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Using an optimized seeding technology allows cells to cooperate positively with biomaterials, resulting in successful reconstructive surgery. In this study, we used four different types of seeding methods in a scaffold of small intestinal submucosa (SIS). The efficiency of the sandwich co\u2010culture, layered co\u2010culture, static\u2010agitation seeding, and centrifugation seeding methods were compared. It was demonstrated that dynamic seeding methods, such as static\u2010agitation and centrifugation seeding, had superior cell\u2010matrix infiltration and mechanical properties. The seeding time could be reduced by 5\u201310 min using the centrifugation method. Furthermore, functional assessment of the barriers revealed that this function was better in the centrifugation seeding method than in any other method. Our study suggests that both the static\u2010agitation and centrifugation methods are suitable for cell seeding on SIS. There is no significant change in surface area of SIS with different seeding methods. These methods reinforce the physiological and mechanical properties of biomaterials and allow for the future <jats:italic>in vivo<\/jats:italic> study of tissue\u2010engineered urethral reconstruction. \u00a9 2015 Wiley Periodicals, Inc. 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