This work reports the micro- and nano-fabrication of substrates obtained by coupling conventional photolithography and
layer-by-layer (LbL) self-assembly technologies, and the application for building in vitro cell culture scaffolds. Using photolithography, cell culture scaffolds of various shapes (circles, squares, and rectangles) and sizes (10 to 300 μm) were obtained using direct and soft lithography methods in SU-8, polydimethylsiloxane (PDMS), and Poly(methyl methacrylate) (PMMA). Using LbL technology, patterned polyelectrolyte ultrathin multilayer films, containing gelatin to promote cell adhesion, were then deposited on or between raised microstructures, depending on the intended application of the cell culture scaffolds. Fluorescent indicator dyes are integrated onto the microstructures to allow
monitoring changes of pH and oxygen in the cell culture media. Thus, cell activity may be monitored by using the fluorescent film properties, enabling a high-throughput cell-based in vitro screening system. The findings indicate that combining conventional photolithography and LbL technologies is an efficient method to fabricate micro- and nanoscale patterns as substrates to modulate the surface properties of in vitro cell culture scaffolds.