Formation of microvascular networks in vitro

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.

Harnessing developmental processes for vascular engineering and regeneration

“…The possibility of creating pre-vascularized tissue constructs is an exciting application of vascular engineering and regeneration. Biomimetic models to simulate angiogenic sprouting morphogenesis in vivo have also been developed using a tissue-molding technique (Morgan et al, 2013;Nguyen et al, 2013). The development of highly organized in vitro vascular constructs also presents a powerful screening platform for studying the effects of different angiogenic factors.…”

“…The microfabrication technique has been widely used to create artificial tissue constructs, including microvascularized networks embedded within a 3D hydrogel, which can be applied as a platform to mimic in vivo vascular microenvironments. Fischbach-Teschel and colleagues recently developed a novel approach to create fully enclosed and perfusable blood vessels in collagen I hydrogels using microfabrication, which allows the control of nutrients and oxygen transfer, and of blood flow rate and pressure (Zheng et al, 2012;Morgan et al, 2013). This technique can be used to study vascular function across multiple parameters, such as mass transfer, which is the transfer of nutrients and oxygen across the vessel wall, physical parameters, such as blood flow and pressure, and drug screening (Rodriguez-Rodriguez et al, 2012;Griep et al, 2013;Westein et al, 2013).…”

State-of-the-Art Methods for Evaluation of Angiogenesis and Tissue Vascularization

“…Other variations of such models have been to create tube channels artificially in 3D matrixes to enable user-defined geometries of the vascular network and then seed the channels with ECs for the creation of tube networks in the absence of EC-driven tubulogenesis and vascular guidance tunnel formation. 22,23 Of note, these systems have so far not been tested by many laboratories, and the robustness and practicability at a larger scale remain to be established.…”

Tumour-on-a-chip: microfluidic models of tumour morphology, growth and microenvironment

“…Using microfluidic technology, capillary lumen structures have been fabricated to mimic the microvasculature in tissues [94]. Common methods to create capillary lumen structures as microvasculatures include moulding the capillaries in hydrogels by needles or rods [95][96][97][98], by photoresists [99][100][101], by sacrificial carbohydrates [102] or creating lumens based on viscous fingering instabilities [103,104]. Alternatively, an endothelial vascular network as the microvasculature can be formed by endothelial sprouting in hydrogels [105][106][107][108][109][110][111][112], monolayer on ECM hydrogel [113][114][115] or on a porous membrane [116,117], and monolayer in microchannels [118,119] (figure 2l ).…”