2.3. Vascular Vessel Models

The fabrication process of the vascular vessel model is shown in Figure 2. The device consisted of five channels (Figure 2A). To set a bottleneck of ion current flows due to cells, PDMS bottlenecks were prepared, as shown in Figure S1. HUVECs (8.0 × 10⁶ cells/mL) were suspended in a precursor solution of fibrin/collagen gel (2.5 mg/mL fibrinogen (Sigma-Aldrich, Burlington, MA, USA), 0.15 U/mL aprotinin (Sigma-Aldrich), 0.2 mg/mL collagen (Corning Inc., Corning, NY, USA), and 0.5 U/mL thrombin (Sigma-Aldrich), and Dulbecco’s phosphate-buffered saline (D-PBS, Nacalai, Japan)) and the solution was then introduced into Channel (Ch.) 3 (Figure 2B). Owing to the surface tension, only Ch. 3 was filled with the solution. In addition, hLFs (5.0 × 10⁶ cells/mL) were suspended in the precursor solutions, and the solutions were introduced into Chs. 1 and 5. The solutions were incubated at 37 °C for 15 min to allow gelation. Then, Chs. 2 and 4 were filled with the culture medium of ECGM2, and these cells were cultured for 2 d to induce vasculogenesis at Ch. 3. After culturing for 2 d, the device was maintained in a tilted position, and additional HUVECs (5.0 × 10⁶ cells/mL) were introduced from Ch. 2 to be seeded on the side wall of the gel at Ch. 3. After 1 h to allow cell attachment, additional HUVECs (5.0 × 10⁶ cells/mL) were introduced from Ch. 4 to prepare endothelial monolayers on the opposite side of the gel. The cells were then cultured to induce angiogenesis, and the culture medium was changed daily, when cellular debris was observed to judge the perfusability of the channel separated by the fibrin/collagen gel. The network formation of HUVECs was subsequently observed under a fluorescence microscope (Eclipse Ts2, Nikon, Tokyo, Japan).

Fabrication of the vascular vessel models. (A) Device outline. (B) Cell culturing. (i)–(iv) HUVECs were cultured in gel in Ch. 3 for vasculogenesis. (v)–(vi) Additional HUVECs were seeded onto the gel sidewalls to prepare monolayers and were cultured for angiogenesis. Not to scale. Although only a simple vessel is illustrated here, in actuality a network of vessels is formed.