Advances in on-chip vascularization

Abstract

Microfluidics is invaluable for studying microvasculature, development of organ-on-chip models and engineering microtissues. Microfluidic design can cleverly control geometry, biochemical gradients and mechanical stimuli, such as shear and interstitial flow, to more closely mimic in vivo conditions. In vitro vascular networks are generated by two distinct approaches: via endothelial-lined patterned channels, or by self-assembled networks. Each system has its own benefits and is amenable to the study of angiogenesis, vasculogenesis and cancer metastasis. Various techniques are employed in order to generate rapid perfusion of these networks within a variety of tissue and organ-mimicking models, some of which have shown recent success following implantation in vivo. Combined with tuneable hydrogels, microfluidics holds great promise for drug screening as well as in the development of prevascularized tissues for regenerative medicine.

Keywords: angiogenesis; microfluidics; microvasculature; organ-on-a-chip; permeability; regenerative medicine; tissue-engineering; vasculogenesis.

Figure 1.. Vascular network formation in vivo.

(A) Vascular progenitor cells directly form the dorsal aorta and cardinal vein (intra-embryonic) or coalesce into endothelial lined blood islands, which subsequently fuse into a primary plexus (extra-embryonic) and together form primary vasculature. Upon assembly, arteries and veins inosculate into smaller arterioles and venules (80–100 μm), and subsequent branching forms smaller capillaries and venules (10–15 μm). (B) Angiogenesis ensues and vessels mature and remodel, directed by a variety of angiogenic factors and cytokines (acidic FGF, bFGF, TGF-α), TFG-β, HGF, TNF-α, angiogenin, IL-8 and the angiopoietins [3]). (C) Pressure drives the perfusion of blood through capillaries, resulting in an efficient method for exchange of O₂ and small molecules across the endothelium, and larger molecules, such as drugs, delivered through the endothelium. Connection to postcapillary venules drives the inward transport of CO₂ and waste. Mechanical factors such as wall shear stress and axial strain also direct angiogenesis. (D) EC-secreted factors, such as TGF-β1, recruit mural cells during angiogenic remodeling. Pro-angiogenic factors are released from stromal cells such as fibroblasts, directing migration and sprouting of ECs. (E) Along with mural cells, and a continuously remodeling BM, ECs largely comprise the endothelium. Specialized organelles, WPBs, contained within ECs respond to various agonists (thrombin, histamine, VEGF, epinephrine, among others) by secretion of a variety of factors involved in wound healing and thrombosis. The most abundantly secreted glycoprotein is vWF. Secretion of vWF into circulating blood results in its binding to a blood-clotting factor, VIII, in its inactivated state. In the presence of thrombin, VIII is released, and vWF binds to glycoprotein Ib allowing for platelet adhesion and plug formation in sites of vascular injury. BM: Basement membrane; EC: Endothelial cell; WPB: Weibel Palade bodies; vSMC: Vascular smooth muscle cell; vWF: von Willebrand Factor. Concepts for Figure 1A & B are adapted with permission from [2].

Figure 2.. Examples of patterned network formation in vitro.

(A) Patterned vascular channels in polydimethyl siloxane. Low and high shear flows are simulated and examined in bifurcated channels lined with ECs. (B) Networks cast in hydrogel. Shown are networks of ECs lining channels in collagen embedded with pericytes (green, top and left). Pro-angiogenic factors caused pericyte localization and angiogenic sprouting into the gel. Bottom right shows the network perfused with 70 kDa dextran. (C) An enlarged region of cerebral cortex vasculature generated from a confocal image stack (red) was used to generate a (green) biomimetic microfluidic network by image-guided laser based degradation of PEGDA hydrogel. (D) Sacrificial scaffold of carbohydrate glass used as a model for vascular networks embedded in hydrogels. Right image shows a representative EC-lined channel (Human umbilical vein endothelial cell, mcherry) surrounded by mural cells (10T1/2 mouse fibroblasts, EGFP) after 9 days in culture. (E) Multi-layered implantable vascularized organ-on-chip, AngioChip. Shown (from left to right) is a scanning electron microscopy image of the porous scaffold, an entirely perfused chip, staining of ECs for CD31 on day 2 of culture in the device, and the sprouting of vessels through microholes. ECM: Extracellular matrix; EC: Endothelial cell; PDMS: Polydimethyl siloxane. (A) Adapted from [14], under a creative commons license. (B) Adapted with permission from [15]. (C) Adapted with permission from [16] © John Wiley and Sons (2016). (D) Adapted with permission from [17] © Macmillan Publishers Ltd: Nature Materials (2012). (E) Adapted with permission from [18] © Macmillan Publishers Ltd: Nature Materials (2016).

Figure 3.. Examples of self-assembled in vitro networks.

(A) A single-gel channel model with posts. Bottom shows a self-assembled network of HUVEC (green) and mural cells - bone marrow derived hMSCs (red) after 1-week culture with VEGF + Ang-1 supplemented media. (B) Self-assembled networks of (top) perfused microtissue array, with vascular networks formed by normal human lung fibroblasts and endothelial cells (CD31, green; nuclei, blue) in fibrin gel (bottom shows one region from the top array). BM: Basement membrane; hMSC: Human mesenchymal stem cell; HUVEC: Human umbilical vein endothelial cell. (A) Adapted with permission from [39] © The Royal Society of Chemistry. (B) Adapted with permission from [40] © The Royal Society of Chemistry.

Figure 4.. Permeability is an important indicator of network function in vivo and in vitro.

(A) Schematic diagram demonstrating the three-pore model, including normal ECs, TJ breaks and leaky AJs, as reviewed in [57]. Besides vesicular transport, shear flow and regulated pressure differences across the BM are necessary to maintain the EC intercellular barrier to flux of small solutes and large macromolecules. Leaky junctions result from low flow as well as high shear forces; regulated by complex expression of junctional proteins such as vascular endothelial-cadherin and occludin. ECs release nitrous oxide in response to shear, which increases L_p, but not P_e to solutes. Increased VEGF expression and changes in the cortical actin network (via RhoA) are known to correspond with an increased P_e. The glycocalyx plays a role in mechanostransduction of shear as well as forms a selective barrier to plasma proteins, breaks in which result in leaky ECs [58]. (B–D) Author's unpublished results. (B) Self-assembled vascular network of human umbilical vein endothelial cells (green) after 7 days in culture with normal human lung fibroblasts in fibrin in a single-gel microfluidic model (similar to that in Figure 3A). Scale bar is 200 μm. (C) Single vessel, or network, P_e can be measured by perfusion with fluorescent tracers. Shown here is the network in (B) perfused with 70 kDa fluorescent Dextran. (D) Shown is a linear increase in measured fluorescence intensity of extracellular perfusate over time. Shown are representative values (mean + standard mean error) from regions surrounding the perfused network. AJ: Adherens junction; Avg.: Average; BM: Basement membrane; EC: Endothelial cell; L_p: Hydraulic permeability; NO: Nitrous oxide; P_e: Effective permeability (to solutes); TJ: Tight junction.