Penicillin streptomycin p s

The TkDN4-M (4M) hiPSCs line was a kind gift from Dr. M Ohtsu at The Institute of Medical Science, The University of Tokyo. 4M cells were cultured as previously described with minor modifications [23 (link)]. They were maintained on mitomycin C (MMC; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan)-treated SNL feeder cells in hiPSCs medium (DMEM/Ham`s F12 (FUJIFILM Wako) supplemented with 20% Knockout Serum Replacement (KSR; GIBCO BRL, Palo Alto, CA, USA), 1x MEM nonessential amino acids (FUJIFILM Wako), 0.5x penicillin streptomycin(PS; FUJIFILM Wako), 55μM 2-melcaptoethanol (2ME Gibco) and 7.5μg/ml basic fibroblast growth factor (FGF2; Peprotech, Rocky Hill, NJ, USA). For passage, 4M colonies were detached using CTK solution, chipped by pipetting, and seeded onto MMC-treated SNL feeder (ECACC, Salisbury, UK) in hiPSCs medium once a week. The 15M63 hiPSCs line was kindly provided from the Center for iPS Cell Research and Application of Kyoto University (Kyoto, Japan) and maintained on vitronectin (Invitrogen, CA, USA) coated dishes in StemFit. For passage, 15M63 was dissociated with accutase (Innovative Cell Technologies, San Diego, USA) by pipetting and seeded on vitronectin coated dishes in StemFit (Ajinomoto, Tokyo, Japan) supplemented with 10μM Y27632 (Cayman Chemical, Ann Arbor, MI, USA).

Primary human bone marrow stem cells (hBMSCs) were purchased from ATCC (Manassas, Virginia, USA). The hBMSCs were cultured in alpha-MEM containing 15% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, Missouri, USA), 1% penicillin/streptomycin (PS, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) at 37°C, 5% CO₂, and 99% humidity until 80% confluence. Cells at passage P3 were used in the experiments.
For osteogenic differentiation, hBMSCs (5 × 10³ cells) were seeded onto the electroactive surfaces (1 cm²) and incubated for 24 h to allow cell adhesion. The cells were then maintained in the normal culture medium supplemented with 10 mM β-glycerophosphate (β-GP, Sigma-Aldrich), 100 µM L-ascorbic acid phosphate (Sigma-Aldrich), and dexamethasone sodium phosphate (10⁻⁸ M), for 14 days [40 (link)].

Bovine myoblast cells were cultured by passaging at 6- or 7-day intervals in DMEM with 10% FBS, 1% Penicillin-Streptomycin (P/S, Fujifilm Wako Pure Chemicals, 168-23191) in 10 cm culture dishes. The bovine myoblast cells were cultured in TRCDs to prepare the cell sheets. The cells were seeded at 5 × 10⁶ cells/dish in 3.5 cm TRCDs, CellSeed, CS3017), coated with laminin-511 (Easy iMatrix-511, Matrixome, 892018), along with 2 ml of DMEM supplemented with 10% FBS and 1% P/S. Laminin-511 coating was performed by spreading Easy iMatrix-511 on the bottom of the dish and incubating at 37 °C for at least 1 h. The cells were then cultured in a CO₂ incubator at 37 °C. The medium was changed on the day after seeding and again on day 4. Bovine myoblast cell sheets were detached from the TRCDs after incubating at 20 °C for 30 min in an incubator on the day after seeding day 1, day 3, or day 7.
Large-sized bovine myoblast cell sheets were prepared using 10 cm TRCDs (CellSeed, CS3017). The cells were seeded at 3 × 10⁷ cells/dish in 10 cm TRCDs along with 10 ml of DMEM supplemented with 10% FBS and 1% P/S. After 1 day culturing in a CO₂ incubator at 37 °C, followed by 20 °C for 30 min, bovine myoblast cell sheets were detached from the TRCDs.