Establishment of an in vitro vascular anastomosis model in a microfluidic device

Abstract

The demand for organ transplantation has rapidly increased over the past few decades due to a large number of patients with chronic organ failure. However, organ shortage is a critical problem in medical transplantation, including liver and kidney (Caplan, 2016; Hong et al., 2014). To overcome this problem, it is necessary to construct transplantable organ/tissue grafts in vitro. Over the last decade, researchers have attempted to construct transplantable bioengineered tissue grafts in vitro (Kasuya et al., 2012; Palakkan et al., 2013; Stevens et al., 2017). Despite significant efforts, these studies remain in the early stages of research due to poor graft survival after transplantation (Caralt et al., 2015; Damania et al., 2014). Abstract Formation of vascular anastomoses is critical for the development of transplantable tissue-engineered grafts, because rapid blood perfusion is required for the maintenance of implanted tissue grafts. However, the process of vascular anastomosis remains unclear due to difficulties in observing vascular anastomosis after transplantation. Although several groups have developed in vitro models of vascular anastomosis, there is a lack of a suitable in vitro anastomosis model that includes perivascular cells. Therefore, we aimed to establish an in vitro vascular anastomosis model containing perivascular cells by a combination of human umbilical vein endothelial cell (HUVEC) monoculture and HUVEC-mesenchymal stem cell (MSC) coculture in a microfluidic device. We found that vascular formation was inhibited when HUVECs were seeded on both sides of gel scaffolds, but HUVECs formed vascular networks when they were seeded on one side only. Next, we tested a series of HUVEC:MSC ratios to induce vascular anastomoses. The results demonstrated that addition of MSCs induced vascular anastomosis. In particular, the number of vascular anastomoses was significantly increased at a HUVEC:MSC ratio of 2:8. The process of vascular anastomosis was further investigated by live-cell imaging of green fluorescent protein-expressing HUVECs, which revealed that vascular anastomoses with continuous lumens were constructed during days 8–10. Computational simulation of VEGF concentrations suggested that local VEGF gradients play important roles in vascular formation while the addition of MSCs was critical for anastomosis. This anastomosis model will provide insights for both the development of tissue-engineered grafts and for the construction of large tissues by assembling multiple tissue-engineered constructs.