Isolation and Culture of Human Tendon Cells

Human tendon cells were isolated from healthy human hamstring (semitendinosus) tendons (excess anterior cruciate ligament autograft material) as previously described [20 (link)]. Male (N = 2; age 28 ± 1.4) and female (N = 2; age 37 ± 5.7) donors provided written informed consent and the protocol was approved by University of British Columbia Clinical Research Ethics Board (certificate number H10-00220). Equal number of females and males were used in each experiment to exclude the effect of sex as a factor. The tendon cells were dissociated from the tissue using mechanical (chopping with surgical scissors) enzymatic (collagenase) methods. To expand the tendon cell culture, the isolated tendon cells were grown in high glucose Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% calf serum, 2 mm L-glutamine, and 1x Antibiotic/Antimycotic solution in a humidified incubator containing 5% CO2 at 37 °C. Then, 10% calf serum was replaced with 5% Lipoprotein Deficient Serum from fetal calf (Sigma, USA, #S5394) before and during incubation with oxLDL. Recombinant TGF-β protein (PeproTech, USA, #100 − 21) at concentration of 5ng/ml was added during the incubation with oxLDL.