Osteocyte-Osteoblast Coculture Protocol

Ocy454 cells, a murine osteocyte line, were kindly provided by Prof. Paola Divieti Pajevic (Boston University, Henry M. Goldman School of Dental Medicine). MC3T3-E1 cells were purchased from The Cell Bank of Type Culture Collection of the Chinese Academy of Sciences. Ocy454 cells were differentiated in α-minimum essential medium (α-MEM, Gibco; Thermo Fisher Scientific, Inc.) containing 10% fetal bovine serum (Gibco) at 37 °C for 10 days. Cells were then trypsinised and subcultured in 6-well plates (2 × 10⁵ cells per well) or 96-well plates (1 × 10⁴ cells per well) for 12 h before subsequent experiments. For coculture experiments, Ocy454 cells were seeded onto 0.4-mm-thick polycarbonate inserts (Costar Corning, Life Sciences, Acton, MA, USA) in 24-well plates while MC3T3-E1 cells were plated in 24-well plates in α-MEM medium. Ocy454 and MC3T3-E1 cells were cocultured in a mixed medium (α-MEM and osteogenic differentiation medium, 1:1 ratio; Cyagen) for 28 days. The medium was replaced every 2 days, and the inserts seeded with Ocy454 cells were replaced every 4 days to prevent unwanted effects associated with excessive cell density.