<p>This paper reports a novel method of using air column to temporarily change microfluidic structure for cell seeding and culture in a microfluidic chamber. In conventional on-chip culture system, the medium flow has to be carefully controlled to prevent from excessive shear stress that washes out cells. We propose a method to directly seed cells into a stagnation area so that the cells can be stably cultured inside a microfluidic chamber without being affected by the flow. Air column is intentionally introduced during the seeding, and it forms a virtual wall, which temporarily changes the flow pattern. The changed flow allows the cell to flow into the stagnation area for seeding. The idea has been experimentally tested, and cells are successfully seeded and cultured in a single inlet chamber.</p>