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The study of immune cell migration is important for understanding the immune system network, which is associated with the response to foreign cells. Neutrophils act against foreign cells before any other immune cell, and they must be able to change shape and squeeze through narrow spaces in the extracellular matrix (ECM) during migration to sites of infection. Conventional in vitro migration assays are typically performed on two-dimensional substrates that fail to reproduce the three-dimensional (3-D) nature of the ECM. Here we present an in vitro method to simulate the 3-D migration of neutrophils using an electrospun nanofibrous membrane, which is similar to the ECM in terms of morphology. We examined the properties of neutrophil movement and the effects of gravity and the presence of IL-8, which has been widely used as a chemotactic attractant for neutrophils. The number of neutrophils passing through the nanofibrous membrane were higher, and their movement was more active in the presence of IL-8. Also, we confirmed that neutrophils could migrate against gravity toward IL-8 through a nanofibrous membrane.