Scientists have developed a technology for generating living, fully functional 3-D microvascular networks that represents an in vitro platform for the study of vascular behavior during angiogenesis and thrombosis, and for investigating the effects of drug candidates. The microvascular networks (μVNs) are effectively created by seeding human endothelial cells (ECs) into a preformed collagen scaffold comprising a network of microfluidic channels that have an inlet and outlet to allow delivery of the both the cells and appropriate medium. Within a few weeks the seeded ECs proliferate and spread throughout the channels, generating a continuous endothelium lining the channels.
Ying Zheng, Ph.D., and colleagues at the University of Washington, Seattle, together with collaborators at Puget Sound Blood Center and Cornell University, used the microvasculature setup to study angiogenesis and vascular behavior in response to different stimuli. They report their findings in PNAS in a paper titled “In vitro microvessels for the study of angiogenesis and thrombosis.”
The microfluidic collagen scaffold was created using a lithographically etched silicon stamp. The two halves of the pressed collagen mold were effectively sandwiched together and sealed, generating an enclosed microstructure with an inlet and outlet. The channels were then flushed through with human umbilical vein endothelial cells and growth medium.
Within two weeks the cells had proliferated and formed an endothelial lining to the channels. Analyses confirmed the cells retained an endothelial phenotype and formed extensive communicating intercellular junctions, including focal adherens junctions, overlapping junctions, and complex junctions. The endothelium also acted as a barrier to the transfer of solutes from the lumen of the vessels into the scaffold matrix, and generated pinocytotic vesicles for the active transport of molecules from the lumen into the collagen matrix.
The team then introduced vasculogenic medium through the μVNs to mimic a proangiogenic environment that might be present around ischemic tissue or solid tumors. This resulted in the vessels becoming more leaky with less well-organized cell-cell contact, and the endothelial cells sprouted out into the surrounding matrix, generating the beginnings of new blood vessels.
In a second set of experiments the researchers looked at the effects of thrombotic factors on blood flow through the μVNs. When the vessels were perfused with whole human blood, the platelets flowed through without sticking to the endothelial lining. However, when blood was passed through μVNs that had been prestimulated with phorbol-12-myristate-13-acetate, the platelets formed large aggregates on the endothelial surface, partially filling the lumen. Some of these broke off into the flowing blood. And within just an hour white blood cells were seen to attach to the luminal surface of the vessel walls and begin migrating through the endothelium into the collagen matrix.
“The versatility of the μVN platform, with respect to its structure, cellular composition, and in situ control of the endothelial microenvironment, opens opportunities to address biophysical and biological questions that are difficult to access either in vivo or in conventional, planar cultures in vitro,” the authors write. Potential fields of study include the effects of geometry, hydrodynamic stresses, and transport processes on vessel stability, angiogenesis, and the interaction of the vessel with blood cells. “As our platform matures, we anticipate that μVNs can be used to test drugs and drug-delivery strategies that target microvascular function ... Scaffolds with μVNs could also provide a starting point for the growth of vascularized tissues in vitro for applications in regenerative medicine.”