INTRODUCTION
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 [1].
Several research groups have reached some important results. For example, nowadays it is known that the extra-cellular space, where the embryo starts to develop, is a highly dynamic chemical environment composed of multiple signalling protein gradients with overlapping spatial and temporal expression profiles [1]. In the stage of development, more precisely the period which spans from the moment of an egg fertilization to the end of the 8th week of gestational age after which the embryo is called a fetus, selected groups of cells within the embryo secrete signalling proteins into the extracellular environment. These secreted proteins diffuse away from source cells, forming chemical gradients that induce proliferation [2], differentiation [3], or migration [4] in other cells. In immune response mechanisms, biomolecule gradients provide immune cells with the directional cues they need to rapidly migrate to the infection site [5]. In nervous systems, during both embryonic development and regeneration following nerve injury, extending axons must navigate to appropriate targets. This operation is mediated by the enlarged tip of the axon called growth cone which senses complex gradients formed by diverse extracellular guidance cues and integrates their inputs throughout extensive migration paths [6].
Introduction
It is clear enough that a complete understanding of gradient effects on cellular behaviour is not terminated 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.
Furthermore, wide interest was also devoted to the investigation of the physical stimulation of cells: several types of substrates were presented in literature reproducing selected aspects of the extracellular environment, confirming that the bare topography affects cell [7] adhesion, morphology, orientation, differentiation and phagocytotic activity [8]. Moreover, the phenomenon of cell alignment, called contact guidance, was found to depend on different parameters of micro/nanofabricated structures (i.e. grooves depth and width) and on cell types: one example is given by Recknor et al. [9], who developed a permissive substrate for in vitro neural stem cell differentiation, using a photolithographic technique to pattern periodical physical barriers with adsorbed laminin on polystyrene dishes. This combination of chemical (adsorbed protein) and physical (microgrooves) cues succeeded in the directional guidance of Rat type-1 astrocytes.
A wide interest is thus devoted to the development of systems for high resolution analysis and control of cellular physiology, where the cell development can be controlled not only by the physical and chemical properties of the substrate but also delivering drugs and chemical agents in a precise manner.
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
Introduction
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 (μ-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.
Introduction
6 REFERENCES
[1] T. M. Keenan and A. Folch, Biomolecular gradients in cell culture systems Folch, Lab Chip, 8: 34-57, 2008.
[2] R. Raballo, J. Rhee, R. Lyn-Cook, J. F. Leckman, M. L. Schwartz and F. M. Vaccarino, Basic fibroblast growth factor (Fgf2) is necessary for cell proliferation and neurogenesis in the developing cerebral cortex, J. Neurosci., 20(13): 5012–5023, 2000.
[3] H. L. Ashe and J. Briscoe, The interpretation of morphogen gradients, Development, 133(3): 385–394, 2006.
[4] F. Gilbert, Developmental Biology, Sinauer Associates, Inc. Publishers, Sunderland, MA, 8th edn, vol. xviii, 817, 2006.
[5] C. Janeway, Immunobiology: the immune system in health and disease, 2001.
[6] C. J. Wang, X. Li, B. Lin, S. Shim, G.L. Ming and A. Levchenko, A microfluidics-based turning assay reveals complex growth cone responses to integrated gradients of substrate-bound ECM molecules and diffusible guidance cues, Lab Chip, 8: 227-237, 2008.
[7] V. Raffa, V. Pensabene, A. Menciassi and P. Dario, Design criteria of neuron/electrode interface. the focused ion beam technology as an analytical method to investigate the effect of electrode surface morphology on neurocompatibility, Biomed Microdevices, 9(3): 371-383, 2007.
[8] A. S. Andersson, J. Brink, U. Lidberg, and D. S. Sutherland, Influence of systematically varied nanoscale topography on the morphology of epithelial cells, IEEE Transaction on Nanobioscience, 2(2): 49-57, 2003.
[9] J. B. Recknor, J. C. Recknor, D. S. Sakaguchi and S. K. Mallapragada, Oriented astroglial cell growth on micropatterned polystyrene substrates, Biomaterials, 25: 2753-2767, 2004.
