Bone marrow-derived mesenchymal stromal cells (RWTH Aachen University)

Rat femur and tibia bones were washed in 70% ethanol, and then in sterile PBS and complete DMEM low-glucose medium, supplemented with L-glutamine, heat-inactivated FCS (10%), penicillin (100 IU/ml), streptomycin (100 μg/ml), and amphotericin B (2.5 μg/ml), at 4°C. Bone metaphyses were cut off, and the diaphyses were flushed with complete medium. Cells were then passed through a 21-G needle several times, spun down at 1200 rpm (4°C for 5 min), followed by a washing step in PBS and resuspended in complete medium. Cells were then seeded into six-well plates and cultured at 37°C in a humidified atmosphere that contained 5% CO₂. Medium was changed after 2 d and every 3 d thereafter. Nonadherent hematopoietic cells were removed when medium was changed. After a mean 6 d, cells reached subconfluence and were detached with trypsin/EDTA, reseeded at 4 × 10³ cells/cm², and were used for experiments after the third passage.