Bone-marrow-on-a-chip allows the monitoring of HSPCs in contact with mesenchymal stem cells in 3D hydrogels. (A) Plan of the microfluidic chip: it comprises the endosteal (2) and the vascular (4) compartments, the HSPC injection channel (3), the cytokine-secreting fibroblast compartment (5), and channels for medium circulation (1 and 6). The inset describes the organization of the central channels loaded with cells. (B) Left: Transmitted light image of the three central channels. Right: Colorized individual trajectories of HSPCs during a time-lapse sequence. Scale bar = 200 µm. (C) Maximum projection of 10-µm-wide Z stack confocal images of HSPCs in the endosteal (upper panel) and vascular (lower panel) compartments. HSPCs (CD34⁺) appear in green, actin structures in red, DNA in blue, and centrosomes in white. Scale bar = 10 µm. (D) Selected Z stacks of HSPCs in the endosteal and vascular compartments are presented in the upper and lower panel, respectively. The HSPC centrosome can be defined relative to the point of contact of the HSPC with the osteoblast or endothelial cell (marked with a white arrow) as proximal (left), in an intermediate position (middle), or distal (right). Actin appears in green, centrosome in white, and DNA in blue. Scale bar = 10 µm. Right: Distribution of HSPCs with proximal, intermediate, or distal centrosome in the vascular (n = 73) and endosteal (n = 199) compartments from three independent experiments. Error bars represent SD.