Abstract <p>The extracellular matrix (ECM) synthesized by multipotent mesenchymal stromal cells (MSCs) plays a critical role in regulating the cellular microenvironment and is widely used in the development of biomaterials for regenerative medicine. The present study demonstrated the influence of cell culture conditions on ECM collagenous compartments secreted by umbilical cord-derived MSCs (MSCs-UC), including the geometric parameters, structure complexity, and production of collagenous proteins. The effects of gelatin coating, cell culture duration, fetal bovine serum concentration, and ECM-stimulating supplements (sodium 2-phospho-L-ascorbate and carrageenan) were examined. The optimized protocol included cultivation of MSCs-UC on gelatin-coated plates for 11 days in α-MEM supplemented with 10% FBS and 50 μM sodium ascorbate. These conditions provided effective cell adhesion and supported the structural integrity of the cellular layer, as well as the formation of a collagen-rich ECM network. Image analysis revealed that the collagenous fibers under these conditions exhibited a uniform distribution, enhanced fiber alignment and an increased number of branching points, reduced lacunarity, and pronounced anisotropy. The relative collagen content under these conditions was significantly higher than under other culture setups. The results support the proposed protocol as an effective and reproducible platform for generating biomimetic ECM scaffolds suitable for applications in cell engineering and tissue therapy.</p>