Isolation and Characterization of Tendon Stem/Progenitor Cells

Eight-week-old healthy male Sprague–Dawley rats were used to isolate TSPCs. All animal experimental protocols were approved by the Animal Research Committee of Tohoku University (approval number: 2020DnA-009). The TSPCs were isolated according to previously established procedures [36 (link)]. Briefly, the midsubstance of Achilles tendon tissues was collected. The collected tissues were minced and digested for 2.5 h at 37 °C with 3 mg/mL of type I collagenase (Sigma-Aldrich, St. Louis, MO, USA). Afterward, the cells were passed through a cell strainer (70 μm pore size, Becton, Franklin Lakes, NJ, USA) to obtain a single-cell suspension. The isolated cells were washed and centrifuged to resuspend in low-glucose Dulbecco’s Modified Eagle’s medium (FUJIFILM Wako Pure Chemical Co., Osaka, Japan) containing 10% fetal bovine serum (FBS; Gibco, Waltham, MA, USA) and 1% penicillin/streptomycin solution (Nacalai Tesque Inc., Kyoto, Japan) as the complete medium at 37 °C in a 5% CO₂ incubator. The cells at passages 3 and 5 (P3, P5) were used for all subsequent experiments. P5 cells served as a control group, and the stemness of TSPCs was identified by measuring the mRNA expression of CD44, CD90, and Nanog using a qPCR. The culture medium was replaced every 3 days during the experiments.