This study investigates the effect of nanoscale topography on human corneal epithelial cells. The human corneal basement membrane has a felt-like surface with features ranging from 20 nm to 200 nm. The researchers designed lithographically defined substrates to test if nanotopography influences cell behavior. They found that cells elongated and aligned along grooves and ridges as small as 70 nm, while on smooth substrates, cells were mostly round. The percentage of aligned cells was consistent across different substrate sizes but increased with groove depth. The presence of serum in the culture medium increased the percentage of cells aligning along the topographic patterns. Actin microfilaments and focal adhesions aligned along the substrate topographies, with the width of focal adhesions determined by the width of the ridges. The study shows that nanoscale topographic features can significantly affect epithelial cell behavior. The results suggest that nanoscale topography is a relevant stimulus for epithelial cells, and that the alignment of cells is influenced by the depth and width of the grooves. The study also highlights the importance of topography in cell culture, tissue engineering, and the development of implantable prosthetics. The findings indicate that nanoscale topography can act synergistically with other cellular inputs to influence cell behavior. The study provides insights into the mechanisms of contact guidance and the role of focal adhesions in cell alignment. The results have implications for understanding the role of topography in cell behavior and for the design of cell culture systems.This study investigates the effect of nanoscale topography on human corneal epithelial cells. The human corneal basement membrane has a felt-like surface with features ranging from 20 nm to 200 nm. The researchers designed lithographically defined substrates to test if nanotopography influences cell behavior. They found that cells elongated and aligned along grooves and ridges as small as 70 nm, while on smooth substrates, cells were mostly round. The percentage of aligned cells was consistent across different substrate sizes but increased with groove depth. The presence of serum in the culture medium increased the percentage of cells aligning along the topographic patterns. Actin microfilaments and focal adhesions aligned along the substrate topographies, with the width of focal adhesions determined by the width of the ridges. The study shows that nanoscale topographic features can significantly affect epithelial cell behavior. The results suggest that nanoscale topography is a relevant stimulus for epithelial cells, and that the alignment of cells is influenced by the depth and width of the grooves. The study also highlights the importance of topography in cell culture, tissue engineering, and the development of implantable prosthetics. The findings indicate that nanoscale topography can act synergistically with other cellular inputs to influence cell behavior. The study provides insights into the mechanisms of contact guidance and the role of focal adhesions in cell alignment. The results have implications for understanding the role of topography in cell behavior and for the design of cell culture systems.