## 3 Results and Discussion
### Evaluation of Cell Behavior on Micro-Structured Surfaces  
Above designed and characterized PDMS-based samples were next exposed to human lung epithelial cells (BEAS-2B) with cellular behavior being evaluated after 24, 48 and 72 h, respectively. Such analysis aimed to understand whether the (1) contact orientation can be minimized when structures are distributed to restrict cell spreading, migration and proliferation; (2) intensity, dynamic and reciprocal interactions between cells and topographic models can be achieved when accessibility and flexibility of the substrate are controlled [44]. Using optical microscopy, the influence of the topography on morphological profiles of the cells as well as on the cell numbers was evaluated. Analysis of the surface effects on cell attachment and growth showed a surface-dependent behavior, with trends towards specific cellular density adaptations as related to the LDSU, MDSU or HDSU surface topographies, respectively (Figure 5a-f). Figure 4: **(a)** Histogram showing the contact angle values of PDMS samples. **(b)** Histogram showing the values of surface energies for the PDMS replicates. The distribution of attached cells seemed to conform to the surface's axis of elongation [45]. Briefly, analysis showed that BEAS-2B cells spread on the microstructures of medium heights, avoiding the highest primary units. On a stiff substrate, such as control glass, cells are known to lengthen in the direction of the developing major axis, while on PDMS, a more radial distribution was observed. Cells grown on top of HDSU also seemed to assume a more individual distribution profile, while cells grown on top of LDSU and MDSU had more conglomerated profiles (Supporting Figure S4). The individual or conglomerate profiles were presumably determined by the overall topography and the amount of interactions between individual cells and respective surfaces. Specifically, close interactions between cells and substrates are known to be essential for improved cellular responses, especially when considering the development of tissue constructs, enhanced cytoskeletal organization, cell growth and cell proliferation, respectively [46]. When comparing the cell numbers for the different surface morphologies (Figure 5f), analysis showed that there was generally a relatively homogenous response of the exposed cells. Specifically, the number of the cells grown on different surfaces showed no significant differences after 24, 48 or 72 h, respectively, all relative to controls. After 24 h, cell activity, however, seemed to decrease on PDMS, regardless of the presence or absence of topographic features; however, at 72 h, the HDSU replica appeared to favor cell proliferation, as shown in Figure 5f and  
Figure 5: Fluorescence microscopic images (40\(\times\)) of Hoechst-labeled BEAS 2B cell nuclei (blue); (**a**) control cells, cells on control (**b**) PDMS, (**c**) LDSU, (**d**) MDSU, and (**e**) HDSU samples, respectively. (**f**) Quantification of BEAS-2B live cells seeded on PDMS microstructures with different topographies (\(n=4\)). supported by the lower variation in the cell numbers as reported relative to MDSU and LDSU surfaces, respectively [47; 48]. Our analyses support that a multi-dimensional design controls cell attachment and implicitly, cell density; moreover, our studies emphasize that, in order to trigger a more localized cellular response, one could use different modulation factors to direct cellular growth and, further, cell proliferation and differentiation. For instance, one could envision topographical features with different growth factors or stimulants to be released upon established cellular contact, to thus provide a localized cell fate response.