Outcomes obtained in patients with two-tendon rotator cuff tear submitted to repair reinforced with porcine small intestinal submucosa (SIS) have not been as encouraging as those observed in animal models. We verify the capacity of SIS to be used as a physical support for a culture of cuff cells. During arthroscopic repairs of large rotator cuff tears, we removed a fragment of supraspinatus tendon. Samples were treated for obtaining a cuff cell culture. Daily microscopic analysis, to observe adhesion to substrate, replication and cell shape was performed. A confluent monolayer was obtained in 1 week. Cells at the second passage were collected and seeded onto scaffold and cultured for 7-30 days. A morphological and immunohistochemical evaluation was performed. After 1 week, a monolayer of tendinous-like cells lay along the surface of the SIS. Within two weeks, a multicellular layer was observable in many foci of the scaffold. After a month, the cells completely invaded the numerous splits of the SIS and were positive to monoclonal anti-type I collagen antibody. Our experimental study has proved that a cuff cell culture can be performed using SIS as substrate. The culture covers the SIS surface, therefore it may reduce immune or non-specific inflammatory reactions.