Effects of continuous passaging on mineralization of MC3T3-E1 cells with improved osteogenic culture protocol

The murine-derived MC3T3-E1 cell line provided by the American Type Culture Collection (ATCC) is a well-known osteogenic cell culture model system to test materials in vitro. However, the effect of passaging on its mineralization capacity has never been described and their culture supplements can be further optimized. Therefore, we evaluated the influence of the passage number and different osteogenic culture supplements, including ascorbic acid (AsAP) and dexamethasone (Dex) on the osteogenic capacity of MC3T3-E1 cells. This capacity was measured by the deposited calcium, the alkaline phosphatase activity, and the expression of osteogenic-related genes, including bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN). The results indicated that the mineralization capacity of MC3T3-E1 cells significantly decreased during passaging and got exhausted at passage 34, as assessed by measuring calcium deposition after 28 days of osteogenic induction. Moreover, the combination of AsAP and Dex triggered significantly more mineralization in MC3T3-E1 cells than the ATCC recommended addition of AsAP alone, as indicated by increased calcium deposition and higher expression of BSP and OPN. However, Dex alone could not trigger this effect, but only in combination with the AsAP, which indicates that Dex has no direct effect on mineralization. In conclusion, the passage number of MC3T3-E1 cells is of great importance and the use of cells above 30 passages should be avoided. In addition, the favored osteogenic supplements providing an improved osteogenic differentiation of MC3T3-E1 cells are the combination of AsAP and Dex.