Aim: Biomaterials that mimic the nanofibrous architecture of the natural extracellular matrix (ECM) are in the focus for stem cell hosting or delivery in tissue engineering of multilayered soft tissues such as skin, mucosa, or retina. Synthetic nanofibers for such ECM are usually produced by single-syringe electrospinning with only one needle-jet at very low production rates of 0.005-0.008?g筩n(-1). The aim of this study was to utilize a novel industrial needle-free multijet electrospinning device with the potential for mass production of nanofibrous ECM (NF-ECM) exhibiting a controlled three-dimensional (3D) morphology for large-scale applications such as large area skin regeneration in patients with burns. Methods: The novel NanoSpider頎S200, an industrial apparatus originally designed for electrospinning of nanofibrous textile meshes, was used to fabricate 3D NF-ECMs of the following synthetic and natural biopolymers: collagen, gelatin, poly(caprolactone) (PCL), and poly(l-lactide-co-glycolide) (PLGA). Different concentrations of Gelatin polymer solution were electrospun under varying processing conditions, namely speed of spinning electrode rotation (u) and electric field intensity (E) by altering applied voltage (v) or the distance between electrodes (h) to achieve homogeneous desirable 3D morphology. Nanofiber diameters were assessed by scanning electron microscopy (SEM). Biocompatibility was tested by WST-1 (water-soluble tetrazolium salt) proliferation assay of seeded human mesenchymal stem cells (HMSCs). Biological performance of HMSCs on 3D PLGA NF-ECM was compared to two-dimensional (2D) PLGA film controls via SEM and confocal microscopy. Western blotting addressed the expression of surface adhesion proteins; focal adhesion kinase (FAK), phosphorylated FAK (pY397), a-tubulin, paxillin, vinculin. and integrin subunits; a5, av, and ߱ proteins. Results: Large-scale mass production of NF-ECM membranes with a highly homogenous nanofiber morphology and 3D architecture could be produced with an extremely high production rate of 0.394ᰮ013?g筩n(-1)筨-1) when compared to standard procedures. This was achieved by electrospinning a 20% (wt)/(v) gelatin solution, in an electric field intensity of 0.381?kV筭(-1). The nanofibers possessed diameters of around 180ᴰ?nm with 28% deviation. HSMCs proliferation was significantly improved on NF-ECMs derived from collagen, gelatin, and PLGA when compared to PCL or flat coverglass controls (p<0.01). PLGA NF-ECM in 3D nanofibrous architecture possessed significantly superior biocompatibility when compared to flat 2D PLGA film (p<0.05). Furthermore, on 3D PLGA NF-ECMs, HSMCs expressed a higher amount of a-tubulin and paxillin compared to the HMSCs cultured on a 2D PLGA film (p<0.05). HMSCs exhibited a complex multifaceted morphology on all NF-ECMs, where cells appeared to be integrated into the 3D NF-ECMs niches with complex cell filopodia extending into to all directions. In contrast, HMSCs on flat 2D films of the same materials or on coverglass displayed a simple flattened, monolayered structure. Conclusion: Needle-free multijet electrospinning can be used to mass produce artificial ECMs with intrinsic biocompatibility and desirable integration of stem cells for large-scale applications.