Comparison of Mixed and Lamellar Coculture Spatial Arrangements for Tissue Engineering Capillary NetworksIn Vitro (original) (raw)
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Assessing the Permeability of Engineered Capillary Networks in a 3D Culture
PLoS ONE, 2011
Many pathologies are characterized by poor blood vessel growth and reduced nutrient delivery to the surrounding tissue, introducing a need for tissue engineered blood vessels. Our lab has developed a 3D co-culture method to grow interconnected networks of pericyte-invested capillaries, which can anastamose with host vasculature following implantation to restore blood flow to ischemic tissues. However, if the engineered vessels contain endothelial cells (ECs) that are misaligned or contain wide junctional gaps, they may function improperly and behave more like the pathologic vessels that nourish tumors. The purpose of this study was to test the resistance to permeability of these networks in vitro, grown with different stromal cell types, as a metric of vessel functionality. A fluorescent dextran tracer was used to visualize transport across the endothelium and the pixel intensity was quantified using a customized MATLAB algorithm. In fibroblast-EC co-cultures, the dextran tracer easily penetrated through the vessel wall and permeability was high through the first 5 days of culture, indicative of vessel immaturity. Beyond day 5, dextran accumulated at the periphery of the vessel, with very little transported across the endothelium. Quantitatively, permeability dropped from initial levels of 61% to 39% after 7 days, and to 7% after 2 weeks. When ECs were co-cultured with bone marrow-derived mesenchymal stem cells (MSCs) or adipose-derived stem cells (AdSCs), much tighter control of permeability was achieved. Relative to the ECfibroblast co-cultures, permeabilities were reduced 41% for the EC-MSC co-cultures and 50% for the EC-AdSC co-cultures after 3 days of culture. By day 14, these permeabilities decreased by 68% and 77% over the EC-fibroblast cultures. Cocultures containing stem cells exhibit elevated VE-cadherin levels and more prominent EC-EC junctional complexes when compared to cultures containing fibroblasts. These data suggest the stromal cell identity influences the functionality and physiologic relevance of engineered capillary networks.
Nephrology Dialysis Transplantation, 2005
Background. There are conflicting results regarding the role of periendothelial mural cells in angiogenesis. In the current study, we investigated the role of mesangial cells (MCs) in endothelial vascularization by using a three-dimensional co-culture system in basement-membrane reconstruct gel (Matrigel). Methods. Human umbilical vein endothelial cells (ECs) and human MCs were used. In the contact co-culture system, ECs and MCs were mixed and then plated together onto Matrigel. In the non-contact co-culture system, MCs were cultured within an intercup chamber, which prevented direct physical contact with the ECs on the Matrigel but allowed both cell types to share culture medium. To visualize ECs and MCs, the cells were labelled with two different fluorescent dyes prior to the co-culture. A capillary-like network formation was observed under a fluorescent microscope and confocal microscope, and the length of the network formation was quantified by the image analyzer. Results. ECs barely formed capillary-like networks when cultured alone in growth factor-free medium. However, ECs cultured with MCs in a contact condition remarkably formed capillary-like networks (9.10±0.96 vs 0.20±0.07 mm/mm 2 at 6 h, contact vs ECs alone, P<0.001). Direct contact between ECs and MCs was clearly demonstrated by confocal microscopy. Differentiation into branching capillarylike structures was also observed in the non-contact co-culture system (3.02±1.21 mm/mm 2 at 6 h, P<0.001 vs ECs alone), but less prominently than in the contact co-culture condition. Vascular endothelial growth factor (VEGF) was secreted from MCs, as determined by enzyme-linked immunosorbent assay and immunofluorescent study. Adding neutralizing antibodies against VEGF into the co-culture system partially inhibited capillary network formation. Conclusions. Our data indicate that MCs help ECs differentiate toward vascularization, in which the direct cell-cell contact between ECs and MCs plays an important role. VEGF is a mediator in this process.
Microvascular Research, 2010
Angiogenesis is a complicated natural process, and understanding the mechanism by which it occurs is important for medical, pharmaceutical, and cell biological sciences. Many techniques for investigating angiogenesis have been reported. In this study, we introduced a novel application of a cell culture technique that can be used in in vitro and in vivo vascular biology research. Cultivated endothelial cells (ECs) were harvested from temperature responsive culture dishes by reducing the temperature, without the need for a proteinase treatment. For this technique, the direct contact of ECs with fibroblasts was important for the formation of a capillary-like network in vitro. Moreover, layered cell sheets containing EC-networks produced lumen and vascular structures in the three-dimensional constructs, as well as in the construct transplanted into a living body. Thus, our culture technique was able to create cell sheets and three-dimensional constructs containing ECnetworks, because they preserved normal and intrinsic cell-cell direct contact and various cell adhesive factors. Moreover, the thickness of these three-dimensional (3-D) constructs could be controlled by the number of layered cell sheets. These observations indicated that our novel technology contributed to the progress of vascular biology and lead to a new tool that can be used in in vivo and in vitro vascular biology research.
Formation of microvascular networks in vitro
Nature Protocols, 2013
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 userdefined geometries of the vascular network and microfluidic perfusion to control mass transfer and hemodynamic forces. these microvascular networks (mVns) 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.
Transport-mediated angiogenesis in 3D epithelial coculture
The FASEB Journal, 2009
Increasing interest has focused on capturing the complexity of tissues and organs in vitro as models of human pathophysiological processes. In particular, a need exists for a model that can investigate the interactions in three dimensions (3D) between epithelial tissues and a microvascular network since vascularization is vital for reconstructing functional tissues in vitro. Here, we implement a microfluidic platform to analyze angiogenesis in 3D cultures of rat primary hepatocytes and rat/human microvascular endothelial cells (rMVECs/hMVECs). Liver and vascular cells were cultured on each sidewall of a collagen gel scaffold between two microfluidic channels under static or flow conditions. Morphogenesis of 3D hepatocyte cultures was found to depend on diffusion and convection across the nascent tissue. Furthermore, rMVECs formed 3D capillary-like structures that extended across an intervening gel to the hepatocyte tissues in hepatocyte-rMVEC coculture while they formed 2D sheet-like structures in rMVEC monoculture. In addition, diffusion of fluorescent dextran across the gel scaffold was analyzed, demonstrating that secreted proteins from the hepatocytes and MVECs can be exchanged across the gel scaffold by diffusional transport. The experimental approach described here is useful more generally for investigating microvascular networks within 3D engineered tissues with multiple cell types in vitro.-Sudo, R., Chung, S., Zervantonakis, I. K., Vickerman, V., Toshimitsu, Y., Griffith, L. G., Kamm, R. D. Transportmediated angiogenesis in 3D epithelial coculture. FASEB J. 23, 2155-2164 (2009)