Murine MSC Tenogenesis Protocol

Murine MSCs (C3H10T1/2, ATCC, Manassas, VA), a model MSC used in prior studies investigating tenogenesis and tendon injury [21 (link), 29 (link), 35 (link)], were cultured and supplemented with TGFβ2 to induce tenogenesis as previously described [14 (link)]. Briefly, cells were expanded in standard growth medium (Dulbecco’s Modified Eagle’s Medium (DMEM), 10% fetal bovine serum (FBS), and 1% Penicillin/Streptomycin) until 70% confluent and used between passages 5 and 13. MSCs were trypsinized and seeded into each well of a 24-well plate. Cells used for 15 minutes (min), 30 min, 1 hour (h), and 24 h timepoints were seeded at 25,000 cells/cm². Cells for 3 and 7 d timepoints were seeded at 5000 cells/cm². Cells were incubated for 24 h to allow for initial cell attachment, and then washed with warmed phosphate-buffered saline (PBS) (Gibco, Grand Island, NY). The medium was switched to low-serum medium (DMEM, 1% FBS, 1% Penicillin/Streptomycin) and allowed to equilibrate for 24 h. Cells were rinsed with warm PBS and cultured for 15 min, 30 min, 1 h, 24 h, 3 d, 7 d, or 14 d in low-serum medium with the corresponding amount of sterile water (vehicle controls) or low-serum medium supplemented with 50 ng/mL recombinant human TGFβ2 (PeproTech, Rocky Hill, NJ). The medium was changed every third day. Experiments were repeated a minimum of 3 times.