The physical properties of a cell's environment are important factors in determining cell behavior and ultimately, phenotype. Two key factors that have been associated with major changes in cell morphology and behavior are (1) spatial organization of extracellular matrix (ECM) molecules and (2) rigidity. In order to understanding how cells sense these factors at the nanoscale and how these factors affect cell function, we have developed new nanofabricated surfaces in which these physical characteristics of the ECM are simulated.

The first type of surface combines nanoimprint lithography with selective biofunctionalization to precisely control the placement and geometric arrangement of integrin binding sites. The binding sites consist of sub-10 nm metallic nanodots functionalized with ECM binding ligands, designed so that each site can accommodate only a single integrin molecule. Cell spreading and motility assays were performed using 3T3 fibroblasts on arrays in which binding site spacing, density and number were independently varied. Cell spreading efficiency was markedly enhanced for clusters comprising at least 4 liganded sites spaced ≤ 60 nm apart, with little or no dependence on global density. This points to the existence of a minimal matrix adhesion unit defined in space and stoichiometry.

A second type of surface consists of elastomeric substrates with locally variable rigidity. We have found that exposure of poly(dimethylsiloxane) (PDMS) to an electron beam alters the rigidity of the elastomer, with the modulus of the exposed regions increasing with the applied electron dose. In addition to planar surfaces, pillared substrates can be patterned with no measurable change to the pillar dimensions. Immortalized mesenchymal stem cells plated on soft PDMS surfaces patterned in this manner displayed a distinct preference for the more rigid, exposed regions, forming focal adhesion nearly exclusively there. Furthermore, focal adhesion formation diminished significantly as the size of the exposed features was reduced below 1 μm, indicating that there is a length scale for cellular rigidity sensing, with the critical length in the range of a few hundred nanometers.

By adapting the tools of nanomanufacturing to cellular systems, we are able to define important parameters that can control aspects of cell function and behavior and will help identify conditions under which these functions may be altered. Potential applications range from therapeutic treatments that block metastasis to the development of new adoptive immunotherapies, as well as the development of new guidelines for the design of tissue scaffolds that can optimize healing without scarring.

One of the main challenges for the robust in-vitro studies is to obtain adaptable 3D culture systems that may mimic the tissue environment. Unfortunately the universal condition used in 2D cell culture techniques may hinder the full functionality of cells and generate misleading results.

Fabrication of firm and flexible micro-structures from organic polymers offers benefits for making smart 3D environments capable of driving cell behavior and surpassing the limitations of the 2D systems. These 3D bio-scaffolds can be employed to study various aspects of cell biology. Furthermore upon functionalization with the extra-cellular matrix proteins or signaling molecules they can be used as platforms for governing stem cell differentiation into the specialized cell types.

Here we present the straightforward approach to generate 3D bio-scaffolds that can facilitate cell growth under controlled geometrical and chemical conditions.

The technique involves UV cross-linking of the polymeric precursors to create the micro-well structures. The geometrical features of the structures are obtained by introducing a physical mask in contact with a liquid precursor, therefore restricting the region of the polymerization. We used PDMS mold as a physical mask to direct the polymerization of the PEG-DA and epoxy based polymers. However the technique can be used with various UV-sensitive polymeric materials.

The chemical and geometrical properties of the structures were characterized by XPS and microscopic techniques.

The features of the scaffolds lead to the development of a geometrically defined neuronal network when applied as platforms in a primary-neuron culture. Cell morphology and expression of the neuronal markers were characterized by fluorescent microscopy.