Abstract
Fabrication of vascular networks is essential for engineering three-dimensional thick tissues and organs in the emerging fields of tissue engineering and regenerative medicine. In this study, we describe the fabrication of perfusable vascular-like structures by transferring endothelial cells using an electrochemical reaction as well as acceleration of subsequent endothelial sprouting by two stimuli: phorbol 12-myristate 13-acetate (PMA) and fluidic shear stress. The electrochemical transfer of cells was achieved using an oligopeptide that formed a dense molecular layer on a gold surface and was then electrochemically desorbed from the surface. Human umbilical vein endothelial cells (HUVECs), adhered to gold-coated needles (ϕ600 μm) via the oligopeptide, were transferred to collagen gel along with electrochemical desorption of the molecular layer, resulting in the formation of endothelial cell-lined vascular-like structures. In the following culture, the endothelial cells migrated into the collagen gel and formed branched luminal structures. However, this branching process was strikingly slow (>14 d) and the cell layers on the internal surfaces became disrupted in some regions. To address these issues, we examined the effects of the protein kinase C (PKC) activator, PMA, and shear stress generated by medium flow. Addition of PMA at an optimum concentration significantly accelerated migration, vascular network formation, and its stabilization. Exposure to shear stress reoriented the cells in the direction of the medium flow and further accelerated vascular network formation. Because of the synergistic effects, HUVECs began to sprout as early as 3 d of perfusion culture and neighboring vascular-like structures were bridged within 5 d. Although further investigations of vascular functions need to be performed, this approach may be an effective strategy for rapid fabrication of perfusable microvascular networks when engineering three-dimensional fully vascularized tissues and organs.
Highlights
Regenerative medicine has attracted increasing attention as a new therapy that circumvents the shortage of donor organs for transplantation and can potentially cure various severe diseases by using a patient’s own cells or other immunologically matched stem cells [1,2]
We examined the effects of phorbol 12-myristate 13-acetate (PMA) and shear stress on sprouting as well as their synergistic effects in our system with the aim of finding a new approach for engineering vascularized 3D large and thick tissues and organs
Fabrication of endothelialized microchannels in collagen gel Human umbilical vein endothelial cells (HUVECs) were seeded on gold-coated single needles modified with oligopeptides and were grown until they reached confluence
Summary
Regenerative medicine has attracted increasing attention as a new therapy that circumvents the shortage of donor organs for transplantation and can potentially cure various severe diseases by using a patient’s own cells or other immunologically matched stem cells [1,2]. Thin and avascular tissues such as skin [3], cartilage [4], and the cornea [5] have been successfully treated using engineered cellular replacements. Engineering of thick and cell-dense organs such as the liver and pancreas have lagged behind. Thick organs in the body rely on the supply of oxygen and nutrients from vascular networks. A reliable strategy for the fabrication of spatially aligned vascular networks in vitro is desired in order to engineer three-dimensional (3D) thick tissues and organs for regenerative medicine
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