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This study examined the replicative senescence of primary-culture human vocal fold fibroblasts, in terms of changes in gross chromosomal structure with sub- culturing, and population doubling time (PDT). The mRNA expressions of 14 target genes were also examined. The objectives were to identify the onset of senescence for establishing the acceptable limit of sub-culturing, and to better understand the effect of cellular aging on matrix protein regulation in the vocal fold. Gross chromosomal changes in vocal fold fibroblasts from a 58-year-old woman were karyotyped with Giemsa stain. Proliferation of the fibroblasts was determined with cell recovery and PDT calculation. Transcript levels of the target genes were found by RT-PCR. Onset of significant chromosomal anomalies was seen with passage 5. For mRNA expressions, significant increases with passaging were observed in collagenase, macrophage elastase, lysyl oxidase and fibromodulin, whereas significant downregulation was detected in decorin, procollagen I, hyaluronic acid-synthase 2 and collagen III. This modulation pattern suggested that fibroblasts underwent in vitro aging, consistent with the significant increase in PDT. The inception of senescence did not occur until passage 5. These findings may facilitate the development of representative in vitro models for testing tissue engineering approaches involving primary-culture fibroblasts.