Aggregate formation of bone marrow stromal cells by rotation culture

The rotation culture method adopted in our experiments induced numerous aggregates of bone marrow stromal cells (BMSCs); this method augments the frequency of cell–cell contacts and thereby facilitates cell aggregation. Cell condensation plays an important role in differentiation and development. Cell adhesion molecules and the extracellular matrix (ECM) are important regulators of cell adhesion and apoptosis. Therefore, we investigated not only whether aggregates of BMSCs could be formed by rotation culture, but also whether expression of N-cadherin and β1-integrin was involved in the formation of cell aggregates and in the avoidance of apoptosis. The culture medium was supplemented with fibroblast growth factor-2 (FGF-2) or transforming growth factor-β1 (TGF-β1), expected to prompt the formation of cell aggregates. This study enabled the formation of numerous aggregates of BMSCs by rotation culture. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that FGF-2 and TGF-β1 increased expression of N-cadherin and β1-integrin. Morphological analysis by light microscopy indicated that FGF-2 and TGF-β1 increased cell–cell adhesion and formation of cell aggregates. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling (TUNEL) assay indicated that FGF-2 and TGF-β1 prevented apoptosis. These results suggest that numerous aggregates of BMSCs were formed efficiently by rotation culture since FGF-2 and TGF-β1 increase cell–cell adhesion and rotation increases cell–cell contact.