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Understanding cell migration in a 3D microenvironment is essential as most cells encounter complex 3D extracellular matrix (ECM) in vivo. Although interactions between cells and ECM have been studied previously on 2D surfaces, cell migration studies in 3D environment are still limited. To investigate cell migration under various degrees of confinements and coating conditions, 3D platforms with micropost arrays and controlled fibronectin (FN) protein coating were developed. MC3T3-E1 cells spread and contacted the top surface of microposts if FN was coated on top. When FN was coated all over the microposts, cells were trapped between microposts with 3 μm spacing and barely moved. As the spacing between microposts increased from 3 to 5 μm, cells became elongated with limited cell movement of 0.18 μm/min, slower than the cell migration speed of 0.40 μm/min when cells moved on top. When cells were trapped in between the microposts, cell nuclei were distorted and actin filaments formed along the sidewalls of microposts. With the addition of a top cover to introduce cell confinement, the cell migration speed was 0.23 and 0.84 μm/min when the channel height was reduced from 20 to 10 μm, respectively. Cell traction force was monitored at on the top and bottom microposts with 10 μm channel height. These results show that theMC3T3-E1 cellmorphology, migration speed, and movement position were affected by surface coating and physical confinement, which will provide significant insights for in vivo cell migration within a 3D ECM. [ABSTRACT FROM AUTHOR]