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The mechanism that cells use to recognize micro-patterned topographies was clarified. First, 3D double-layer poly(ε-caprolactone) scaffolds equipped with honeycomb-patterned micro-pores ("honeycomb films") were prepared by simply casting a polymer solution of water-immiscible solvent under high relative humidity of about 80% at 20±1 °C. Then, porcine aortic endothelial cells (PAECs) were cultured on these scaffolds for 1-6 h in serum-free medium. Finally, their initial spreading process was investigated by using tapping-mode atomic force microscopy and confocal laser scanning microscopy. On a honeycomb film with micro-pore of 3 μm PAECs showed morphology similar to that on a flat film as a control irrespective of culture time. On the contrary, the attachment and spreading of PAECs on honeycomb films having either 6- or 16-μm pore diameters, resulted in voids within the cell cytoplasm, which correspond with the size and location of the honeycomb micropores. This is the first report of this unique morphology. The number of cells with this morphology decreased with increasing culture time. This dependence of morphology on film pore size and culture time suggests a spreading process of PAECs in which the cells spread, trying to sense suitable sites on which to adhere. Using thick filopodia, the cells spread along the rim of the film and produced pores by close contact between two spreading filopodia. Evidently, these pores became filled in during culture, presumably as the cells began to reorganize their cytoplasma. Moreover, the cells migrated into the honeycomb pores and expanded along the bottom layer through interconnecting channels of the film. In conclusion, the spreading of PAECs on a honeycomb film gives insight into the mechanism of a honeycomb film to control cellular behavior. |