Human osteogenic differentiation media

Human bone marrow-derived mesenchymal stem cells (hMSCs, RoosterBio Inc., MD, USA) were used to fabricate spheroids. Cells at passages from 4 through 7 were used. hMSCs were cultured in mesenchymal stem cell expansion media (R&D Systems, MN, USA) at 37 °C in 5% CO₂ in a humidified sterile incubator. To prepare hMSC only spheroids, the expanded hMSCs were trypsinized, centrifuged, and transferred to each well of a U-bottom 96-well plate (Greiner Bio One, Austria) with 15,000–20,000 cells per well. Spheroids were obtained with a size of 400–800 μm as we reported before (11 ). Bioprinted spheroids were then cultured in human osteogenic differentiation media (Cell Applications Inc., USA) for 28 days post-bioprinting to enable their differentiation into an osteogenic lineage. The differentiation media was changed every 2–3 days.

For in-vitro study, human mesenchymal stem cells (hMSCs, RoosterBio Inc., MD, USA) were cultured using MSC expansion media (R&D Systems, MN, USA) (growth media; GM) supplemented with Penicillin (100 IU/ml) - Streptomycin (100 μg/ml) (cat. no. 30–002-CI, Corning Life Sciences, Lowell, MA) until usage. As an osteogenic media (OM), human osteogenic differentiation media (Cell Applications, USA) added with Penicillin-Streptomycin was used.
For in-vivo study, as implantation of hMSCs was not possible into immunocompetent rats, allogenic rBMSCs were extracted from inbred 4-week-old male Fischer white rats (F344/DuCrl, Charles River Laboratories, Wilmington, MA) [25 ]. All animals were cared in the animal facility (Millennium Science Complex, PSU) and euthanasia procedures were according to American Association for Laboratory Animal Science (AALAS) and The Institutional Animal Care and Use Committee (IACUC protocol #46591). Isolated rBMSCs were plated on 6-well cell culture plates in Minimal Eagle’s Medium, Alpha modification (αMEM; cat. no. 10–022-CV, Corning Cellgro^®, Manassas, VA) supplemented with 10% fetal bovine serum (FBS) (cat. no. 35–010-cv, Thermo Fisher Scientific, Waltham, WA), PS and 2.5 μg/ml Fungizone (cat. no. 15–290-026, Thermo Fisher Scientific) and incubated at 37 °C in a humidified 5% CO₂ incubator.