Recruitment of pericytes to the ablumenal surface of EC-lined tubes within vascular guidance tunnels using a new model of EC-pericyte tube coassembly in 3D collagen matrices. EC-pericyte cocultures were allowed to assemble randomly within a 3D collagen matrix for 5 days, fixed, and processed for immunofluorescence or cross-sectioning (HUVECs-bovine pericytes). (A) ECs were stained with the endothelial specific marker, CD31, whereas pericytes were GFP labeled. Bar equals 25 μm. (B) Plastic sections show association of pericytes with endothelial tubes on the abluminal face. Bar equals 25 μm. (C-D) EC-pericyte cocultures were allowed to form, and a thin plastic section of the monolayer was examined along an EC tube surface. ECs form a continuous layer with pericytes recruited to the basal surface. Black arrows indicate the border of the vascular guidance tunnel and the entrance of pericytes within these borders. Bar equals 50 μm. (E) Randomly placed ECs and GFP pericytes were allowed to form within the 3D collagen matrices for 5 days. These cultures were then immunostained with an anti-collagen type I antibody. (F) Images of the GFP pericytes were also obtained and overlaid with the corresponding collagen type I stain. Images show the clear presence of pericytes within vascular guidance tunnels. Bar equals 50 μm.