Generation of cell-derived three dimensional extracellular matrix substrates from two dimensional endothelial cell cultures

Within the cellular microenvironment, extracellular matrix (ECM) proteins are critical nonsoluble signaling factors that modulate cell attachment, migration, proliferation, and differentiation. We have developed a simple method to isolate and process ECM from endothelial cell cultures to create a three-dimensional (3D) ECM substrate. Endothelial cell monolayers were chemically lysed and enzymatically digested to isolate a thin, two-dimensional (2D) ECM substrate. This thin 1.8 μm 2D ECM was collected and applied to a solid support to produce 12-16-fold thicker 3D ECM substrates with average thicknesses ranging from 21 to 29 μm. The biological activity of isolated ECM was assessed by cell culture. Neural progenitor cells were cultured on endothelial-produced ECM, and unlike the thin 2D ECM, which was quickly remodeled by cells, 3D ECM substrates remained in culture for an extended period (>7 days), suggesting that a continuous signaling cue for in vitro experiments may be provided. This simple method for creating 3D ECM substrates can be applied to a variety of cell culture models for studies aimed at identifying the signaling effects of the ECM within cellular microenvironments.