Primary tenocytes rapidly undergo senescence and a phenotypic drift upon in vitro monolayer culture, which limits tendon research. The Ink4a/Arf locus encodes the proteins p16 and p14 (p19 in mice) that regulate cell cycle progression and senescence. We here established an immortalized cell line using tenocytes isolated from Ink4a/Arf deficient mice (Ink4a/Arf). These cells were investigated at three distinct time points, at low (2-5), intermediate (14-17) and high (35-44) passages. Wild-type cells at low passage (2-5) served as controls. Ink4a/Arf tenocytes at all stages were comparable to wild-type cells regarding morphology, expression of tenogeneic genes (collagen type 1, 3 and 5, Scleraxis, Tenomodulin and Tenascin-C), and surface markers (CD29, CD44 and CD105) and form 3D tendon-like structures. Importantly, Ink4a/Arf tenocytes maintained their phenotypic features and proliferation potential in culture for more than 40 passages and also following freeze-thaw cycles. In contrast, wild-type tenocytes underwent senescence starting in passage 6. These data define Ink4a/Arf tenocytes as novel tool for in vitro tendon research and as valuable in vitro alternative to animal experiments.
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http://dx.doi.org/10.1038/s41598-023-28318-4 | DOI Listing |
Sci Rep
January 2023
Veterinary Tissue Engineering and Regenerative Medicine Lab, Equine Surgery Unit, Department of Companion Animals and Horses, University of Veterinary Medicine Vienna, Veterinaerplatz 1, 1210, Vienna, Austria.
Primary tenocytes rapidly undergo senescence and a phenotypic drift upon in vitro monolayer culture, which limits tendon research. The Ink4a/Arf locus encodes the proteins p16 and p14 (p19 in mice) that regulate cell cycle progression and senescence. We here established an immortalized cell line using tenocytes isolated from Ink4a/Arf deficient mice (Ink4a/Arf).
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