Serial Passage of MC3T3-E1 Cells Alters Osteoblastic Function and Responsiveness to Transforming Growth Factor-β1 and Bone Morphogenetic Protein-2

Abstract The murine-derived clonal MC3T3-E1 cell is a well-studied osteoblast-like cell line. To understand the effects of serial passages on its cellular function, we examined changes in cell morphology, gap junctional intercellular communication (GJIC), proliferation, and osteoblastic function between early passage ( 65) cells. MC3T3-E1 cells developed an elongated, spindle shape after multiple passages. Intercellular communication decreased significantly (33%) in late vs. early passage cells. Transforming growth factor-β1 (TGF-β1) stimulated cell proliferation in early passage cells and induced c- fos expression, while it inhibited proliferation in late passage cells. Using alkaline phosphatase (ALP) activity and osteocalcin (OC) secretion as markers for osteoblastic function and differentiation, we demonstrated that both markers were significantly reduced after multiple cell passages. Bone morphogenetic protein-2 (BMP-2) significantly enhanced ALP activity and OC secretion in early passage cells while TGF-β1 exerted an opposite effect. Both BMP-2 and TGF-β1 had minimal effects on late passage cells. We conclude that serial passage alters MC3T3-E1 cell morphology, and significantly diminishes GJIC, osteoblastic function, TGF-β1-mediated cell proliferation, and responsiveness to TGF-β1 and BMP-2. Cell passage numbers should be clearly defined in functional studies involving MC3T3-E1 cells.