Biomolecular concentration gradients play relevant roles in many biological phenomena including development, inflammation, wound healing, and cancer growth and spreading. In order to better investigate and elucidate these aspects, several in vitro systems have been defined for exposing cells to chemical gradients. In combination with in vivo studies, these methods have revealed gradient signalling to be an intricate, highly-regulated process, in which the ultimate cellular response is determined by the concentration, and spatiotemporal characteristics of the gradients to which the cells are exposed. A complete understanding of gradient on cellular behaviour is still on going and more technological platforms are required to increase the knowledge of many cellular mechanisms. Thus it is a big technological challenge the realization of systems that makes the in vitro environment more similar to the in vivo one, including spatially and temporally organized signals supplemented with sensors for monitoring cellular parameters.This work is focused on the realization of a microfluidic chip for controlled cell culture, integrating a gradient generator and a micropatterned substrate. Exploiting the advantages of soft lithographic techniques, a polymeric structure in polydimethylsiloxane (PDMS) is conceived with two orthogonal sets of fluidic channels and active components, such as switching valves. The system is designed to be applied on a substrate, previously patterned by non-conventional micro contact printing of adhesion proteins. Finally, the developed system guarantees the production of a complex user-defined gradient, with tailored spatial and temporal profiles; secondly, it allows immobilizing and observing a selected small population of adherent cells.
The remainder of this thesis is organised as follows. Chapter 1 presents a brief description of microfluidic gradient generators, highlighting the limits of the published architectures. In Chapter 2 a novel microfluidic gradient generator is proposed. This system is designed to allow generating multiple concentration gradients. The microfluidic network is combined with a glass substrate which presents cell-adhesive and cell-repellent areas. Materials and methods for the fabrication of the chip and for the substrate micropatterning are presented in Chapter 3. Specifically, Multilayer Soft Lithography (MLSL), used for the microfluidic chip fabrication, and micro-contact printing (micro-CP), required for the substrate micropatterning are described in detail. In Chapter 4 all the specific fabrication protocols and an extensive experimental characterization and validation of the system are reported and discussed. In particular, three major results are presented: i) demonstration of complex gradient generation, ii) micro-patterning of fibronectin/PLL-g-PEG on glass substrates, iii) immobilization on the microfabricated systems. Chapter 5 outlines obtained results and contributions and suggests directions for the future work.