Establishing a simple protocol to induce the osteogenic differentiation of MC3T3-E1 cells in 2D and its transfer to 3D spheroid cultures.

The murine cell line MC3T3-E1 is used in many in vitro studies in bone-related research, but different protocols to induce its osteogenic differentiation have been reported. The aim of this study was to identify the best mixture of osteogenic supplements to induce osteogenic differentiation of MC3T3-E1 subclone 4 cells in a two-dimensional cell culture setup. As spheroids as three-dimensional cell aggregates are of increasing importance, we also present a simple method to generate osteogenic differ.entiated spheroids on this basis. Three different mixtures of osteogenic supplements were used to induce osteogenic differentiation for up to 28 days. Osteogenic differentiation was monitored by alizarin red and von Kossa staining, energy dispersive X-ray (EDX) analysis, and real-time quantitative PCR analysis of osteogenic marker genes. Spheroids were generated from osteogenic differentiated cells by liquid overlay technique. The use of 5 mM β-glycerophosphate, 10 nM dexamethasone, and 50 µg/mL ascorbic acid was able to induce osteogenic differentiation of MC3T3-E1 cells within 14 days, as shown by strong positive signals in both staining methods. Scanning electron microscopy revealed extracellular secretions on the membranes of differentiated cells with a significantly increased calcium content of 16.4 ± 2.4% and a phosphorus content of 10.1 ± 1.1%, as shown by energy dispersive X-ray analysis. Differentiated MC3T3-E1 cells could be detached by incubation in AccuMax for 10 min and spheroids were generated from this cell suspension on day 14. Significant upregulation of the osteogenic markers Sp7, osteocalcin, and bone sialoprotein was detected by real-time quantitative PCR analysis of these spheroids. In addition to other reports in the literature describing osteogenic differentiation of spheroids, we were able to show that it is also possible to generate spheroids from osteogenically differentiated two-dimensional cell cultures, which are easier to handle. Thus, there are indeed several ways to generate osteogenic differentiated MC3T3-E1 spheroids.