Introduction: Tendon ruptures are increasingly common, repair can be difficult and healing poorly understood. Tissue engineering approaches often require expansion of cell numbers to populate a construct, and maintenance of cell phenotoype is essential for tissue regeneration.

Methods: In this study we characterise the phenotype of human Achilles tenocytes and assess how this is affected by passaging. Tenocytes, isolated from tendon samples from 6 patients receiving surgery for rupture of the Achilles tendon, were passaged 8 times. Proliferation rates and cell morphology were recorded at passages 1, 4 and 8. Total collagen, the ratio of collagen types I and III and decorin were used as indicators of matrix formation and expression of the integrin ‘alpha’1 subunit as a marker of cell-matrix interactions.

Results: With increasing passage number, cells became more rounded, were more widely spaced at confluence and confluent cell density declined from 18700 /cm2 to 16100 /cm2 (P=0.009). No change to total cell layer collagen was observed but the ratio of type III to type I collagen increased from 0.60 at passage 1 to 0.89 at passage 8 (P< 0.001). Decorin expression significantly decreased with passage number, from 22.9 ± 3.1 ng/ng DNA at passage 1, to 9.1 ± 1.8 ng/ngDNA at passage 8 (P< 0.001). Integrin expression did not change.

Conclusion: We conclude that the phenotype of tenocytes in culture rapidly drifts with progressive passage.