Micropatterning of Glass Substrates Using Electron‐Beam Lithography for Morphological Analysis of Human Umbilical Vein Endothelial Cells

This study presents a micropatterning technique for glass substrates aimed at investigating the morphological adaptations of human umbilical vein endothelial cells (HUVECs). This method employs electron‐beam lithography technology to produce sacrificial structures on glass prior to gas‐phase deposition of polyethylene glycol‐bearing silanes. HUVECs seeded on the patterned substrates exhibit a pronounced visible difference in adhesion, alignment, and morphological characteristics depending on the type of pattern, specifically influenced by the line dimensions and the spacing between them. The technique proves to be effective for generating high‐resolution patterns with minimal alignment errors, making it particularly suitable for prototyping and small‐scale studies. By removing the necessity of a photomask and enabling rapid design modifications, this method offers a robust platform for exploring the biochemical and morphological dynamics of cells. Notably, cell orientation is obtained only on patterns with inter‐line distances of at least 10 μm, suggesting that effective directional guidance requires minimum spacing. Moreover, cells cultured on patterns with line widths below 20 μm lose their alignment after 24 h, and the increase in spacing from 10 to 15 μm has a significantly greater impact on cell morphology than the corresponding change from 15 to 20 μm.