Electrospinning technology is a versatile method for fabricating three-dimensional (3D) nanofibrous scaffolds using a wide range of polymeric materials for tissue engineering and regenerative medicine. However, a major concern regarding 3D electrospun scaffolds is that the densely packed layers hinder an even cellular distribution and in-depth infiltration. Here, we describe a new 'all-at-once' method enabling scaffold fabrication and cell seeding simultaneously, in which the medium bath containing cells is rotated eccentrically at high speed (>1500 rpm). The unstable flow of culture medium under hydrodynamic conditions resulted in a skein-shaped 3D structure and enhanced the even cellular distribution and in-depth infiltration. Cellular distribution and infiltration analyses confirmed that our method was superior to static and dynamic seeding methods. Moreover, we showed that our method facilitated long-term (14 days) proliferation. (C) 2015 Elsevier B.V. All rights reserved.