To assess cellular behavior within heterogeneous tissues, such as bone, skin, and nerves, scaffolds with biophysical gradients are required to adequately replicate the in vivo interaction between cells and their native microenvironment. In this study, we introduce a strategy for depositing ultrathin films comprised of laminin-111 with precisely controlled biophysical gradients onto planar substrates using the Langmuir–Blodgett (LB) technique. The gradient is created by controlled desynchronization of the barrier compression and substrate withdrawal speed during the LB deposition process. Characterization of the films was performed using techniques such as atomic force microscopy and confocal fluorescence microscopy, enabling the comprehensive analysis of biophysical parameters along the gradient direction. Furthermore, human adipose-derived stem cells were seeded onto the gradient films to investigate the influence of protein density on cell attachment, showing that the distribution of the cells can be modulated by the arrangement of the laminin at the air–water interface. The presented approach not only allowed us to gain insights into the intricate interplay between biophysical cues and cell behavior within complex tissue environments, but it is also suited as a screening approach to determine optimal protein concentrations to achieve a target cellular output.
