Fig 1.
Fabrication of perfusable vascular-like structures by using an electrochemical reaction.
(A) Modification of the multi-needle surface with zwitterionic oligopeptides to mediate endothelial cell adhesion. (B) Placement of the HUVEC-coated needle in a culture chamber. Pouring and gelation of collagen in the chamber. Application of electric potential to transfer HUVECs to the internal surface of the microchannels within the collagen gel and desorption of the molecular layer. (C) Needle removal and culture medium perfusion. (D) Gold-coated multi-needle (9 needles; diameter, 500 μm; pitch, 500 μm; length, 1 cm).
Fig 2.
Endothelial cell sprouting in collagen gel under stationary culture conditions in a culture dish.
(A) SEM image of HUVECs on a gold rod prior to cell transfer. The inset is a magnified view. (B) HUVECs transferred onto the internal surface of a microchannel in collagen gel after 48 h of culture. (C, D) HUVECs migrating into the collagen gel at 14 d. The box in (C) indicates the location of (D). (E, F) Image analysis. Sprouts were traced and quantified using image analysis software (E, S1 Movie). The sprout length was quantified at 21 d (F).
Fig 3.
Effects of PMA on endothelial cell vascularization in collagen gel.
(A) Schematic diagrams of the experimental design. (B) Phase-contrast microscopy images of HUVECs exposed to various PMA concentrations. HUVECs did not sprout into the collagen gel in the absence of PMA, forming a monolayer on the surface. When exposed to 20–500 ng/mL PMA, HUVECs formed typical luminal structures. At 1000 ng/mL PMA, sprouting was inhibited and cells became detached. Scale bar, 1 mm.
Fig 4.
Effects of shear stress on endothelial cell alignment.
(A–C) HUVEC layers electrochemically transferred to the internal surfaces of microchannels and cultured in chambers for 12 h in the presence or absence of culture medium perfusion. HUVECs were partially detached in the absence of perfusion (A, arrows), but continued to cover the surface at a flow rate of 1 μL/min (B) and 10 μL/min (C). Scale bars, 500 μm. (D–G) Quantification of HUVEC orientation with respect to flow direction after 12 h of culture in the presence or absence of perfusion. The angle and mean amplitude at 0 μL/min (D), 1 μL/min (E), and 10 μL/min (F), and comparisons of the orientation intensity (G). *P < 0.05, **P < 0.01. Error bars indicate standard deviations calculated from three independent experiments.
Fig 5.
Synergistic effects of PMA and perfusion on endothelial cell sprouting.
(A) Cross-sections of 3D-aligned HUVEC-enveloped microchannels. GFP-HUVECs were transferred to the internal surfaces by using the multi-needle. (B) HUVEC sprouting after 48 h of culture under perfusion at 10 μL/min and 50 ng/mL PMA. (C) Sprouts extended toward neighboring microchannels and bridged them after as few as 7 d. (D) Confocal microscopy image of luminal structures. (E) Quantification of the sprout length. Static culture in the absence of PMA (○), perfusion culture in the absence of PMA (Δ), and perfusion culture in the presence of PMA (■). Error bars indicate standard deviations calculated from three independent experiments. Scale bars indicate 500 μm in (A, C), and 100 μm in (B, D).