Microvascular Networks and Models, In vitro Formation

  • Ulrich Blache
  • Julien Guerrero
  • Sinan Güven
  • Agnes Silvia Klar
  • Arnaud ScherberichEmail author
Living reference work entry
Part of the Reference Series in Biomedical Engineering book series (RSBE)


The microvasculature involves the part of the vascular system made of vessels with diameters inferior to 100 μm. There are many culture models allowing for the formation of microvascular networks in vitro, developed either to study cellular and/or molecular aspects of angiogenesis and vasculogenesis or to prevascularize engineered tissues. In this chapter, we describe the cellular (Sect. 2) and material (Sect. 3) components used to generate such in vitro models. Innovative, advanced bioengineering processes, based on bioprinting or microfluidics, to create microvascular networks are also described (Sect. 4).


Bioprinting Human Umbilical Vein ECs (HUVECs) Sprouting Angiogenesis Microvascular Network Formation Endothelial Progenitor Cells (EPCs) 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

1 Introduction

The microvasculature constitutes the largest organ in the body and the part of the vascular system that comprises the smaller vessels (diameter < 100 micrometers). Precise control of blood flow and vascular permeability is central to tissue and organ performance and is accomplished by the microvasculature. Most of the gas, solute and hormone exchange between blood and tissues occurs through the microvasculature. It has the highest remodeling capacity and is consequently the most active vascular compartment through angiogenic processes.

1.1 The Microvasculature

Microvasculature is the most distal part of the vascular tree (Fig. 1). It has to be noted that, in addition to blood vessels, the microcirculation also includes lymphatic capillaries and collecting ducts. This lymphatic system has numerous functions; first, the lymphatic vasculature system maintains homeostasis of tissue fluid by regulating lymph transport. Second, it controls the absorbance of nutrients including proteins and lipids from blood vasculature into peripheral tissues. Lastly, lymphatic vessels are used as a route of immune cell trafficking from peripheral tissues into lymph nodes (Kim and Song 2017). For more details, see “Approaches for Generation of Lymphatic Vessels”. The microvascular network supports metabolic activity and defines a microenvironment inside healthy or pathological tissues (Jain 2003). In addition to the delivery of oxygen, nutrients and removal of carbon dioxide (CO2) and other metabolic wastes, the microcirculation also regulates blood flow and tissue perfusion, thereby affecting blood pressure and responses to inflammation including edema (swelling) (Looney and Matthay 2009). This microvascular network is composed of terminal arterioles, capillaries (forming the capillary bed), and post-capillary venules, all of which drain the blood flow to tissues. The vessels on the arterial side of the microcirculation are called the arterioles and present a diameter of 10–100 μm. The arterioles are well innervated and surrounded by smooth muscle cells. Blood is next carried to the capillary bed with a diameter of 5–8 μm. Such micro-vessels present an endothelial lining, are not innervated and have no smooth muscle around, but are stabilized by numerous pericytes (Sims 1986; Sims 1991).
Fig. 1

The microvasculature is the most active vascular compartment during angiogenesis. Pericytes have been shown to mediate and stabilize blood vessel maturation. Nerves are often located close to vessels and can modulate vascular tone as well as vessel formation (Adapted from Kreuger and Phillipson 2016)

This thin layer represents a minimal diffusion barrier permitting only water and solutes lower than 3 nm in molecular radius to freely diffuse. Larger molecules, upper than 3 nm, must be selectively transported across the endothelium. This permeability allows a high blood-tissue exchange rate (Clough 1991). The exchange surface area is further increased by a very high density of capillaries inside the vascular bed. Next, blood flow goes out of the capillary bed through the venules, which have diameters ranging from 10 to 200 μm. Venules are also innervated but supported only by a limited amount of smooth muscle cells.

1.2 Dynamics of Microvasculature Networks

The development of new blood vessels can occur through two distinct processes: vasculogenesis and angiogenesis, the latter happening through 2 different mechanisms, namely sprouting or intussusceptive angiogenesis (Fig. 2). The initial step in the development of new blood vessels (in embryos and in adults) is called vasculogenesis and involves the differentiation of ECs from precursor angioblast cells, assembling into a primitive plexus of capillaries, which next is remodeled and grown by angiogenesis. Angiogenesis (Eming et al. 2007) occurs during normal development of blood vessels as well as in pathologic conditions by vessel growth from existing capillaries by sprouting or intussusception (= splitting angiogenesis). These two processes are driven by a combination of growth factors, chiefly vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF) and transforming growth factor-β (TGF β)) and their receptors.
Fig. 2

Vasculogenesis and two types of angiogenesis are shown: sprouting and intussusceptive angiogenesis (ten Dijke and Arthur 2007)

In sprouting angiogenesis , ECs proliferate behind the tip cell of a growing branch in response to growth factors, mainly VEGF and lumens can then form by vacuole fusion. Such sprouting angiogenesis is the best characterized angiogenic process at the molecular level and is defined by the outgrowth of new branches from existing vessels (Djonov et al. 2003). Sprouting angiogenesis can also promote blood vessels into tissue parts previously devoid of blood vessels.

Intussusceptive angiogenesis involves splitting and growth of vessels in situ in a metabolically efficient manner. Vessel splitting occurs through formation of transluminal pillars. The subsequent growth results in a subdivision of the vessel and a remodeling of the local vascular network (Risau 1997). The control of intussusceptive angiogenesis is poorly understood as compared to sprouting angiogenesis, in part due to its relatively late discovery in 1986 (Caduff et al. 1986). Intussusception has been described in three distinct processes of vascular growth and remodeling: (i) Intussusceptive microvascular growth for rapid expansion of the capillary plexus, providing a large endothelial surface for metabolic exchange, (ii) Intussusceptive remodeling to modify the size, position and organization of a vascular network into the typical tree-like arrangement, (iii) Intussusceptive branching remodeling to modify the branching geometry of supplying vessels, optimizing pre-capillary and post-capillary flow properties.

None of the processes during intussusceptive angiogenesis requires the immediate proliferation of ECs but rather the rearrangement and remodeling of existing ones. Therefore intussusception allows a more rapid increase in the number of capillaries without extensive EC proliferation or basal membrane formation. However, intussusceptive angiogenesis induce mainly the formation of new capillaries where vascular networks already exist.

1.3 The Microvasculature in Engineered Tissue

The prevascularization of tissue-engineered constructs with microvascular network is expected to improve their engraftment and connection of graft’s vasculature to recipient host microvasculature, while accelerating vascular ingrowth. This strategy involves de novo formation of capillaries and vessels (vasculogenesis), by ECs or by endothelial progenitor cells, rather than from an existing vasculature (angiogenesis).

There are different approaches to engineer vascularized tissues , including: (i) stimulating rapid vessel growth in avascular implants by using pro-angiogenic factors (Ehrbar et al. 2008; Sun et al. 2011), (ii) seeding biodegradable polymeric scaffolds with ECs and pro-angiogenic factors (Novosel et al. 2011), and (iii) prevascularizing the artificial tissue before implantation (Phelps and Garcia 2010). All of these methods have shown promising results and will be described later in this chapter.

In order to mimic the complexity of in vivo microvascular networks in in vitro vascularized engineered tissues and thereby to improve the engraftment of engineered tissues, a combination of cellular components (Sect. 2, Cells and Model Systems to Generate a Capillary Network) and materials components (Sect. 3, Scaffold Materials that Enable the Formation of 3D Micro-Capillary Networks) is critical.

2 Cells and Model Systems to Generate a Capillary Network In Vitro

2.1 Role of Different Cell Types in Capillary Formation and Stabilization

2.1.1 Endothelial Cells

ECs play major roles in angiogenesis, vasculogenesis as well as in the control of blood flow, thrombosis, vessel permeability, and immunity (See Sects. 1.1 and 1.2). A monolayer of ECs lines the entire vascular system providing a barrier between the tissue and blood. Interestingly, there is phenotypic variation between ECs at different levels of the vascular tree, e.g. between arterial and venous cells, which express different markers and can also differentially respond to stimuli (Gallagher and Sumpio 1997). Primary, mature ECs can be isolated from veins and arteries of adult tissues as well as from umbilical cord veins (fetal tissues), by using the pan-endothelial marker CD31 (Fig. 3). Among the ECs isolated from different tissues, human umbilical vein ECs (HUVECs) show relatively high proliferative potential. However, the vasculogenic potential of endothelial progenitor cells (EPCs) is higher than of mature ECs. This is particularly important for therapeutic angiogenesis.
Fig. 3

Mature and progenitor endothelial cells (Modified from Levenberg et al. 2005)

Various EPCs were characterized, with variable potential to restore long-lasting organ vascularization and function in a clinical setting (Rafii and Lyden 2003) (See Sect. 2.3: Origin of Cells). Asahara and co-workers showed in a pioneering work that CD34+ and Flk-1+ progenitor cells can be isolated from peripheral blood, differentiated into ECs and incorporated into vascular structures in ischemic in vivo models (Asahara et al. 1997a). EPCs can also be isolated from bone marrow or cord blood using CD133 and CD34 markers (Fig. 3).

2.1.2 Pericytes

Pericytes , also known as perivascular, mural or Rouget cells, reside alongside ECs within the vascular basement membrane (Armulik et al. 2005a). They are recruited in growing cords, and once the endothelial-pericyte complex is established, extracellular basement membrane proteins are secreted to stabilize the newly formed vasculature (Betsholtz et al. 2005). Mature pericytes are thus embedded inside the basement membrane of microvessels. There is no single pan-pericyte marker but different markers commonly expressed by pericytes are used to identify them. In vivo, pericytes can be distinguished by their close association to the microvasculature as well as their marker expression profile.

A direct pericyte–EC contact is established via so called peg–socket junctional complexes, located at sites where the basement membrane is missing (Fig. 4). To form these complexes, pericytes form pegs, which are cytoplasmic fingers that are inserted into endothelial invaginations (sockets). Adhesion plaques are other important contact sites between pericytes and endothelial cells. They contain fibronectin and resemble adherence junctions at the ultrastructural level. Moreover, gap junction-like structures have also been reported between those two cell types (Diaz-Flores et al. 2009).
Fig. 4

A schematic overview of the endothelial-pericyte interactions in microvessels. Pericytes surround endothelial cells and share with them the basement membrane. A direct pericyte-endothelial contact is formed by peg-socket junctional complexes (Adapted from Armulik et al. 2005b)

In vitro culture models with ECs and pericytes were developed to study specific interactions between those two cell types. Conditioned medium (CM) from pericytes was shown to increase the proliferation and migration of ECs (Watanabe et al. 1997). Furthermore, CM from pericytes stimulates angiogenesis of different ECs (retinal ECs and HUVECs) in vitro at the same extent as basic fibroblast growth factor (bFGF). Hence, pericytes likely play an important role in angiogenesis through secretion of FGF-like molecules. Pericytes also regulate the maturation, migration, proliferation and survival of ECs. Perivascular cells from different anatomical locations show differences in the morphology, marker expression, and function, e.g. pericytes in the brain maintain the stability of the blood-brain barrier and regulate blood flow. Different tissues also show distinct pericytic coverage. While the vasculature of the central nervous system (CNS) is known to be the most highly covered by pericytes, with a 1:1–3:1 ratio between ECs and pericytes (Mathiisen et al. 2010), human skeletal muscle shows a 100:1 endothelial to pericyte ratio (Diaz-Flores et al. 2009).

Pericyte influence ECs by different signaling mechanisms such as TGFβ, PDGF-BB, and SDF-1α (Fig. 5) (See also Sect. 1.2). TGFβ has been directly implicated in pericyte proliferation and differentiation, as well as in the regulation of differentiation and proliferation of endothelial cells. TGFβ acts mainly on two receptors on pericytes: Alk-1 and Alk-5. While Alk-1 induces cell proliferation and migration, Alk-5 promotes vessel maturation through differentiation and extracellular matrix (ECM) formation (Armulik et al. 2011). Moreover, perivascular cells secrete angiopoietins, which act on Tie receptors expressed by endothelial cells and influence vascular development and remodeling (Gaengel et al. 2009).
Fig. 5

A scheme representing cell-cell interactions in angiogenesis and neovascularization. Signaling pathways between the different cell types are indicated by thin arrows. Bold, solid arrows show cell differentiation. Dashed lines represent rolling and adhesion of endothelial progenitor cells (EPC) to endothelial cells (EC) (Adapted from Melchiorri et al. 2014)

2.1.3 Mesenchymal Stem Cells

Mesenchymal stem cells, or MSCs, are multipotent stromal cells derived from the mesoderm able to differentiate into bone, cartilage, fat, or muscle. These multipotent cells can be isolated from bone marrow, adipose tissue, and umbilical cord blood. MSCs are found in close proximity to the microvasculature of various organs (Crisan et al. 2008). They have a supportive role in the formation of vascular networks and therefore might serve as a potential perivascular cell source in prevascularized constructs for tissue regeneration. Three-dimensional co-culture systems were established to assess the effect of mesenchymal cells on angiogenesis in vitro. It has been reported that the paracrine interaction between endothelial and mesenchymal cells potentiates angiogenesis in vitro (Tille and Pepper 2002). The interaction between MSCs and ECs requires, however, a precise spatial and temporal regulation. Interestingly, the recruitment of MSC to the site of neovascularization starts after the formation of nascent EC microstructures (Fig. 5) (Au et al. 2008a). It has been shown that the presence of MSCs induces quiescence of EC and vascular maturation in two- and three-dimensional in vitro culture systems, as well as after subcutaneous in vivo implantation (Pedersen et al. 2014).

2.1.4 Smooth Muscle Cells

Smooth muscle cells (SMCs) are defined by the expression of SMC-specific contractile proteins including alpha smooth muscle actin (α-SMA), tropomyosin, desmin or calponin. Smooth muscular layer lines arteries to regulate the blood flow and vasoconstriction. Relaxation of the smooth muscle increases the internal diameter in a process called vasodilation. When cultured under appropriate in vitro conditions, SMCs differentiate into vascular smooth muscle cells (vSMCs). Hence, they might be used for in vitro tissue engineering applications to support the development of functional engineered microvessels. ECs of the developing vessels produce PDGF-BB, which binds to PDGF receptor β expressed by developing SMCs and the surrounding mesenchyme (Hellstrom et al. 1999) (Westermark et al. 1990). Previous studies also demonstrated an important role of other soluble factors involved in this process: bFGF (Montesano et al. 1986) and heparin binding-epidermal growth factor (HB-EGF) (Higashiyama et al. 1993), produced by ECs. Furthermore, endothelial cells can mediate induction of other cells such as mouse fibroblasts (10 T1/2) toward a SMC-lineage in vitro as evidenced by the expression of α-SMA, tropomyosin, desmin, and calponin (Hirschi et al. 1998).

2.1.5 Hematopoietic Cells and Inflammation

The balance between pro- and anti-angiogenic factors determines the rate of blood vessel formation in a tissue. During inflammation, angiogenesis is generally favored. Inflammatory response activates the recruitment of numerous inflammatory cells including circulating leukocytes, platelets and also activates macrophages and mast cells, which produce pro-angiogenic factors, including VEGF and cytokines (Naldini and Carraro 2005).

Macrophages differentiate from bone marrow–derived monocytes and are key modulator and effector cells in the immune response. They were shown indispensable for angiogenesis (Chung et al. 2009), wound healing (van Amerongen et al. 2007) and tumor growth (Condeelis and Pollard 2006). Two forms of macrophage phenotypes can be distinguished: pro-inflammatory M1 or “killer” macrophages, which play a critical role in fighting infections and destroying foreign organisms and M2, regenerative macrophages. M1 macrophages produce reactive oxygen species and pro-inflammatory cytokines and chemokines. In the resolution phase of inflammation, macrophages polarize into an M2-phenotype and contribute to debris scavenging, angiogenesis, and wound healing. M2 macrophages produce various angiogenic growth factors such as VEGF-A, bFGF, insulin-like growth factor-1 (IGF-1) or placental growth factor, which accelerate the blood supply of the regenerating tissue (Jetten et al. 2014). M2 macrophages also play a key role in angiogenesis of developing tissues (Pollard 2009). A recent study showed that distinct subsets of M2 macrophages - M2a and M2c may promote angiogenesis through different signaling pathways (Jetten et al. 2014). However, only an orchestrated response of both M1 and M2 macrophages efficiently supports angiogenesis and collagen scaffolds vascularization in vivo (Spiller et al. 2014). Accordingly, mouse embryos lacking tissue macrophages (McKercher et al. 1996) showed significantly reduced number of vascular networks (Fantin et al. 2010) and macrophage depletion also significantly impaired wound healing in an autologous corneal model in mice by reducing angiogenesis and wound closure (Li et al. 2013).

Mast cells are another type of bone marrow-derived granulated hematopoietic cells, which reside in nearly all tissues. They provide rapid response to stimuli followed by a degranulation and release of different inflammatory mediators such as histamine, leukotrienes, and tumor necrosis factor (TNF) (Kunder et al. 2011). They promote localized and systemic inflammatory responses through EC activation and vascular leakage. As mast cells are found predominantly in close proximity to both blood and lymphatic micro-vessels, they can act directly on vascular endothelium and promote local and long-distance effects. Furthermore, mast cells have a function in vascularisation through secretion of VEGF.

2.2 In Vitro Models of Vasculogenesis and Angiogenesis

2.2.1 Vasculogenesis Models

Vasculogenesis occurs during both embryonic development and adult vascular growth by angioblast mobilization (Carmeliet 2000) (see also “Angiogenesis: Basics of Vascular Biology”). Several in vitro systems have been developed in order to investigate the physiological and cellular events involved in vasculogenesis. Differentiation assays with embryo-derived mesodermal cells and ES cell cultures allowed investigating vasculogenesis in vitro. In vitro vasculogenesis can be observed when blastodisc cells are grown in suspension and form three-dimensional spheroids (Krah et al. 1994). Adherent cultures of dissociated cells from blastodiscs can also generate both hematopoietic and ECs that aggregate into characteristic blood islands and give rise to vascular structures in long-term culture (Flamme and Risau 1992; Flamme et al. 1993). The murine ES cell-derived EB formation assay is another promising system. In this in vitro model, a primitive vascular plexus is formed and provides an attractive tool to investigate the mechanisms involved in vasculogenesis: angioblast cells proliferation, migration and differentiation, but also the formation of a primitive plexus of capillaries (microvasculature). Blood island formation and many aspects of normal endothelial differentiation and growth, leading to the formation of vascular channels have been reported during ES-derived EB development (Doetschman et al. 1985; Risau et al. 1988; Wang et al. 1992). Microscopic analysis has revealed that the vascular structures found within the walls of cystic EB consist of ECs that form tubular channels with typical endothelial junctions (Wang et al. 1992). More recently, studies have indicated that endothelial development in mouse EB follows a systematic sequence of genetic events that recapitulates murine vasculogenesis and leads to the formation of vascular structures recalling a primitive vascular network (Vittet et al. 1996).

These observations indicate that this in vitro system contains most of the endothelial differentiation program and probably reflects the events taking place during in vivo endothelial differentiation in the embryo, which constitutes a significant technical advance for the study of vasculogenesis for tissue engineered constructs. Genetic modifications can be easily introduced in vitro into totipotent ES cells. The differentiation of genetically modified ES cells, in which gain-of-function or loss-of-function mutations are introduced, offers excellent models to analyze the consequences of specific mutations on the process of vascular development, especially when these mutations are lethal (Bautch et al. 2000; Schuh et al. 1999; Vittet et al. 1997). Analysis of flk-1−/− ES cell differentiation in vitro allowed determining that flk-1 deficiency does not affect endothelial differentiation but rather impairs subsequent EC migration and organization into a vascular network (Schuh et al. 1999), issues that cannot be easily addressed in flk-1–deficient embryos. Similar in vitro differentiation experiments, performed with heterozygous or homozygous VEGF-A mutant ES cells, allowed for the characterization of a stage-specific differentiation step at which vasculogenesis is blocked by VEGF-A deficiency (Bautch et al. 2000). EB may also be useful for the development of genetically manipulated EC lines carrying lethal gene mutations (Balconi et al. 2000). Indeed, purified EC progenitors and ECs can easily be isolated from EB at different maturation steps (Balconi et al. 2000; Hirashima et al. 1999a).

2.2.2 Angiogenesis Models

Angiogenesis in vitro was observed for the first time more than 30 years ago (Folkman and Haudenschild 1980). After long-term culture of capillary ECs, spontaneous organization of these cells into a microvascular network was observed. The presence of a lumen was confirmed by phase contrast and transmission electron microscopy. This first report of angiogenesis in a culture dish provided a basis for the definition of in vitro endothelial angiogenesis: all the subsequently published assays referred to the presence of a lumen as a criterion for the validation of an in vitro model of microvasculature. From a physiological point of view, an ideal in vitro model should recapitulate all the representative steps of in vivo angiogenesis (Angiogenesis: Basics of Vascular Biology). Furthermore, it should be easy to use, reproducible, and easily quantifiable (length of capillaries, area covered by the capillary network, number of tubes and complexity of the network). The models described to date can be classified into two categories: 2D models, in which cells develop tubular structures on the surface of a substrate and 3D models, in which cells can invade the surrounding 3D matrix.

2D models present a cellular network with a planar organization, parallel to the surface of the culture plate (Vernon and Sage 1995). Many reports state that microvascular network formation could be observed spontaneously in long-term planar cultures (Folkman and Haudenschild 1980; Feder et al. 1983). Later, it was observed and well documented in such 2D models that distinct extracellular matrix components can promote microvascular network formation. ECs are simply seeded onto plastic culture dishes, coated or not with adhesive proteins (Feder et al. 1983; Ingber and Folkman 1989; Madri and Williams 1983; Pelletier et al. 2000). Alternatively, they can be loaded on top of a hydrogel made of either collagen (Vernon et al. 1995), fibrin (Vailhe et al. 1997) or Matrigel (Sage and Vernon 1994).

ECs can either proliferate, when seeded on Type I or III collagen, or differentiate, when seeded on Type IV or V collagen (Madri and Williams 1983). Matrigel, a laminin-rich matrix, was shown to support rapid formation of microvascular networks by ECs (Kubota et al. 1988). Similarly, ECs seeded on fibrin reorganized into microvascular networks (Vailhe et al. 1997; Olander et al. 1985). These assays can be subdivided into two groups: short-term (Ingber and Folkman 1989; Madri and Williams 1983; Vailhe et al. 1997; Kubota et al. 1988) and long term (Folkman and Haudenschild 1980; Feder et al. 1983; Pelletier et al. 2000; Vernon et al. 1995; Maciag et al. 1982) 2D cultures. In short-term models, microvascular network are observed after 1 to 3 days of culture (Vailhe et al. 1997) and essentially recapitulate the morphogenesis step of angiogenesis but not the proliferation and migration steps. In long-term cultures, the complex processes and factors involved in the formation of microvascular networks are less characterized (Vernon et al. 1995) and take place on top of a confluent monolayer of cells (Vernon et al. 1995), with morphogenetic pathways that require synthesis of extracellular matrix by cells (Sage and Vernon 1994). However, they are more convenient for the observation of stable tubular structures forming slowly over a long period of culture. For both short-term and long-term two-dimensional models, a limited number of co-culture systems were developed (Co-culture Systems for Vasculogenesis). 2D angiogenesis models have thus significantly increased our understanding of the role of extracellular matrix in vascular morphogenesis, but they obviously do not reflect all steps of physiological angiogenesis. In particular, the pattern of cellular organization observed in these assays might be more representative of intussusceptive rather than sprouting angiogenesis (Vernon and Sage 1995).

3D angiogenesis assays are based on the capacity of ECs to grow-up and/or invade three-dimensional substrates. The matrix may consist of collagen gels, fibrin, Matrigel or mixtures of them, or can also consist of solid materials. These models are described later in this Chapter (Scaffold Materials that Enable the Formation of 3D Micro-Capillary Networks).

2.3 Origin of Cells Used in In Vitro Models

EC populations are an attractive cell source for neo-vascularization of engineered or ischemic tissues. An important characteristic of ECs is their ability to dedifferentiate into stem cells when stimulated to undergo endothelial-mesenchymal transition, which makes them a potential cell source for regenerative strategies (Susienka and Medici 2013). However, phenotypical differences exist between EC populations due to the various origins of this cell type, resulting in high variability of functional behavior in angiogenesis assays. Moreover, mature ECs present a limited proliferative potential (Sieveking et al. 2008). Therefore, alternative sources of EC progenitors have emerged.

2.3.1 Embryonic Stem Cell

Embryonic stem (ES) cells represent a promising cell source for regenerative medicine approaches, thanks to their pluripotency and self-renewal potential. However, the origin of ES also raises ethical concerns. ES cells are extensively used to investigate molecular mechanisms and gene function involved in mammalian development (Rathjen et al. 1998). Both mouse and human ES cells are derived from the inner cell mass (ICM) of blastocysts or morula stage embryos. When maintained under specific culture conditions, ES cells acquire an immortal characteristic and can be propagated stably in an undifferentiated state in vitro. Upon withdrawal of leukemia inhibitory factor (LIF) from the culture medium, ES cells will spontaneously and irreversibly differentiate and form colonies or EB. These aggregates resemble early post-implantation embryos and contain precursors of all three germ layer lineages including those of the hematopoietic and endothelial systems (Smith 1992; Itskovitz-Eldor et al. 2000), arising from a common precursor, called the hemangioblast (Choi 2002).

The group of Levenberg (Levenberg et al. 2002) described the expression of endothelial-specific genes during differentiation of human ES cells cultured as EB without additional growth factors (Fig. 6 ). ECs derived from human ES cells expressed specific endothelial markers such as CD31, VE-cadherin, vWF and N-cadherin, and were capable of Dil-Ac-LDL uptake. Moreover, the process of vasculogenesis can be induced in ES-derived EB (Hirashima et al. 1999b). The endothelial development within these bodies was identified by the onset of specific gene expression, followed by an ordered sequence of events that reflect in vivo vasculogenesis and leads to the formation of a primitive three-dimensional vascular network (Risau et al. 1988; Vittet et al. 1996). However, differentiation of ES cells into ECs does not require EB formation, and can also be induced by seeding ES cells on feeder cells or inside an ECM (Zhang et al. 2005). Mouse endothelial Flk-1+ progenitors were isolated following differentiation of ES cells on collagen (Nishikawa et al. 1998). ES cell–derived Flk-1 positive cells were shown to differentiate into both endothelial and mural cells (pericytes and smooth muscle cells) and reproduce the complex vascular organization process (Risau 1991; Yamashita et al. 2000). However, growth factors such as VEGF are needed to achieve endothelial-mural cell interaction within the sprouting vessels. Interestingly, human ES cells can differentiate into CD34+ cells, when seeded on stromal feeder cells (bone marrow and yolk sac), with 50% of the CD34+ cells also expressing CD31 (Kaufman et al. 2001).
Fig. 6

Development of human embryonic stem cells into vascular cells. Hemangioblasts are common precursors to hematopoietic and endothelial progenitor cells (EPC). Vascular progenitor cells (VPCs) can differentiate to mural cells (SMC and pericytes) and endothelial cells. EPC from cardiovascular progenitor cells may give rise to hematopoietic progenitor cells. The differentiation of smooth muscle cells (SMC) is dependent on the location in the embryo. In addition, EPC can differentiate into SMC during vessel formation

2.3.2 Endothelial Progenitor Cells

The discovery of putative endothelial progenitor cells (EPCs) in 1997 (Asahara et al. 1997b) contributed to enlarge the knowledge about EC populations. These cells exist in blood circulation and were described to organize into vessels after mobilization from bone marrow and to participate in neovascularization of ischemic sites (Asahara et al. 1999). Since several years, EPCs are routinely isolated from peripheral or umbilical cord blood (Asahara et al. 1997b; Ingram et al. 2004; Fuchs et al. 2006a, b; Au et al. 2008b). Moreover, other sources of endothelial progenitors were described more recently including bone marrow (Asahara et al. 1999; Shi et al. 1998; Nolan et al. 2007; Chen et al. 2012a), amniotic fluid (Zhang et al. 2009) and adipose tissue (Lin et al. 2008; Szoke et al. 2012; Klar et al. 2016; Klar et al. 2014). Independently of their origin, these progenitor cells seem to be mobilized into the circulation and contribute to neo-vascularization processes (Rafii and Lyden 2003; Asahara et al. 1997b; Asahara et al. 1999). Numerous studies on EPCs are available, defining EPCs by surface markers such as CD133 and CD34 (Gehling et al. 2000; Peichev et al. 2000; Salven et al. 2003), and by characteristics such as low density lipoprotein (LDL)-uptake (Shi et al. 1998) and binding of ulex europaeus agglutinin (UEA) (Asahara et al. 1997b; Kalka et al. 2000). The ability to form vascular structures in pro-angiogenic matrices in vitro, as well as the potential to contribute to vascularization in vivo was defined as functional validation of EPCs.

Two types of EPCs were characterized, early EPCs and late EPCs, also called outgrowth endothelial cells (OECs) (Sieveking et al. 2008; Lin et al. 2000; Medina et al. 2010). Early EPCs are described as BM-derived cells sharing surface markers with hematopoietic stem cells, like CD14, CD45 and CD133 (Asahara et al. 1997b; Medina et al. 2010; Masuda and Asahara 2003; Doulatov et al. 2012), can differentiate into phagocytic macrophages and possess myeloid progenitor cell activity (Yoder et al. 2007; Shi et al. 2014). These cells are evidenced after 4–7 days in culture and are thought to secrete pro-angiogenic factors, acting in a paracrine manner, whereas OECs (late EPCs) appear only after 14–21 days of culture, exhibit typical endothelial characteristics and incorporate into resident vasculature (Sieveking et al. 2008; Mukai et al. 2008). Moreover, early EPCs were shown to preferentially express genes involved in immune response and inflammation, while OECs express genes involved in development and angiogenesis, including the angiopoietin receptor Tie2, endothelial nitric oxide synthase (eNOS), ephrins and transforming growth factor-β (TGF-β) (Cheng et al. 2013). A more recent study also showed that umbilical cord blood (UCB)-derived OECs secrete a broad spectrum of pro-inflammatory and angiogenic cytokines, including angiogenin, angiopoietin (Ang)-2 and platelet-derived growth factor (PDGF)-BB (Liu et al. 2012), one of the four homodimeric glycoproteins of the PDGF family.

Therefore, endothelial progenitors (both EPCs and OECs), in particular from peripheral or cord blood, have raised great interest, their potential autologous use constitutes a major advantage for clinical applications.

2.3.3 Primary Endothelial Cells

Human umbilical vein ECs (HUVECs) , which are isolated from macrovascular endothelium, have been extensively studied and developed into commercially available primary cell culture and cell lines. Comparing to human microvascular endothelial cells (HMECs) from dermal tissues, HUVECs are more readily available and easily extractable from newborn’s umbilical cords (Park et al. 2006). However, all primary cultures present the limitations of variability in characteristics and lifespan of the cells from different donors. Immortalized cell lines are established by transfecting some viral vectors or hybridizing ECs (HMECs or HUVECs) with cancerous cells to make them as in vitro model systems for primary cells. EA.hy926 and HMEC-1 are two well characterized, immortalized and commonly used cell lines showing similar traits as primary ECs (Park et al. 2006; Bouis et al. 2001; Unger et al. 2002).

2.3.4 Endothelial Cells from Adult Tissue (Lung, Brain, Colon, Mucus Membrane, Skin)

Other tissue-resident progenitor cells capable of differentiating into ECs have been reported. Distinct progenitors known as “side population cells (SPs) “, defined according to active efflux of a Hoechst dye, have been found in various tissues , including skeletal muscle (Majka et al. 2003), lung (Irwin et al. 2007), and heart (Oyama et al. 2007), and were shown to differentiate into ECs and can be used for in vitro models.

2.3.5 EC-iPSCs

The production of vascular endothelial cells from iPS cell lines has recently been demonstrated using EB-differentiation protocols (Feng et al. 2010; Choi et al. 2009; Taura et al. 2009; Homma et al. 2010), however in vivo function of the derived cells has yet to be evaluated. Thus, human iPSC-derived endothelial cells (iPSC-ECs) represent a potentially valuable tool for the development of robust and reproducible vascular tissues for disease modeling and screening applications (Wetmore et al. 2013; Tice et al. 2013; Kleinstreuer et al. 2011).

2.3.6 Adipose Stromal Vascular Fraction/SVF-ECs for Prevascularization Approaches

The non-adipocytic fraction of adipose tissue is also rich in endothelial and progenitor cells (Scherberich et al. 2010) and represents an abundant source of autologous vasculogenic progenitors for engineering vascularized tissues. It was indeed shown that adipose-derived stromal cells expanded as monolayer can differentiate into ECs and become vasculogenic (i.e., blood vessel forming) cells. Two teams (Miranville et al. 2004; Planat-Benard et al. 2004) simultaneously showed the presence of a cell population, positive for the progenitor marker CD34, but negative for the mature endothelial marker CD31 inside the freshly isolated human adipose-derived cells, called the stromal vascular fraction (SVF) of adipose tissue, exhibiting characteristics of endothelial progenitor cells both in vitro and in a hindlimb ischemia model in nude mice. A population of CD34 and CD31 positive cells from the SVF, lost upon monolayer expansion, was shown to have an endothelial phenotype and could form blood vessels when implanted in vivo (Scherberich et al. 2007). These results suggest that adipose-derived cells, freshly isolated or expanded, constitute an abundant reservoir of relevant vascular progenitors.

2.3.7 Bone Marrow Stromal Vascular Fraction

A study demonstrated that the whole human bone marrow population (Guerrero et al. 2015), without selection or expansion, can be used to generate microvascular networks. The whole bone marrow contains a population of cells expressing endothelial markers, like CD31, CD34, CD146, and CD29. An endothelial microvascular population of cells from human bone marrow and its purification was described (Rafii et al. 1994). This cell source is therefore suitable for MSC-based vascularization strategies in tissue-engineering applications, without preliminary cultures.

3 Scaffold Materials that Enable the Formation of 3D Micro-Capillary Networks

3.1 Lesson from the Pioneers and the ECM

Engineering microvascular networks in vitro not only requires appropriate cellular components but also suitable scaffolds allowing for the formation of microvascular structures by ECs in 3D. Inside the body, the native scaffolding material for microvasculature is the ECM, which essentially is a hydrated meshwork of glycoproteins and glycosaminoglycans. Therefore, the initial attempts to reproduce vascular structures in vitro have been carried out with isolated ECM components as scaffolds. In fact, the first material that enabled scientists to successfully achieve a 3D microvascular network in vitro was the chick plasma clot in 1982 (Nicosia et al. 1982). Nicosia et al. placed rat aortic rings in plasma clot cultures and within 2 weeks obtained capillary-like outgrowth and lumen formation of ECs as well as perivascular remodeling of the ECM (Nicosia et al. 1982; Nicosia et al. 1986; Nicosia and Madri 1987). In parallel, studies by Montesano et al. proved that scaffolds consisting of individual ECM components such as collagen and fibrin hydrogels are sufficient to promote the formation of 3D microvascular networks (Montesano et al. 1983, 1985, 1987 Montesano and Orci 1985). The great success of the ECM-based pioneering work was not only due to the general biocompatible features of ECM but also to the specific role of the ECM in vascular organization. In fact, ECM acts as a key regulator of many steps during vascular development ranging from early endothelial lumen formation to late vessel stabilization and maturation (Davis and Senger 2005; Davis et al. 2011). Therefore, fundamental insights into microvessel formation and regulation have been achieved using 3D platforms based on ECM proteins. Moreover, since they readily allow for the formation of 3D microvascular networks, ECM hydrogels became the most commonly used biomaterial in tissue prevascularization strategies. However, the natural origin of ECM possesses downsides, like batch-to-batch variability, the risk of material contamination by bioactive substances or poor mechanical properties. This has motivated scientists to integrate synthetic modifications to ECM components or even to develop synthetic ECM analogous materials. In this section, both natural and synthetic biomaterials that support the formation of microvascular structures in vitro will be discussed.

3.2 Natural Materials: Protein Polymers

Natural materials are polymers assembled by glycoproteins or glycosaminoglycans (GAGs) that mainly contribute to the ECM in vivo. The most frequently used class of materials for vascular engineering is ECM protein polymers. The ECM components are extracted from tissues or cell cultures and can form hydrogels that reproduce properties of native ECM. These highly biocompatible hydrogels are of homogenous or heterogeneous origin and can be assembled under cell-friendly conditions.

Matrigel is a reconstituted gel of the basement membrane components laminin, collagen IV, entactin (nidogen-1) and heparin sulfate proteoglycan (Kleinman et al. 1982; Kleinman et al. 1986). Matrigel is derived from Engelbreth-Holm-Swarm (EHS) mouse sarcoma cells and contains many growth factors in addition to its structural components. In vitro, matrigel forms a hydrogel under cell-friendly conditions in a temperature-driven process. Since it resembles the basement membrane, it is a very potent substrate for in vitro capillary-like tube formation by ECs and has become the standard 2D angiogenesis assay (Kubota et al. 1988; Grant et al. 1989; Kleinman and Martin 2005; Arnaoutova and Kleinman 2010). It has also been applied as gelatinous cell carrier for the prevascularization of porous scaffolds (Levenberg et al. 2002; Levenberg et al. 2003; Levenberg et al. 2005; Caspi et al. 2007).

Fibrin is a fibrous protein polymer formed at terminal step of the blood coagulation cascade. The polymer formation is initiated by the serine protease thrombin that activates soluble fibrinogen to polymerize and is completed by the enzyme FXIIIa that covalently cross-links fibrin by transglutamination (Weisel 2005; Janmey et al. 2009). Fibrin has viscoelastic properties and is a cell-friendly scaffold allowing for cell growth and migration in wound healing and angiogenesis (Lord 2007; Weisel 2008). The concentration of soluble fibrinogen in human blood is approximately 2–4 mg/ml (Lord 2007; Weisel 2008; Kamath and Lip 2003; Tennent et al. 2007). Therefore fibrin hydrogels have been applied in vitro with this physiological concentration and enabled 3D microvascular structures in various reports (Montesano et al. 1987; Kniazeva and Putnam 2009; Ghajar et al. 2010; Kachgal and Putnam 2011; Kachgal et al. 2012; Brudno et al. 2013; Smith et al. 2013). These studies addressed questions in terms of 3D behavior of ECs and the formation of microvascular structures. However, it is conceivable that the matrix density of fibrin exerts a direct influence on the cellular behavior including microvascular network formation and stabilization. In two studies using microbeads as cell carriers for ECs, fibrinogen concentrations of 2.5 mg/ml, 5 mg/ml and 10 mg/ml were compared (Kniazeva and Putnam 2009; Ghajar et al. 2008). The onset of capillary network formation was delayed and the total length of the networks was decreased with increasing fibrin density (Kniazeva and Putnam 2009; Ghajar et al. 2008). Nevertheless, capillary networks were observed in 10 mg/ml fibrin and ECs are known to form vacuoles and lumens at this concentration (Bayless et al. 2000; Bayless and Davis 2002). Importantly, studies that applied fibrin as scaffolding material for prevascularization were carried out by using 10 mg/ml fibrinogen (Klar et al. 2014; Ghajar et al. 2008; Chen et al. 2009; Chen et al., 2010; Montano et al. 2010; Marino et al. 2014).

Collagen I is a fibrillar ECM protein polymer present in various connective tissues. In vitro, the polymerization of collagen I can be induced in an entropy-driven process by increasing the temperature of a neutralized collagen solution. Therefore, hydrogels can be formed from tissue-derived collagen I at physiological conditions. Due to its natural origin collagen I is intrinsically cell-friendly and was shown by Davis and co-workers to be an excellent in vitro scaffold for lumen containing capillary-like structures (Bayless and Davis 2002; Davis et al. 2000; Koh et al. 2008; Stratman et al. 2009; Stratman et al. 2011). Similarly to fibrinogen, 1.5–4 mg/ml collagen concentrations have been applied to enable ECs forming 3D microvascular networks in vitro (Bayless and Davis 2002; Marino et al. 2014; Davis et al. 2000; Koh et al. 2008; Stratman et al. 2009; Stratman et al. 2011; Nguyen et al. 2013; Sieminski et al. 2004). Although limited prevascularization and poor mechanical stability of the scaffolds have been mentioned (Montano et al. 2010; Marino et al. 2014), collagen hydrogels were used at concentrations of 3–4 mg/ml for in vitro generation of microvascular networks followed by their successful inosculation into an host vasculature (Shepherd et al. 2004; White et al. 2014). Interestingly, in one of these studies, White et al. compared pure collagen scaffolds with collagen-fibrin scaffolds (1:1 mass ratio) and showed that collagen scaffolds lead to a more efficient formation of vasculature in vivo (White et al. 2014). However, a comprehensive in vitro study by Rao and colleagues found that within a gradual series of collagen-fibrin composite scaffolds, the amount of collagen was negatively correlated with the microvascular network length while positively with the mechanical properties of the scaffold (Rao et al. 2012).

Silk fibroin is a protein polymer that differs from the proteins described above in being not part of the mammalian ECM. It is produced by arthropods and mostly obtained from the silk worm. Silk consists of the proteins fibroin and sericin. Fibroin is separated from sericin by a degumming step (Kundu et al. 2013). In a series of studies, Kirkpatrick and co-workers investigated the potential of fibroin fiber nets for vascular tissue engineering. It was shown that fibroin nets are compatible with various cell types including also ECs when fibroin was coated with ECM proteins (Unger et al. 2004a, b). By co-culturing ECs with supporting cells in silk fibroin scaffolds, 3D microvascular structures were successfully formed in vitro (Unger et al. 2007; Fuchs et al. 2009). Moreover, in vitro prevascularized networks were functional in vivo after subcutaneous implantation of the vascularized fibroin scaffolds by anastomosis with the host vasculature (Unger et al. 2010).

3.3 Tuning Protein Polymers: Modified Collagen

Despite displaying many native features needed for cell adhesion, cell survival and scaffold remodeling, protein polymers do not always offer the optimal range of scaffold properties. One of the problems of protein polymers like collagen is their poor mechanical stability. The conventional solution of increasing the material concentration not just leads into stiffer scaffolds but also into a denser matrix and an increased number of biological effectors present in the scaffold. To uncouple the physical properties from material concentration and biological effectors, Singh et al. modified collagen I by photo-cross-linkable poly(ethylene glycol) (PEG) diacrylamide PEGDA-based linkers (Singh et al. 2013). Thereby, they developed collagen-PEG hybrid materials in which until a certain degree of cross-linking, the material stability was increased without impeding the formation of microvascular networks (Singh et al. 2013). Unfortunately, the microvascular networks generated in collagen-PEG hybrids were not tested for their in vivo functionality. In contrast, Khademhosseini and co-workers proved the great potential of modified natural materials for vascular tissue engineering in vivo by using gelatin-based materials (Chen et al. 2012b). Gelatin is a mixture of denatured proteins, mainly collagens, able to form a non-covalent gel below temperatures of 35 °C (Xing et al. 2014). By methacrylic anhydride modification, gelatin becomes photo-cross-linkable gelatin methacrylate (GelMA) that upon radical polymerization generates chemically crosslinked hydrogels (Van Den Bulcke et al. 2000), which are suitable for cell-based tissue engineering approaches (Chen et al. 2012b; Nichol et al. 2010; Benton et al. 2009). Chen et al. proved that the features of GelMA enabled the formation of both 3D microvascular networks in vitro and functional microvessels in vivo (Chen et al. 2012b).

While collagen in vascular tissue engineering is widely applied as hydrogel it can be prepared as porous sponges too. For instance, sponge scaffolds have been generated from collagen by mixing collagen I and III with chitosan and chondroitin sulfate, followed by freeze-drying. The resulting sponge materials were shown to be suitable constructs for capillary network formation in vitro (Black et al. 1998; Tremblay et al. 2005a; Berthod et al. 2006) and the inosculation of prevascularized structures in vivo (Tremblay et al. 2005b).

3.4 Modified Natural and Synthetic Polymers

The requirements that turn a material into an adequate scaffold for micro-capillaries adapt to the features of the natural ECM and include biocompatibility, cell adhesion, physical support and especially MMP-mediated remodeling (Slaughter et al. 2009; Park and Gerecht 2014). In contrast to protein polymers, synthetic polymers but also glycosaminoglycans lack some of these features and need to be tailored to act as cell-friendly scaffolds.

Glycosaminoglycans (GAGs) are carbohydrate polymers consisting of repetitive disaccharide units built by an amino sugar (N-acetylglucosamine or N-acetylgalactosamine) and an uronic acid (or galactose). When chemically modified, GAGs can readily form materials suitable for tissue engineering approaches. Towards cell-based vascularization strategies, GAGs need to be further modified since they do not possess potent intrinsic cell-attachment sites and are not sensitive to MMP-mediated degradation. In particular, two GAGs have been modified accordingly and resulted in favorable platforms for microvascular network formation. (1) Hyaluronic acid (HA) is an essential part of the ECM and unique among the GAGs as it is non-sulfated (Almond 2007; Burdick 2011). HA was acrylated and modified by the cell attachment RGD motif before hydrogel polymerization by means of dithiol-containing MMP-sensitive peptide cross-linkers (Khetan and Burdick 2010). These tailored HA-constructs were shown to facilitate microvascular networks in vitro and their inosculation in vivo (Hanjaya-Putra et al. 2011; Hanjaya-Putra et al. 2012; Kusuma et al. 2013). (2) Similarly, Heparin, a strongly sulfated and thus very negatively charged GAG, was engineered to become a suitable material for in vitro vasculogenesis. Heparin-maleimide was tuned by the RGD-motif and cross-linked via thiol-containing MMP-sensitive multi-arm PEG-peptide conjugates (Tsurkan et al. 2013). In this PEG-heparin hydrogels, very robust microvascular networks were generated and stable for up to 4 weeks in vitro (Chwalek et al. 2014a). However, the in vivo functionality of microvessels formed in PEG-heparin hydrogels remains to be tested.

Poly(ethylene glycol) (PEG) is a synthetic polymer of ethylene glycols and can have various molecular weights based on its chain length. When covalently cross-linked in aqueous solutions, PEG polymers swell and form biocompatible hydrogels. PEG is biological inert and therefore must be tuned towards specific requirements. Modification of PEG by introducing MMP-sensitive peptide sequences and the cell attachment RGD motif turned PEG hydrogels into ECM-free materials suitable for cell culture and tissue engineering approaches (Gobin and West 2002; Lutolf and Hubbell 2003; Lutolf et al. 2003a, b). In a landmark study, Moon and West used MMP-sensitive, RGD-modified photo-cross-linkable PEG hydrogels to generate 3D networks of capillary-like structures inside this fully synthetic environment (Moon et al. 2010). The finding that ECM-free synthetic PEG hydrogels are sufficient to allow for microvascular networks in vitro was supported in another biologically-modified, enzymatically cross-linked PEG matrix (Sala et al. 2011; Blache et al. 2016). Interestingly, 3D microvascular networks generated inside PEG matrices were shown to support mass transport in vitro (Cuchiara et al. 2012). However, despite the in vivo biocompatibility of PEG enabling the ingrowth of the host vasculature (Moon et al. 2010; Phelps et al. 2010; Hsu et al. 2015) as well as the establishment of microvessels by PEG-delivered cells (Vigen et al. 2014), evidence for the inosculation of in vitro prevascularized construct remains to be shown.

Poly(L-lactic acid) (PLLA) and poly(lactic-co-glycolic acid) (PLGA) are synthetic polymers of lactic and glycolic acid, biodegradable by hydrolytic cleavage of their ester bonds. In contrast to most of the materials discussed above, which represent essentially hydrogels with viscoelastic properties, PLLA and PLGA are solid materials of comparable, high mechanical strength. Due to their non-susceptibility to proteolytic degradation, scaffolds fabricated from PLLA and PLGA are designed as sponges with large pore sizes (>200 um) to facilitate 3-dimensional cell seeding, distribution and migration. Nevertheless, cells seeded into porous sponges are faced with 2D surfaces. Moreover, PLLA and PLGA lack cell attachment sites. To ensure both their 3D orientation and their attachment, cells are typically delivered within matrigel, collagen, or fibrin carrier gel solutions into synthetic PLLA and PLGA sponges. In a seminal series of studies, Levenberg and co-workers used 50:50 PLLA:PLGA scaffolds in combination with matrigel (or fibrin) to vascularize porous sponges in vitro and in vivo (Levenberg et al. 2002, 2003, 2005; Caspi et al. 2007; Lesman et al. 2011). They showed that human ES cells-derived ECs within the PLLA:PLGA scaffolds form perfused microvessels in vivo (Levenberg et al. 2002) and that PLLA:PLGA scaffolds can be applied in vitro for the engineering of vascular tissue construct (Caspi et al. 2007). Most importantly, in vitro prevascularized networks were shown to be functional in vivo after implantation as they were perfused and did anastomose to the host vasculature (Levenberg et al. 2003, 2005).

4 Advanced Bioengineering Approaches

Novel bioengineering tools such as 3D bioprinters, lithography techniques, acoustic and microfluidic systems demonstrate systematic approach to build microscopic and macroscopic vascularization. Biofabrication of functional vascular network models is providing control of the physical, chemical and biological parameters of the process and the material composition, enhancing the reproducibility and the standardization of the final product. With the advances of bioengineering, novel artificial matrices (Loessner et al. 2016) can be synthesized and computational models developed to mimic the dynamics of the newly formed/engineered blood vessels (Piskin et al. 2015) and implement the effect of time on tissue engineered products, introducing a 4D concept in bioengineering (Gao et al. 2016). In vitro fabricated vascular structures over time will replace the animal models, provide more relevant platforms to study the basic biological questions regarding the vascularization and angiogenesis, while such technologies can implement high-throughput systems for drug discovery and tissue engineering (Rouwkema and Khademhosseini 2016).

4.1 3D-Printing of Blood Vessels

Bioprinting is an emerging bottom-up tissue engineering approach which provides micro/macro control of spatial organization over placement of cells and other biological components such as ECM proteins, nutrients, therapeutic drugs and growth factors in order to build artificial tissues and organs (Arslan-Yildiz et al. 2016). Although native vascular organization can be mimicked in scaffold-based approaches (Peck et al. 2012; da Graca and Filardo 2011), there are several drawbacks such as limited cell-to-cell contact, scaffold elasticity, residual deposition upon biodegradation, and adverse immune responses. Bioprinting technology offers highly controlled, reproducible and robust platforms to generate large and small size vascular networks. Scaffold-free bioprinting techniques aim to create 3D vasculature using living cells or cell aggregates (Skardal et al. 2010; Cui and Boland 2009; Norotte et al. 2009; Visconti 2010). Norotte et al. reported microtubular constructs with 500 μm inner diameter that can function as an intermediate vasculature and can connect bioprinted vessels and capillary network. Significant efforts have been performed, pioneering the fabrication of artificial blood vessels using bioprinting approach (Mironov et al. 2008; Sooppan et al. 2016; Bertassoni et al. 2014). Computer assisted design allows for the fabrication of large blood vessels by using the medical images of target organs (Kucukgul et al. 2015).

Bioprinters can be designed to use different types of bioinks to generate heterogeneous tissue architectures. 3D, perfusable vascular structures were bioprinted with multiple materials to biomimic the vascular ECM and cellular composition (Fig. 7) (Bertassoni et al. 2014; Kolesky et al. 2014). In order to fabricate the lumen of the vessels, Bertassoni et al. first bioprinted agarose rods, which serve as micro-molds for the formation of a template for capillaries and then cast cells with methacrylated gelatin (GelMA) on top. After mechanical removal of agarose rods with light vacuum, the perfusable microchannels served as capillaries. Kolesky et al. developed a strategy to form microvasculature by bioprinting Pluronic F127 as fugitive ink to generate microchannels and GelMA as cell-laden ink. The difference in gelation temperatures of these two materials assisted the formation of perfusable capillaries.
Fig. 7

(a, b) Illustration of the top-down and side views of a multi-compartmental bioengineered construct. (c) Microscopic image of the 3D bioprinted tissue construct. (d) Demonstration of the spanning and out-of-plane architecture of the 3D printed construct. (e) Removal of fugitive ink. (f) Composite image (top view) of the 3D printed tissue construct acquired using three fluorescent channels: fibroblasts (blue), HNDFs (green), HUVECs (red). (g) Viability assay of bioprinted cells (Kolesky et al. 2014)

4.2 Acoustic Bioprinting

Organizing cells and cell spheroids can also be achieved by acoustics. Acoustic waves carry an energy used and transformed into noninvasive cell/tissue assembly techniques for regenerative medicine (Chen et al. 2014). Garvin et al. reported the use of ultrasound standing waves for vascularization of collagen hydrogels by forming multilayered EC networks (Garvin et al. 2011). Prior to exposure to ultrasound standing waves, ECs were mixed with a collagen gel and placed on an acoustic transducer covered with a reflector. When the sound field propagated through the transducer, ECs were arranged in multilayer bands inside the collagen and remained stable after gelation. Another acoustic bioprinting approach by Chen et al. showed the assembly of pre-formed spheroids from different cell types using Faraday waves to fabricate 4 cm 2 constructs with 1.6 mm thickness (Chen et al. 2015). The assembly pattern of spheroids can be adjusted by the frequency of standing waves, resulting in highly versatile spatial organizations. The study showed the fusion of bioprinted ECs and fibroblast spheroids in 3 days.

4.3 Microfluidic System

Microfluidic culture devices are platforms for cell culture under continuous perfusion in micrometer-sized engineered environments with the scope of modeling and mimicking physiological functions of desired tissues and organs. The ultimate goal is not to fabricate an entire living organ but rather to achieve minimal functional units that recapitulate tissue/organ-level functions (Wong et al. 2012; Bhatia and Ingber 2014). The integration of technological developments in miniaturized chips is also valuable for advances in 3D cell culture and offers promising tools to investigate vascularization and angiogenesis. Microfluidic devices are useful tools for 3D cell culture and cell-based assays as they provide the ability to spatially and temporally control the medium flow, and thus the biological factor gradients, which make it possible to regulate nutrient and drug concentrations for single-cell or small cell clusters analysis. They display many advantages such as the use of very small amount of samples and reagents, which is associated with reduced cost, the ability to create dynamic micro-scale environments close to the native microenvironments and the tight control of chemical gradients such as gradients of chemokines to study cell migration and avoid animal models.

The dynamics and microarchitecture of vascular structures makes microfluidic platforms ideal to investigate the physiology of vascularization, angiogenesis and organ-level functions. The fabrication of in vitro perfusable, interconnected, long-lasting vascular network in a microfluidic device is well described by Kim et al. (Fig. 8) (Kim et al. 2013). Cell seeding configuration within the designed device enables to study both vasculogenesis and angiogenesis. Shin et al. developed a microfluidic system in which complex gradients of multiple soluble factors such as VEGF and ANG-1 can be regulated to investigate the 3D co-operative sprouting angiogenesis into the ECM from a pre-existing endothelial monolayer (Shin et al. 2011). Besides the physical stimuli originating from the fluid flow, the 3D microenvironment is also critically important for generating vascular network in a microfluidic device. To address how the stiffness and the composition of the hydrogel affects the vascularization in a microchip, Park et al. tested different concentrations of fibrin, collagen type I and their combinations (Park et al. 2014). With an elegant design, multiple biological systems can be integrated and studied on a single microfluidic chip. Alonzo et al. fabricated a four chamber microfluidic chip mimicking the interstitial flow to study homotypic and heterotypic cell-cell interaction under defined pressure and Baker et al. investigated the effect of patterning of microchannels on angiogenic sprouting (Alonzo et al. 2015; Baker et al. 2013).
Fig. 8

Microfluidic system design and endothelial cell seeding configurations for microvascular network and angiogenic sprout formation. (a) Image of the microfluidicchip, channels filled with colored hydrogel for demonstration. (b) Schematic of the microfluidic channels compartmentalized by microposts. (c, d) Cell-seeding configuration for the vasculogenesis where endothelial cells are encapsulated in a 3D fibrin matrix in the central channel. (e, f) Cell-seeding configuration for the angiogenesis experiment. Endothelial cells are seeded on the side of central channel that is filled with empty fibrin matrix (Reproduced from Kim et al. 2013)

Recently Zhang et al. presented built-in vasculature within biodegradable scaffold for organ-on-a-chip bioengineering (Zhang et al. 2016). The developed microfluidic system consists of perfusable 3D-branched microchannel network seeded with ECs and can be implanted in vivo to establish immediate blood circulation. The vessel-like microchannels have nanopores and micro-holes that support the intercellular crosstalk and extravasation of monocytes and endothelial cells upon biomolecular stimulation.

Metastasis of cancer from one organ to another is the essential way of spreading the disease, which typically happens through the microvasculature. Microfluidic platforms that represent a vascularized organ-level function became powerful tools to investigate the extravasation of cancer cells. Groups of Kamm and Moretti developed bone microenvironment in a microfluidic chip with native-like capillary network to study metastatic breast cancer cell extravasation (Jeon et al. 2015). They showed the behavior of cancer cells perfused in bioengineered endothelial capillary in defined microenvironment and the anti-metastatic role of skeletal muscle cells. Further, the developed in vitro microfluidic chip also served as a drug screening platform, which enabled to test the effects of A3AR antagonist on breast cancer extravasation and the role of adenosine in bone tissue.

5 Conclusion and Outlook

  • Microvascular networks can be engineered…

    The vascular system is fundamental to organ function and tissue homeostasis as it supplies tissue with nutrients, oxygen, molecular signals and cells. The long-distance transport of blood between heart and organ tissue is fulfilled by large blood vessels. However, small micro vessels forming the microvascular system ensure the actual exchange of molecules between blood (or lymph) and tissues. Besides few exceptions, like cartilage, the microvasculature permeates all tissues and is required for their functionality. Therefore, it is conceivable that microvascularization has become an important aspect of tissue engineering in many respects. Microvascularization has been (a) integrated into the in vitro pre-establishment of tissues prior to in vivo implantation; (b) reproduced in vitro to investigate the microvasculature itself; and (c) considered when aiming at the generation of faithful in vitro tissue models. Despite focusing on very different scientific goals, all these strategies have in common that they require the formation of microvascular networks in vitro. Therefore, we have discussed in this chapter how the microvasculature is built up, what are the cellular and material components needed to engineer microvascular networks in vitro and how novel engineering approaches such as bioprinting or microfluidics can contribute to the generation of large-scale and perfused microvascularization models.

    Engineering microvascular networks in vitro most often involves a de novo formation of vasculature (vasculogenesis) rather than from existing vasculature (angiogenesis) since starting from defined and available cells is more feasible than from tissue explants. Nevertheless, as soon as vasculogenesis has established primitive microvascular networks, angiogenesis can contribute to the growing and extension of this microvasculature. Obviously, to reproduce these mechanisms in vitro two fundamental starting materials are required: a) appropriate cells and b) appropriate scaffolding materials.

  • … with the correct cellular and material components

    Cellular components for in vitro microvascularization include two big groups of cells, namely ECs and supporting cells. In mono-cultures, ECs have been shown to generate primitive capillary-like phenotypes in vitro for short periods (Stratman et al. 2011; Chwalek et al. 2014a; Salazar et al. 1999; Bell et al. 2001; Zanotelli et al. 2016); however the generation of a robust and mature microvascular network requires supporting cell types that stabilize endothelial capillary-like structures. These supporting cell types surround the endothelial capillary-like structures and therefore are called perivascular cells or pericytes. More recently, also inflammatory cells such as macrophages are, due to their regulatory function of the microvasculature in vivo, considered to be included into in vitro microvascular network formation. However, the benefit of macrophages and their impact on the generation of microvascular structures in vitro remains to be demonstrated.

    Engineering the microvasculature in vitro demands not only the appropriate cellular components but also scaffold materials that favor the formation of microvascular networks outside the body. As discussed in this chapter a variety of biomaterials has been applied to host 3D ramified microvascular networks in vitro. Since it seems promising to mimic in vitro the in vivo environment as closely as possible, scaffolding materials derived from the natural ECM have been used for the initial approaches of microvascular engineering and in fact are until today the most frequently used material class to recreate the microvasculature in vitro. Furthermore, the progress in biomaterial research has led to the development of semi-synthetic and synthetic ECM analogs. Such artificial microenvironments are free of animal or human-derived ECM proteins, chemically well-defined and readily tunable, which taken together make them promising materials for microvascularized in vitro tissue models. However, at least two pivotal features of the natural ECM need to be transferred onto synthetic ECM analogs to allow the in vitro formation of microvascular networks in synthetic materials: cell attachment and MMP-sensitivity. These two biological features are required to enable proteolytically-driven cell migration through the 3D ECM analogs and thereby the self-assembly of endothelial cells into capillaries. Consequently, all (semi-)synthetic materials discussed in this chapter are modified by adhesion and MMP-sensitive sites when applied for microvascular network formation.

5.1 Use of In Vitro Microvascularized Constructs

Once established in vitro, the resulting artificial microvascular networks are used for either further in vitro use or for in vivo transplantation. Since adequate vascularization still ranks among the major bottlenecks of engineered tissue constructs for in vivo applications, the future success of these applications depends on the quality of prevascularization. However, until today the in vivo application of prevascularized tissue has been only realized by rather small constructs. Therefore, it is of fundamental importance to the success of prevascualrization to increase both the size of engineered vessels as well as the size of prevascularized constructs. In this regard, novel bioengineering approaches can become very useful tools to fabricate large vascularized tissue units. Despite the significant progress in engineering macrovascular structures, the in vivo translation of such constructs remains to be accomplished. Besides prevascularizing tissue constructs for in vivo implantation, basic microvascularized tissues models have been described (Blache et al. 2016; Sakaguchi et al. 2013; Bersini et al. 2016). Tissue-specific microvascularized in vitro models could be used to investigate the interaction of micro-capillary structures with specific cell types in their native-like microenvironments. In this context, related cells as well as scaffold materials can be considered from a great toolbox as described in this chapter. Diverse panel of biomaterials allow the modulation of the in vitro microvascularized multicellular tissue models on demand. Developing vascularized tumor models and highly reproducible micro-tissues for drug screening are some potential fields that are going to benefit at most (Chwalek et al. 2014b; Bray et al. 2015; Sobrino et al. 2016; Roudsari et al. 2016). Nevertheless, for biomimicry tissue models, not just the composition of starting materials needs to be highly defined but also their way of biofabrication. Therefore, in this chapter we have also discussed novel bioengineering tools for in vitro vascularization strategies such as 3D bioprinting and microfluidic organ-on-a-chip technologies. Bioengineering approaches can provide advancement in standardization, precision assembly and enhanced control and mimicking of in vitro physical and biochemical environments to address complex questions in microvascularization.


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Copyright information

© Springer International Publishing AG 2018

Authors and Affiliations

  • Ulrich Blache
    • 1
  • Julien Guerrero
    • 2
  • Sinan Güven
    • 3
  • Agnes Silvia Klar
    • 4
  • Arnaud Scherberich
    • 2
    • 5
    • 6
    Email author
  1. 1.University Hospital ZurichZurichSwitzerland
  2. 2.Department of BiomedicineUniversity Hospital Basel, University of BaselBaselSwitzerland
  3. 3.Izmir Biomedicine and Genome Institute and CenterDokuz Eylul UniversityIzmirTurkey
  4. 4.University Children’s Hospital in ZurichZurichSwitzerland
  5. 5.Department of Biomedical EngineeringUniversity of BaselAllschwilSwitzerland
  6. 6.Department of Plastic, Reconstructive, Aesthetic and Hand SurgeryUniversity Hospital BaselBaselSwitzerland

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