AbstractIn biomaterial development, the design of material surfaces that mimic the extra-cellular matrix (ECM) in order to achieve favorable cellular instruction is rather challenging. Collagen-type IV (Col-IV), the major scaffolding component of Basement membranes, a specialized ECM with multiple biological functions, has the propensity to form networks by self-assembly and supports adhesion of cells such as endothelial cells or stem cells. The preparation of biomimetic Col-IV network like layers to direct cell responses is difficult. We hypothesize that the morphology of the layer, and especially the density of the available adhesion sites, regulates the cellular adhesion to the layer. The Langmuir monolayer technique allows for preparation of thin layers with precisely controlled packing density at the air-water (A-W) interface. Transferring these layers onto cell culture substrates using the Langmuir Schäfer (LS) technique should therefore provide a pathway for preparation of BM mimicking layers with controlled cell adherence properties. In situ characterization using ellipsometry and polarization modulation-infrared reflection absorption spectroscopy of Col-IV layer during compression at the A-W interface reveal that there is linear increase of surface molecule concentration with negligible orientational changes up to a surface pressure of 25 mNcenterdotm<sup>-1</sup>. Smooth and homogeneous Col-IV network-like layers are successfully transferred by LS method at 15 mNcenterdotm<sup>-1</sup> onto poly(ethyleneterepthalate) (PET), which is a common substrate for cell culture. In contrast, the organization of Col-IV on PET prepared by the traditionally employed solution deposition method results in rather inhomogeneous layers with the appearance of aggregates and multilayers. Progressive increase in the number of early adherent mesenchymal stem cells (MSCs) after 24 h by controlling the Col-IV density by LS transfer at 10, 15 and 20 mNcenterdotm<sup>-1</sup> on PET is shown. LS method offers the possibility to control protein characteristics on biomaterial surfaces such as molecular density and thereby, modulate cell responses.