Serial passage of MC3T3‐E1 cells down‐regulates proliferation during osteogenesis in vitro

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Abstract
Abstract.  Generally, fibroblast‐like cells and other types of human cells have been used to demonstrate the principles of replicative senescence in vitro and in vivo. These cells go through three stages of proliferation, including vigorous proliferation, declining proliferation and quiescence or no proliferation. Any variation of this process occurring in osteoprogenitor cells may offer insight into the mechanism of age‐related osteopaenia that predisposes individuals to osteoporosis and bone fractures. We selected MC3T3‐E1 cells derived from mouse calvaria to study the mechanism of replicative senescence of pre‐osteogenic cells because: (i) these cells constitute a well‐known model for studying osteogenesis in vitro; (ii) they undergo a developmental sequence of proliferation and differentiation similar to primary cells in culture; and (iii) they show signs of replicative senescence. These cells were aged by multiple passaging before their use for studying growth kinetics and the effects of population density, effect of extracellular matrix (ECM), size and phases of the cell cycle. Our results show that (i) MC3T3‐E1 cells go through the first two stages of proliferation in a manner similar to human cells, but escape the quiescent phase; (ii) the rate of proliferation is similar for low passage (LP) and high passage (HP) cells, but is decreased in very high passage cells (VHP); (iii) growth inhibition is observed using HP cells seeded at high density; (iv) HP ECM stimulates proliferation of both LP and HP cells; (v) a small increase in cell size is observed in HP cells, but no change is seen in the distribution analysis of their cell cycle; (vi) distribution analysis of the cell cycle of VHP cells reveals a decreased and an increased frequency of cells in S and G2 + M phases of their cell cycle, respectively. These results suggest that the mouse MC3T3‐E1 cell line exhibits many of the cellular and molecular markers associated with replicative senescence in culture as defined by human cells, such as fibroblast‐like cells. Alteration in the sensitivity of MC3T3‐E1 cells to intercellular contact and increase in cell size are the primary factors contributing to decreased proliferation of HP cells.