Cell response on microporous membranes is important since the membranes are often used for the applications of tissue engineering. We previously developed the 3D stacked-up culture of hepatic progenitor cells, which are small hepatocytes (SHs), where the cells were cultured on microporous membranes for reconstructing layered tissues in vitro. Since membranes with randomly distributed micropores were used in the culture, the size and distribution of the micropores remained to be optimized. Here we prepared microporous membranes with different pore sizes and spacing to investigate the SH morphogenesis on the membranes. Laser-ablation technique was used to fabricate micropores in desired patterns. Phase-contrast microscopy revealed that the formation of SH colonies was correlative to the micropores. Furthermore, SH colonies exhibited collective cell movements along the micropatterned pores. The correlation enhanced with increasing sizes (>10 μm) and decreasing spacing (<40 μm) of micropores. Scanning electron microscope images showed that SHs located at the colony edge extended protrusions invading into the micropores. Furthermore, immunofluorescent staining revealed that the focal complexes localized intensively around the micropores, suggesting that the SH morphogenesis was regulated by the focal complexes. This study demonstrated that the surface topography of microporous membranes plays an important role in the formation and movement of SH colonies.
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