Exploitation of S-layer Anisotropy: pH-dependent Nanolayer Orientation

Since the spatial arrangement of cells within their microenvironment influences ceil behavior, control over the ceilular organization is highly relevant for in vitro ceil culture methods. In this regard, the fabrication of micro-patterned surfaces is an indispensable töol in ceil biology in terms of influencing and controlling cellular functions such as cell adhesion, migration, proliferation, cell shape and apoptosis, as weil as cell differentiation. We have developed a simple ceil patterning method using self-assembled crystalline protein monolayers that exhibit depending an the orientation either cell adhesive (cytophilic) or ceil repulsive (cytophobic) surface properties. In other words, we exploit the inherent biological anisotropy of the bacterial surface layer (S-layer) protein SbpA isolated from Gram-positive bacterium Lysinibacillus sphaericus CCM 2177, which is capable of interacting with its cytophilic inner side with bacterial ceil wail components, while its outer cytophobic side interacts with the environment. By simply altering the reassembly protocol from basic to acidic pH, the 's-layer orientation can be switched from non-sticky antifouling layers to effectively promote ceil attachment, spreading and proliferation. An important feature of our method is that a single protein solution can be used to generate ceil adhesive as weil as repulsive regions over !arge areas using a simple pHdependent self-assembly procedure. The rapid and reliable establishment of cytophobic and cytophilic SbpA layers ailows for the generation of well-defined surface Patterns that exhibit uniform height with p4 lattice symmetry of SbpA, and simlar potential under cell culture conditions. In addition,biocaompatibility studies revealed strong adhesion preferences of a variety of mammalian cells towards the cytophilic side of the SbpA Layer. The pH-dependent "orientation Switch" of the SbpA Protein nanolayer was combined with micromolding in capillaries (MMIC, Technology to demonstrate ist application for spatial cell pattering using a variety of cell lines (epithelial, fibroblast and endothelial cells).