Clamped nanofibrous fibrinogen scaffolds were used to mimic this basal lamina of bone marrow sinusoids. Both sides of the scaffold were seeded with different cells (HMEC-1 cells, iMSC#3 cells, HSPCs). After a culture period of 7 days, the scaffolds were unclamped, fluorescently stained and mounted for microscopy analysis. The video consists of a 3D reconstruction (maximum intensity projection mode + transparency mode) and a z-stack based on the same microscopy images. The transparency mode provides additional insights as it facilitates the visualization of the cell locations on both sides of the scaffold. For the video preparation images of the CD45 staining (pink), iMSC staining (yellow), F-actin staining (turquoise) and nucleus staining (white) were taken. Additionally, the scaffold autofluorescence is visible in yellow and turquoise. For clarity, the nucleus staining was omitted from the 3D reconstruction videos. The images were taken with a cLSM in the 10x objective using the Airyscan detector in the multiplex mode (SR 4Y). This particular video shows microscopy images of a fibrinogen scaffold seeded with HMEC-1 cells and HSPCs (on the same side of the scaffold).