Engineered Microvessels for the Study of Human Disease

Abstract

The microvasculature is an extensive, heterogeneous, and complex system that plays a critical role in human physiology and disease. It nourishes almost all living human cells and maintains a local microenvironment that is vital for tissue and organ function. Operating under a state of continuous flow, with an intricate architecture despite its small caliber, and subject to a multitude of biophysical and biochemical stimuli, the microvasculature can be a complex subject to study in the laboratory setting. Engineered microvessels provide an ideal platform that recapitulates essential elements of in vivo physiology and allows study of the microvasculature in a precise and reproducible way. Here, we review relevant structural and functional vascular biology, discuss different methods to engineer microvessels, and explore the applications of this exciting tool for the study of human disease.

Fig. 1

(a) Schematic of the systemic circulation. An artery is shown dividing into smaller arteries, arterioles, and capillaries. These drain into postcapillary venules, which collect into veins. A capillary is shown in magnification, consisting of a single layer of endothelial cells supported by scattered pericytes. (b) Circulating blood, including red blood cells, platelets, and a neutrophil are shown within a blood vessel bounded by endothelial cells. Vessels are supported by ECM components within the interstitium. (c) Leukocyte recruitment in response to inflammation. Cytokines released in response to inflammatory stimuli, such as infection, injury, allergen, or tumor, prompt endothelial cells to express leukocyte adhesion molecules including P-selectin and E-selectin. Initial capture occurs when these molecules bind ligands on circulating leukocytes such as P-selectin glycoprotein ligand-1 (PSGL-1) or E-selectin ligand-1 (ESL1). Leukocytes “roll” on these receptors and slow, allowing time for additional ligands on leukocytes to firmly adhere to the endothelium via receptors such as intercellular adhesion molecule (ICAM) 1 and 2. Leukocytes then transmigrate through the endothelium and toward inflammatory stimuli (chemotaxis). (d) Endothelial interactions and primary hemostasis. Upon vessel injury the endothelium rapidly adopts a procoagulant profile, secreting endothelin (which promotes local vasoconstriction), and releasing VWF which binds circulating platelets via their GP1B receptors. VWF is also expressed on subendothelial collagen and is exposed by injury. Platelets “carpet” the endothelium and roll forward until they slow and firmly adhere to exposed collagen. Platelet activation then occurs, which leads platelets to change shape, degranulate, and aggregate, forming the platelet plug of primary hemostasis.

Fig. 2

Methods for formation of engineered microvessels. (a) Needle-removal technique. A needle is embedded in collagen gel and removed to form a channel. This is then seeded with endothelial cells and perfused, as shown. An endothelialized microvessel is shown under magnification on the right side of the figure (scale bar is 100 μm). (Reproduced with permission from Lee et al. [47]. Copyright 2014 by Materials Research Society, based on original figure found in Ref. [48].) (b) Dissolvable matrix technique. A gelatin mesh is formed on a PDMS stamp and then embedded within ECM materials (here, Matrigel). Heating leads to dissolution of the gelatin, leaving behind channels which can be seeded with endothelial cells as shown in the rightward portion of the figure. (Reprinted with permission from Golden and Tien [49]. Copyright 2007 by The Royal Society of Chemistry.) (c) Layering method. A collagen slab is formed on top of a patterned PDMS (i). A separate top collagen is similarly formed (ii). The two collagen slabs are joined, and channels within the collagen are seeded with endothelial cells (iii). The microvascular network is then cultured under flow conditions (iv). At the far right, a fluorescent micrograph shows cultured/stained endothelial cells within engineered microvessels (scale bar 100 μm). Adapted from Ref. [50]. (d) Angiogenesis/vasculogenesis-based technique. HUVECs were placed at either end of a PDMS construct, next to a fibrin-filled channel, and cocultured with fibroblast cells. Over time, perfusable capillaries formed via angiogenesis, spanning this fibrin channel. (Reprinted with permission from Yeon et al. [51]. Copyright 2012 by The Royal Society of Chemistry.)

Fig. 3

(a) Engineered microvessels for the study of thrombosis and microhemodynamics. Engineered microvessels were seeded with HUVEC cells and exposed to phorbol-12-myristate-13-acetate (PMA) to stimulate VWF secretion (left image). Platelet aggregation in these stimulated vessels was compared with control vessels which did not have PMA exposure. In the upper right image, VWF can be seen oriented along stimulated vessels in the direction of flow. VWF aggregates in narrow vessels with high shear stress, strong flow acceleration, or sharp turns, as demonstrated in the lower right image. Figure adapted from Refs. [50] and [69]. (b) Engineered microvessels for the modeling of human thrombotic disease. A branching microfluidic network was created using soft lithography, consisting of a gas channel network separated from a vascular network by a PDMS membrane. Perfusing the vascular network with blood from patients with sickle cell disease and varying the oxygen content of the gas channel network, the authors were able to provoke vascular occlusion events (detailed on the right side of the figure). (Reprinted with permission from Higgins et al. [87]. Copyright 2007 by The National Academy of Sciences of the U.S.A.) A similar construct was endothelialized in later work, for further examination of microangiopathic diseases (see Ref. [88]). (c) Engineered microvessels for the study of tumor metastasis. A microvessel model was created with microfluidic channels flanking either side of a larger chamber filled with fibrin gel matrix. A tricellular culture was created within the center chamber, with HUVECs, human bone human bone marrow-derived mesenchymal stem cells (MSCs), and bone marrow-derived mesenchymal stem cells with partial differentiation toward bone (OB). Growth factors were added and capillary formation occurred over 4 days, spanning the two center chamber and connecting the two flanking chambers. Breast cells were then introduced into these microvessels, and extravasation observed (middle image). The rate of extravasation in this “bone microenvironment” was compared with extravasation in a muscle environment (using myoblast cell line C2C12), or a control of acellular matrix material, and found to be highest in the bone microenvironment (see figure on the right). This is consistent with the clinical tendency of breast cancer to metastasize to bone tissue. (Reprinted with permission from Jeon et al. [89]. Copyright 2015 by The National Academy of Sciences of the U.S.A.)