Micropatterning of functionalized surfaces for the design of cellularized capillary-scale microfluidic devices

This thesis focuses on the development and application of micropatterning techniques for the precise control of endothelial cell adhesion and morphology on glass substrates, with the goal of integrating these surfaces into capillary-scale microfluidic devices. 
A reproducible micropatterning protocol was established, employing electron-beam lithography and aluminium alignment marks to define square adhesive areas ranging from 10 to 30 μm. The method was optimized to ensure adequate cell attachment, alignment, and spreading, providing a standardized platform for cell characterization. Using fluorescence microscopy, human umbilical vein endothelial cells (HUVECs) seeded on micropatterned lines exhibited a clear dependence of adhesion, alignment, and morphology on both line width and inter-line spacing. Directional guidance was observed only on patterns with a minimum inter-line distance of 10 μm, and cells confined to lines narrower than 20 μm lost alignment after 24 h. Notably, the impact of increasing spacing from 10 to 15 μm on cell morphology was greater than that of a corresponding increase from 15 to 20 μm, highlighting non-linear effects of pattern dimensions on cell behaviour.
Extending this approach, single cells were isolated on square patterns ranging from 15 to 30 μm per side to quantify topographical adaptations using Atomic Force Microscopy (AFM). Cells on smaller patterns (15 - 20 μm) exhibited significantly higher average and peak heights, increased roughness, and elevated root-mean-square slopes compared to cells on larger islands (25 - 30 μm). Higher-order statistical analysis revealed a shift from platykurtic height distributions on smaller islands to strongly leptokurtic and positively skewed distributions on larger islands, indicating a flatter and more regular surface morphology for cells on wider adhesive areas. These findings provide precise, quantitative insight into how spatial constraints modulate single-cell surface properties and demonstrate that the combination of micropatterning and AFM might prove valuable to study how cells react to different types of spatial constrains.
The integration of patterned cells into microfluidic devices was explored, with particular focus on channel alignment, bonding, and perfusion. While successful alignment and closure of microchannels were achieved, the development of a fully reliable bonding protocol remained a critical limitation, primarily due to the challenges in controlling adhesive spreading and operator-dependent alignment.