:bold>\n                    : Evaluation of \u03b1\u2010SMA phenotype \u2010 functional analysis of FDMs\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Support Protocol 6<\/jats:bold>\n                    : Lysis of fibroblast\/ECM units or FDM\u2010replated fibroblasts for Western blot analyses\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Support Protocol 7<\/jats:bold>\n                    : Mechanical compression of the fibroblast\u2010derived matrix\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Support Protocol 8<\/jats:bold>\n                    : Solubilization of FDM\u2010derived matrix\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Support Protocol 9<\/jats:bold>\n                    : Isolation of primary fibroblasts by a nonenzymatic procedure\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Support Protocol 10<\/jats:bold>\n                    : Isolation of primary fibroblasts by enzymatic tissue digestion\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Support Protocol 11<\/jats:bold>\n                    : Generation of hTERT lentiviruses for primary fibroblast immortalization\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Support Protocol 12<\/jats:bold>\n                    : Biochemical fractionation of multilayered fibroblast\/ECM units\n                  <\/jats:p>","DOI":"10.1002\/cpz1.70483","type":"journal-article","created":{"date-parts":[[2026,10,21]],"date-time":"2026-10-21T19:14:46Z","timestamp":1792610086000},"source":"Crossref","is-referenced-by-count":0,"title":["Preparation of Extracellular Matrices and Cell\/Matrix Functional Units Produced by Cultured and Primary Fibroblasts"],"prefix":"10.1002","volume":"6","author":[{"given":"Aleksandr","family":"Dolskii","sequence":"first","affiliation":[{"name":"Cancer Signaling &amp; 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