FL cells incorporated into HUVEC-derived tube-like structure as well as connections and enhanced tube formation.

BMMNCs are separated to attached (AT) cells and floating (FL) cells 24 hour after culture with 10%FBS/DMEM medium on temperature-sensitive culture plate followed by further culture with 10%FBS/EBM2 growth factor containing medium for 3 days. The non-separated total adhered cells (TT) were uses as a control. HUVECs and DiI-acLDL-labeled TT cells, AT cells or FL cells are co-cultured with 10%FBS/EBM2 on Matrigel^TM coated 12-well plate. After 24 hours in culture, incorporation of each DiI-ac LDL-labeled cells was observed under a phase-contrast/fluorescent microscope. (A), Phase-contrast images. (B), Merged images of phase-contrast and fluorescent images. Arrows indicate DiI+ cells. One selected field is magnified in the left lower corner in the FL (B) image. Arrow indicates DiI+ cells. (C) Tube length and connection number were measured and counted, respectively, 48 hours after culture. Relative values of tube length to TT cells (C) and that of connection number to TT cells (D) were calculated and averaged. (n = 3) *, P<0.05 vs. AT. All experiments were performed in triplicate and confirmed the reproducibility.