Engineering new microvascular networks on-chip: ingredients, assembly, and best practices

Abstract

Tissue engineered grafts show great potential as regenerative implants for diseased or injured tissues within the human body. However, these grafts suffer from poor nutrient perfusion and waste transport, thus decreasing their viability post-transplantation. Graft vascularization is therefore a major area of focus within tissue engineering because biologically relevant conduits for nutrient and oxygen perfusion can improve viability post-implantation. Many researchers utilize microphysiological systems as testing platforms for potential grafts due to an ability to integrate vascular networks as well as biological characteristics such as fluid perfusion, 3D architecture, compartmentalization of tissue-specific materials, and biophysical and biochemical cues. While many methods of vascularizing these systems exist, microvascular self-assembly has great potential for bench-to-clinic translation as it relies on naturally occurring physiological events. In this review, we highlight the past decade of literature and critically discuss the most important and tunable components yielding a self-assembled vascular network on chip: endothelial cell source, tissue-specific supporting cells, biomaterial scaffolds, biochemical cues, and biophysical forces. This article discusses the bioengineered systems of angiogenesis, vasculogenesis, and lymphangiogenesis, and includes a brief overview of multicellular systems. We conclude with future avenues of research to guide the next generation of vascularized microfluidic models and future tissue engineered grafts.

Keywords: angiogenesis; lymphangiogenesis; microvascular networks; organ-on-a-chip; self-assembly; vasculogenesis.

Figure 1.

We argue that five critical components allow researchers to achieve vascularized MPSs: ECs, supporting cells, growth factors, ECM-mimicking biomaterials, and mechanical forces. As we will review, the combination of some or all of these components results in perfusable, self-assembled microvasculature for drug discovery and screening, healthy and diseased tissue modeling, and material testing for tissue engineering applications.

Figure 2.

Angiogenesis and its MPS models. A) sprouting angiogenesis occurs first through tip cell migration in response to angiogenic stimuli. Stalk cell proliferation causes sprouts to elongate. Vessels mature via pericyte recruitment that forms cell-cell contacts with ECs. Angiogenesis ends when ECs and pericytes mature and lay down basement membrane (denoted BM) and enter a quiescent state. Reproduced with permission.^[139] 2013, John Wiley and Sons. B) traditional microfluidic device designs used in angiogenic self-assembly. i) cylindrical lumen formation via gelation of hydrogels around a removable needle. Reproduced with permission.^[35] 2018, Elsevier. ii) micropost device defined as multiple parallel channels boundaried by microposts forming perpendicular conduits through which cells, materials, and fluids can perfuse throughout a device. Angiogenesis-chips utilizing a micropost device typically coat a fluidic channel with ECs to mimic a parent vessel. Sprouts migrate into a hydrogel channel that remains within microposts due to the gel solution’s high viscosity and surface tension. Reproduced with permission.^[46] 2019, Elsevier. C) Examples of angiogenesis-chips incorporating some or all of the five reviewed components initiating angiogenic self-assembly in MPSs. i) Left: microvascular networks formed via angiogenic sprouting supported by pericytes. Scale bar = 50 μm. Right: Higher magnification and confocal sections. Scale bar = 20 μm. Reproduced with permission.^[27] 2013, Royal Society of Chemistry. ii) VEGF-induced angiogenic sprouting from a parent vessel recreated in a cylindrical lumen device. Reproduced with permission.^[35] 2018, Elsevier. iii) Angiogenic sprouting assay to determine the optimal combination of different, previously reported pro-angiogenic growth factors. Scale bar = 100 μm. Reproduced with permission.^[54] 2019, Springer Nature.

Figure 3.

Vasculogenesis and its MPS models. A) vasculogenesis in embryonic development begins as mesodermal cells aggregate to form a blood island. The outer cells specify to angioblasts, an EPC. As cells differentiate to ECs, tight junctions form and a basement membrane is laid down. Reproduced with permission.^[64] 2010, UBC Press. B) Microfluidic models of vasculogenesis often follow two designs. i) 5-channel micropost device featuring compartmentalization of NHLFs and HUVECs in encapsulating fibrin hydrogels. Reproduced with permission.^[27] 2013, Royal Society of Chemistry. ii) a variation of a micropost device featuring large diamond-shaped hydrogel compartments with small openings to fluidic microchannels. Reproduced with permission.^[70] 2016, Springer Nature. C) Examples of vasculogenesis-chips formed using a combination of the five components of vascular self-assembly in MPSs. i) Vasculogenesis-chip formed by co-culturing encapsulated HUVECs in the same device as encapsulated NHLFs. Network functionality is confirmed via the perfusion of microbeads and fluorescent Dextran. Scale bar = 100 μm. Reproduced with permission.^[27] 2013, Royal Society of Chemistry. ii) Vasculogenesis-chip displaying physiologically relevant tight junctions, von Willebrand Factor (vWF) expression, and collagen IV (basement membrane) deposition after co-culturing with senescent fibroblasts. Scale bar = 50 μm. Reproduced with permission.^[72] 2019, John Wiley and Songs. iii) EC monocultures in vasculogenesis-chips setups are stable and selectively permeable when intraluminal flow is established. Scale bars = 100 μm. Reproduced with permission.^[70] 2016, Springer Nature.

Figure 4.

Lymphangiogenesis and its microfluidic models. A) Lymphangiogenesis in the developing embryo begins as ECs in the anterior cardinal vein begin expressing PROX-1, an event specifying their LEC fate. LECs migrate and form lymph sacs, or precursors to lymphatic vessels. As the lymph sacs mature, lymphangiogenic sprouts bud and form new lymphatic microvasculature. In the adult, lymphangiogenesis is uncommon except in pathological conditions, where sprouting follows similar EC events such as migration and proliferation in response to pro-lymphangiogenic factors such as VEGF-C. Reproduced with permission.^[82] 2010, Company of Biologists. B) Common chip designs in lymphangiogenesis MPSs. i) Micropost device situated across from fibrin-encapsulated NHLFs, similar to early angiogenesis-chips. Reproduced with permission.^[85] 2016, Elsevier. ii) Cylindrical lumen formed through sacrificial molding around solidified hydrogels, followed by endothelialization by LEC coating. Reproduced with permission.^[87] 2020, Royal Society of Chemistry. C) Examples of lymphangiogenesis-chips. i) LECs sprout in response to IF toward a parent lymphatic vessel and a combination of growth factors that alone induce less sprouting. Reproduced with permission.^[85] 2016, Elsevier. ii) Lymphatic vessels reported to exhibit higher lymphangiogenic sprouting in response to laminar flow (denoted LF) within the vessel. Scale bars = 100 μm. Reproduced with permission.^[94] 2017, American Society for Clinical Investigation.