Tissue-engineered 3D microvessel and capillary network models for the study of vascular phenomena

Abstract

Advances in tissue engineering, cell biology, microfabrication, and microfluidics have led to the development of a wide range of vascular models. Here, we review platforms based on templated microvessel fabrication to generate increasingly complex vascular models of (i) the tumor microenvironment, (ii) occluded microvessels, and (iii) perfused capillary networks. We outline fabrication guidelines and demonstrate a number of experimental methods for probing vascular function such as permeability measurements, tumor cell intravasation, flow characterization, and endothelial cell morphology and proliferation.

Keywords: capillary networks; metastasis; microfabrication; microfluidics; microvascular models; self-organization; templating; vessel occlusion.

Figure 1.

Relevant design considerations for templated vascular models. VEGF-vascular endothelial growth factor, PMA- phorbol 12-myristate 13-acetate, bFGF- basic fibroblast growth factor growth factor, HGF- hepatocyte growth factor, cAMP-cyclic adenosine monophosphate.

Figure 2.

Schematic illustration of template synthesis of a microvessel. A single cylindrical channel is formed by casting a solution form of extracellular matrix material (ECM) around a template rod. The rod is removed and the channel is perfused with media and endothelial cells to form a confluent vessel endothelium.

Figure 3.

Microvessel functionality. (A) 3D projection from confocal z-stacks of a HUVEC microvessel fluorescently stained for VE-cadherin (green) and DAPI (blue). Maximum intensity projection of the lower half of the vessel (right). (B) Phase-contrast and fluorescence time-series depicting 70 kDa dextran (green) perfused through a HUVEC microvessel demonstrating retention of the tracer molecule and slow leakage into the surrounding ECM. (C) Corresponding fluorescence image intensity over time, showing a steady increase in tracer molecule accumulation in the ECM. The permeability can be calculated from the intensity profile. (D) Fluorescence time-series showing a focal leak of 70 kDa dextran from the microvessel that dissipates over 8 minutes.

Figure 4.

Imaging endothelial proliferation, cell loss, and morphology within the vessel monolayer. (A) Illustration of the imaging plane focused on the bottom of the vessel and projected in 2D with a representative phase-contrast image (bottom). (B) Time-series images depicting mitosis and apoptosis of VeraVec-GFP endothelial cells in phase-contrast and GFP (green). Arrows indicate mitotic and apoptotic cells. (C) Time-series showing VeraVec alignment in the direction of flow in response to shear stress increased from 7 to 14 dyne cm⁻² over 45 hours. Flow is from left to right in all images.

Figure 5.

Tumor-microvessel models of metastasis. (A) Illustration of platform observed from the side with tumor cells incorporated within the ECM. (B) Representative image of device connected to external tubing and flow. (C) Illustration of platform in 3D with the imaging plane focused in the middle of the vessel. (D) Representative 3D projection obtained from confocal fluorescence microscopy of a vessel lined with VeraVec-GFP (green) endothelial cells and a dual-labeled MDA-MB-231 breast cancer cell (nucleus (green), cytoplasm (red)) located on the periphery. (E) Illustration of a 2D projected image obtained from focusing at the middle of the vessel. (F) Wide field fluorescence images overlaid with phase-contrast focused at the middle of the vessel.

Figure 6.

Time-series images of invasion and intravasation within a tumor-microvessel model. (A) Dual labeled MDA-MB-231 breast cancer cell (nucleus (green), cytoplasm (red) invades into the ECM-microvessel interface. VeraVec-GFP (green) microvessel. (B) Breast cancer cell intravasation and detachment into vessel flow. HMVEC microvessel labeled with BSA-488 (green). (C) An orthogonal view of breast cancer cell invasion into the ECM-microvessel interface and detachment into flow. Image series adapted from reference [75]. HMVEC microvessel labeled with BSA-488 (green).

Figure 7.

Occluded microvessel model. (A) Schematic illustration of microvessel occlusion model. (B) Access rods actuated to occlude microvessel. (C) Phase contrast image of microvessel and access rod before occlusion. (D) Phase contrast image of partially occluded microvessel. (E) Fluorescence image of a VeraVec microvessel before occlusion. (F) Image of an occluded VeraVec microvessel. (G) Fluorescence image of microvessel perfusion with microspheres. (H) Fluorescence image of an occluded microvessel perfused with microspheres.

Figure 8.

Perfused capillary network model. (A) Schematic illustration showing a capillary network formed between two microvessels. (B) Schematic illustration showing flow conditions for optimizing capillary formation. (C) Fluorescence image showing two VeraVec microvessels (prior to capillary formation) with dextran supplemented media in one vessel to confirm independent perfusion. (D) Image showing capillary sprouting from a VeraVec microvessel. (E) Anastomosing capillary, and (F) DAPI stain of the same capillary comprised of ~9 cells. (G) Confocal image showing that capillaries form well defined lumens. (H) Fluorescence image of a long, branched capillary. (I) Fluorescence images showing capillary network (top) and perfusion of the same network with fluorescent microbeads (bottom) to identify perfused, functional capillaries.

Figure 9.

In vitro capillary flow assay. (A) A region of the microvasculature lacking capillaries and sequential fluorescence time-lapse images of dextran perfusion. (B) A region containing a capillary bed. (C) Analysis of fluorescence images showing ~6.5X difference in dextran accumulation in vascularized tissue (blue) compared un-vascularized tissue (red) after 15 minutes. The capillary perfusion ratio (CPR) is about 6.5 at 15 minutes. (D) Analysis showing dextran intensity in the downstream microvessel in vascularized and un-vascularized tissue. When dextran is detected in the downstream microvessel, full perfusion of the capillary bed has occurred.