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Formation of microvascular networks in vitro.

Nature protocols (2013-08-31)
John P Morgan, Peter F Delnero, Ying Zheng, Scott S Verbridge, Junmei Chen, Michael Craven, Nak Won Choi, Anthony Diaz-Santana, Pouneh Kermani, Barbara Hempstead, José A López, Thomas N Corso, Claudia Fischbach, Abraham D Stroock
ABSTRACT

This protocol describes how to form a 3D cell culture with explicit, endothelialized microvessels. The approach leads to fully enclosed, perfusable vessels in a bioremodelable hydrogel (type I collagen). The protocol uses microfabrication to enable user-defined geometries of the vascular network and microfluidic perfusion to control mass transfer and hemodynamic forces. These microvascular networks (μVNs) allow for multiweek cultures of endothelial cells or cocultures with parenchymal or tissue cells in the extra-lumen space. The platform enables real-time fluorescence imaging of living engineered tissues, in situ confocal fluorescence of fixed cultures and transmission electron microscopy (TEM) imaging of histological sections. This protocol enables studies of basic vascular and blood biology, provides a model for diseases such as tumor angiogenesis or thrombosis and serves as a starting point for constructing prevascularized tissues for regenerative medicine. After one-time microfabrication steps, the system can be assembled in less than 1 d and experiments can run for weeks.

MATERIALS
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Product Description

Sigma-Aldrich
Albumin, Bovine Serum, Fraction V, Fatty Acid-Free, Nuclease- and Protease-Free, BSA Fatty Acid-free is designed for use in serological testing, RIA, and hormone response studies. Suitable for use in Molecular Biology applications, such as Northern and Southern blots.
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FGF-2 / basic FGF Protein, Human recombinant, The FGF-2 / basic FGF Protein, Human recombinant is available in a 25 µg format.
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VEGF Protein, Human recombinant, The human recombinant VEGF Protein is available in a 10 µg format.
Sigma-Aldrich
VEGF165 Protein, Human Recombinant Animal Free, The Vascular endothelial growth factor (VEGF or VEGFA) is a potent mediator of both angiogenesis & vasculogenesis in the fetus & adult. Manufactured using all non-animal reagents.