3D-Cultured Vascular-Like Networks Enable Validation of Vascular Disruption Properties of Drugs In Vitro

Abstract

Vascular-disrupting agents are an interesting class of anticancer compounds because of their combined mode of action in preventing new blood vessel formation and disruption of already existing vasculature in the immediate microenvironment of solid tumors. The validation of vascular disruption properties of these drugs in vitro is rarely addressed due to the lack of proper in vitro angiogenesis models comprising mature and long-lived vascular-like networks. We herein report an indirect coculture model of human umbilical vein endothelial cells (HUVECs) and human dermal fibroblasts (HDFs) to form three-dimensional profuse vascular-like networks. HUVECs embedded and sandwiched in the collagen scaffold were cocultured with HDFs located outside the scaffold. The indirect coculture approach with the vascular endothelial growth factor (VEGF) producing HDFs triggered the formation of progressively maturing lumenized vascular-like networks of endothelial cells within less than 7 days, which have proven to be viably maintained in culture beyond day 21. Molecular weight-dependent Texas red-dextran permeability studies indicated high vascular barrier function of the generated networks. Their longevity allowed us to study the dose-dependent response upon treatment with the three known antiangiogenic and/or vascular disrupting agents brivanib, combretastatin A4 phosphate (CA4P), and 6´-sialylgalactose (SG) via semi-quantitative brightfield and qualitative confocal laser scanning microscopic (CLSM) image analysis. Compared to the reported data on in vivo efficacy of these drugs in terms of antiangiogenic and vascular disrupting effects, we observed similar trends with our 3D model, which are not reflected in conventional in vitro angiogenesis assays. High-vascular disruption under continuous treatment of the matured vascular-like network was observed at concentrations ≥3.5 ng·ml−1 for CA4P and ≥300 nM for brivanib. In contrast, SG failed to induce any significant vascular disruption in vitro. This advanced model of a 3D vascular-like network allows for testing single and combinational antiangiogenic and vascular disrupting effects with optimized dosing and may thus bridge the gap between the in vitro and in vivo experiments in validating hits from high-throughput screening. Moreover, the physiological 3D environment mimicking in vitro assay is not only highly relevant to in vivo studies linked to cancer but also to the field of tissue regeneration.

In vitro vascularization of hydrogel-based tissue constructs via a combined approach of cell sheet engineering and dynamic perfusion cell culture

“…On the basis of the confocal microscopy images, the vascular-like network in our collagen hydrogels met this criterion. As expected from previous studies [27,33], no stable HUVEC network was formed in the absence of HDFs. This was presumably related to the rapid loss in activity of the unbound medium-supplemented VEGF and fibroblast growth factor (FGF) as they are known to be unstable in the presence of cells [34].…”

“…Before combining the cell sheet-covered vascular vessel mimic with self-organizing single HUVECs, we studied the network formation capacity of collagenembedded HUVECs in a membrane insert under indirect coculture conditions with HDFs. Previously, the presence of HDFs outside the insert was proved to maintain the long-lived vascular-like network structure of HUVECs seeded in 2D between two collagen layers [27]. Here, we studied the vascular network formation of suspended GFP-HUVECs encapsulated homogenously within a 3D collagen gel.…”

Papain-Based Solubilization of Decellularized Extracellular Matrix for the Preparation of Bioactive, Thermosensitive Pregels

“…Both dECM-Papain and dECM-Pepsin pregels were transparent at acidic pH and exhibited a translucent appearance after gelling at 37 °C in neutral conditions, as indicated by the slightly reduced readability of the @-symbol. The turbidimetric gelation kinetics of dECM-Papain and dECM-Pepsin pregels were spectrophotometrically studied with stiffness-matched samples (see below) at a fixed wavelength of 350 nm at 10 mg/mL and compared to commercial rat-tail collagen type I at a concentration of 2.5 mg/mL, which has been reported to be an optimal collagen concentration for cell-laden hydrogels elsewhere. , The detection principle is based on the increase in turbidity resulting from collagen self-assembly. The normalized, time-dependent absorbance curves for dECM-Papain, dECM-Pepsin, and collagen are shown in Figure C, which exhibit a sigmoidal profile, with the dECM samples gelling after a longer lag phase than pure rat tail collagen.…”

Rapid innervation and physiological epidermal regeneration by bioengineered dermis implanted in mouse

“…Endothelial cells, HUVECs at P4, were seeded as a drop on the surface of the samples, at a high density of about 10.000/15.000 cells for mm 2 . Although the HUVECs are not derived from the dermis, we chose them since their use is very well documented for tissue engineering application due to their capacity to develop capillary-like structures when co-cultured with dermal fibroblasts [ 13 , [29] , [30] , [31] ]. HUVEC seeding was performed into low attach multiwells.…”