Author:
Oreff Gil Lola,Maurer Barbara,ELKhamary Ahmed N.,Gerner Iris,Sexl Veronika,Jenner Florien
Abstract
AbstractPrimary tenocytes rapidly undergo senescence and a phenotypic drift upon in vitro monolayer culture, which limits tendon research. The Ink4a/Arf locus encodes the proteins p16Ink4a/Arf and p14ARF (p19ARF 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.
Funder
University of Veterinary Medicine Vienna
Publisher
Springer Science and Business Media LLC