Advancements in preclinical human-relevant modeling of pulmonary vasculature on-chip

Abstract

Preclinical human-relevant modeling of organ-specific vasculature offers a unique opportunity to recreate pathophysiological intercellular, tissue-tissue, and cell-matrix interactions for a broad range of applications. Lung vasculature is particularly important due to its involvement in genesis and progression of rare, debilitating disorders as well as common chronic pathologies. Here, we provide an overview of the latest advances in the development of pulmonary vascular (PV) models using emerging microfluidic tissue engineering technology Organs-on-Chips (so-called PV-Chips). We first review the currently reported PV-Chip systems and their key features, and then critically discuss their major limitations in reproducing in vivo-seen and disease-relevant cellularity, localization, and microstructure. We conclude by presenting latest efforts to overcome such technical and biological limitations and future directions.

1. Introduction

The ultimate goal of biomedical research is not only to understand human diseases but also to develop new and more effective methods for diagnosis, prevention, treatment, or therapeutic intervention (Benam et al., 2015a). Lung diseases are a leading cause of mortality and commonly associated with high risk of morbidity. In 2019, the World Health Organization (WHO) reported that chronic obstructive pulmonary disease (COPD), respiratory infections, and lung cancers comprise three of top six causes of death in humans globally (WHO, 2020). The public health, economic, and societal burden of emerging respiratory pathogens such as coronaviruses and influenza viruses, that are capable of causing epidemics and pandemics, further underlines the importance of pulmonary disorders. In addition, there are patients with rare pathologies such as pulmonary arterial hypertension (PAH) and idiopathic pulmonary fibrosis (IPF) that are progressive and often present with poor prognosis and lack curing agents (Ainscough et al., 2022; Asmani et al., 2018; Farber and Loscalzo, 2004; Gross and Hunninghake, 2001; Richeldi et al., 2017; Si et al., 2021). Thus, there is a pressing need to better understand mechanisms of disease biogenesis and identify new therapies for human lung diseases.

A great deal of preclinical efforts to date in this space has been focused on the lung epithelium (alveolar or airway), the respiratory interstitium, or the stromal cells (Francis et al., 2022; Niemeyer et al., 2018). However, there is growing evidence on role of pulmonary vasculature in disease genesis and progression, and shaping vascular and inflammatory responses that necessitate understanding and reproducing these cellular and molecular processes in preclinical setting and harnessing such knowledge and experimental tools for therapeutic development. 2D models of lung endothelial cell (EnC) cultures have been widely used by investigators (Griffith and Swartz, 2006; Nawroth et al., 2019). However, despite their simplicity and low-cost, such models do not provide adequate physiological relevance for accurate modeling of biology and functioning of the vasculature. For instance, often cell lines or non-lung cells such as umbilical vein EnCs are used (rather than organ-specific primary cells like donor tissue/organ-derived human pulmonary arterial endothelial cells [hPAEnCs] or human lung microvascular endothelial cells [hLMVEnCs]), the EnCs are cultured and grown on supra-physiologically stiff synthetic materials such as polystyrene, polycarbonate or glass (rather than natural extracellular matrix at pathophysiological rheology range), the EnCs are maintained at static condition (rather than experiencing mechanical forces associated with vascular shear), the models do not recreate in vivo-present intercellular crosstalk between endothelium and perivascular cells, or fail to reproduce cross-sectionally round/oval-shaped geometry of blood vessels which is important for unform shear stress distribution across EnC layer (Vo et al., 2023). Such limitations have led to the use of laboratory animals. However, despite providing relevant physiology with in vivo conditions, animal models are often poor predictor of clinical success due to important inter-species differences in homeostatic functioning and disease-associated pathological processes (Miller and Spence, 2017; Nawroth et al., 2019). For instance, in mice, systemic blood vessels such as the bronchial circulation do not penetrate into the intraparenchymal airways as they do in humans (Rydell-Tormanen and Johnson, 2019). This difference has important consequences in modeling human lung cancer using mouse models as in humans the vascular supply of lung tumors is primarily derived from systemic circulation (Mitzner et al., 2000; Yuan et al., 2012). Moreover, the use of laboratory animals is subject to high-cost, low throughput, and ethical concerns. Bottom-up organoid three-dimensional (3D) cell culture approach, which is based on stem cells, has been recently exploited as an alternative approach for experimental models of lung development (Chen et al., 2017) or lung injury and repair (Kong et al., 2021) with certain successes. However, organoids also suffer from critical limitations including the lack of native organ’s microenvironment and perfusable vascular channels and/or network (Nawroth et al., 2019; Zhang et al., 2022).

Organs-on-Chips are miniaturized biomimetic, perfusable, cell culture systems containing living cells arranged to simulate organ- and tissue-level physiology in a controlled in vitro setting. Their fabrication and cellularization has introduced a cutting-edge technology at the intersection of microfluidics, tissue engineering, and pathophysiological modeling for a diverse range of applications in biomedical science. Organs-on-Chips offers multiple advantages over the traditional 2D in vitro cell cultures, animal models, and organoids (Benam et al., 2015b). These include a more physiologically relevant preclinical model with dynamic fluid (vascular) flow, well-controlled experimental parameters, real-time monitoring capability, and higher throughput (based on device design and manufacturing method). These advantages render Organs-on-Chips particularly useful in various applications including disease modeling (Felder et al., 2019; Li et al., 2023; Nesmith et al., 2014), drug development and testing (Benam et al., 2016b; Mathur et al., 2015; Niemeyer et al., 2018), toxicity testing (Benam et al., 2016a), and personalized medicine (Benam et al., 2015a; Nawroth et al., 2019).

On-chip microfluidic modeling of pulmonary vasculature has been commonly performed as part of Lung-on-a-Chip (hereafter referred to as Lung-Chip) devices which have been primarily developed to recreate and study lung (airway or alveolar) epithelial cells in vitro (Asmani et al., 2018; Barkal et al., 2017; Benam et al., 2016b; Felder et al., 2019; Hassell et al., 2017; Huh et al., 2010; Nesmith et al., 2014; Si et al., 2021; Xu et al., 2013) . Nevertheless, given the importance of lung vascular pathologies and its close link with respiratory diseases, there has been a growing interest in creating standalone Organ-on-a-Chip models of pulmonary vasculature (Nguyen and Ahsan, 2023). However, such focused tissue engineering approaches are still at their infancy with limited published works focusing on three pulmonary vascular diseases (PVDs): pulmonary hypertensions (Ainscough et al., 2022; Al-Hilal et al., 2020; Sarkar et al., 2022), lung fibrosis (Akinbote et al., 2021), and pulmonary thrombosis (Barrile et al., 2018). Thus, this is a unique unmet need for preclinical pathophysiology modeling to create impact on drug development and better understanding of disease mechanisms.

Here, we provide an overview of the existing models of the human pulmonary vasculature on-chip (hereafter referred to as ‘PV-Chip’), and their features such as in vivo-like geometrical considerations, and presence and nature of perivascular niche, 3D extracellular matrix (ECM), and matrix-embedded stromal cells. These properties are key when reproducing diseases-associated pathologies and homeostatic physiology. We also review emerging Organ-on-a-Chip systems that can be applied for modeling pulmonary vasculature at health or disease, and their competitive advantages. We conclude by discussing future directions and major challenges to be tackled.

2. Current models of human pulmonary vascular pathologies on-chip

2.1. Pulmonary arterial hypertension

Pulmonary hypertension (pH) is a major PVD which manifests by increased resistance to blood flow (by > 25 mm Hg at rest or 30 mm Hg on exercise) and consequently enhanced blood pressure in pulmonary arteries (PAs) (Rubin, 1997), that can ultimately lead to heart failure. pH is relatively rare, typically affecting 1 to 2 persons per million people in the general population (Rubin, 1997); however, due to its progressive nature and absence of effective therapy it can cause high mortality. As such, it is critical to better understand underlying pathophysiology and accurately reproduce key disease features in pH preclinically to enable identification of diagnostic biomarkers and development of disease-modifying therapeutics. pH has been categorized into five groups (pulmonary arterial hypertension (PAH), pH due to left heart disease, pH due to lung disease, pH due to blood clot in the lung, and pH due to unknown causes), of which only PAH has been modeled using Organs-on-Chips (Ainscough et al., 2022; Al-Hilal et al., 2020). Ainscough et al. (2022) developed a pulmonary artery-on-a-chip (PA-o-n-a-Chip) adapting the Lung Alveolus-on-a-Chip design from Huh and colleagues (Huh et al., 2010). The model consisted of two microfluidic channels separated by a nanoporous polyethylene terephthalate (PET) membrane, and hPAEnCs and human PA smooth muscle cells (hPASMCs) cultured as single cellular sheets on opposite sides of the membrane to mimic intimal and medial layers of the PAs, respectively (Fig. 1a). Culture media were flowed through the top endothelial channel at a rate that generated shear stress of 6 dyne/cm², which is within physiological range seen in human lung arterioles. To reproduce PAH, the authors knocked down expression of endothelial bone morphogenetic protein receptor 2 (BMPR2) – a major gene linking to multiple PAH pathways, in the hPAEnCs and induced hypoxia on-chip to trigger inflammatory cellular responses seen in vivo. The model was able to capture several important features of the PAH such as hPASMCs activation and proliferation, and intimal and medial layers thickening. In addition, the PA-on-a-Chip was used for testing therapeutic efficacy of Ambrisentan and AZD5153.

Fig. 1.

Current PV-Chip Systems for Preclinical Reproduction of Pulmonary Vascular Pathologies. (a) PA-on-a-Chip model developed by Ainscough et al., for modeling PAH. The model consists of two parallel cross-sectionally rectangular channels separated by a porous membrane. hPAEnCs were cultured over the top surface of the membrane creating vasculature on-chip while hPASMCs were cultured on the undersurface of the membrane to create perivascular space. (b) Multilayer PAH-on-a-chip model developed by Al-Hilal et al., Five consecutive cross-sectionally rectangular channels separated by arrays of pillars were used to respectively create pulmonary vasculature, intima layer, medial layer, adventitial layer, and perivascular layer on-chip. EnCs, SMCs, ADCs were embedded in hydrogel and respectively cast to the intima, medial, and adventitial channels. (c) PV-Chip model for studying IPF developed by Akinbote et al., The model consisted of two perfusable (media) channels separated by a rectangular chamber used to cast ECM hydrogel embedded with EnCs and fibroblasts. Interestingly, EnCs from this model were derived from iPSCs constituting one of the first PV-Chip models utilizing iPSC-derived cells. (d) Vessel-Chip model of PE developed by Barrile et al., The model consisted of two parallel channels separated by a porous membrane, similar to the one developed by Ainscough et al., Note that only one channel was used to create vasculature on-chip. The EnCs were cultured to cover all the four inner surfaces of the vascular channel creating a hollow endothelium lumen that is almost rectangular on cross-section. Human whole blood with full coagulation potential was used in culture instead of culture media to promote clot formation on-chip. (e) PV-Chip model for studying pulmonary vascular remodeling in the context of IPF. The model contains five consecutive channels and chambers: two perfusable rectangular cross-sectional channels alternatively arranged between three non-perfusable chambers. The chambers and channels are separated by arrays of micropillars allowing exchanges of media and nutrients. The two channels are used for perfusing culture media, while the chambers were filled with cell-laden hydrogels, respectively, playing as the vessel’s walls and the connective tissues. Images were reproduced from (Ainscough et al., 2022; Akinbote et al., 2021; Al-Hilal et al., 2020; Barrile et al., 2018; Zeinali et al., 2018).

Al-Hilal and colleagues in seprate work developed another Artery-on-a-Chip model of PAH (Al-Hilal et al., 2020). Their device consisted of five parallel channels separated by micropillar arrays that served as interchannel walls (Fig. 1b). The outermost first and fifth channels were designated as luminal and perivascular layers of PA, respectively. The three inner channels were filled with collagen hydrogel, each loaded with a different cell type present in human pulmonary afteries: EnCs, smooth muscle cells (SMCs), and adventitial cells (ADCs) in channels 2–4 representing intimal, medial, and adverntitial layers, respectively (Fig. 1b). The model allowed morphometric assessment of multicellular behaviors and evaluation of pathology development such as intimal thickening, muscularization, artieral remodeling, and endothelial to mesenchymal transition (EndMT) under effects of vascular shear stress. All the cells cultured on-chip were primary and obtained from healthy and idiopathic PAH donors allowing recapitulation of disease characteristics and gender dependence on-chip, and the model was applied for testing efficacy of several PAH therapies including sexual hormones and anti-PAH drugs.

2.2. Idiopathic pulmonary fibrosis

Idiopathic pulmonary fibrosis (IPF) is a rare, progressive lung disease that is predominantly characterized by excessive deposition of ECM proteins in lung parenchyma and increased tissue stiffness, which cause irreversible scarring and can ultimately lead to respiratory failure and death (Adams et al., 2020; Gross and Hunninghake, 2001). IPF still remains an unmet need as only two anti-fibrotic agents (pirfenidone and nintedanib) have been clinically approved for its treatment. However, these medications only slow the rate of decline in lung function, and they fail to halt or reverse the disease progression (Zhao et al., 2023).

The majority of preclinical studies on fibrotic pathologies in IPF have focused on role of stromal cells, particularly fibroblasts; however, recent reports show that endothelial cells can contribute to fibrosis through multiple mechanisms including becoming senescent, giving rise to myofibroblasts via EndMT, and secreting pro-fibrotic, pro-inflammatory cytokines and chemokines (Sun et al., 2020; Zeisberg et al., 2007). Thus, there has been a growing interest in PV-Chip models that allow study of blood vessel-stroma crosstalk and matrix modeling in vitro. To address this, Akinbote and colleagues developed a 3D PV-Chip recaptipulating lung microvasculatures under induced IPF conditions (Akinbote et al., 2021). Their model consisted of two perfusable parallel channels (media channels) sandwiching by a central rectangular chamber within which cell-laden ECM hydrogel is cast (Fig. 1c). Instead of using pillar arrays (Shin et al., 2012) or porous membranes (Ainscough et al., 2022) to prevent hydrogel leak into the media channels during casting, the authors fabricated rasterized paterns of ¼ channel height along the lateral walls of the hydrogel chamber. Pulmonary EnCs derived from human induced pluripotent stem cells (iPSCs) and primary human lung fibroblasts (hLFs) were mixed with a fibrinogen-based ECM and seeded into the middle chamber allowing the EnCs to self-assemble and form blood microvessels within the matrix. To induce IPF conditions, the cells on-chip were treated with transforming growth factor beta (TGFβ) diluted at varying concentrations in the culture media flowing through the media channels on either side of the ECM chamber. Using this model, the authors demonstrated the role of TGFβ and stromal-endothelial interactions in inducing fibrotic phenotypes including increased ECM stiffness, reduced MMP activity, and increased smooth muscle actin expression. This work also showed feasibility of culturing iPSC-derived cells in PV-Chips for testing patient-specific therapies.

2.3. Pulmonary embolism

Pulmonary embolism (PE) occurs when blood clots, often originated by thrombosis in the deep veins of lower extremity but travelling to the lungs, halt blood flow to the lung arteries. While prompt treatment can greatly reduce the risk of death, PE remains a life-threatening disease due to its sudden blockage of PAs that can result in pH or even heart failure (Lutsey et al., 2022). In an effort to model PE in Organs-on-Chips, Barrile et al., developed Vessel-Chip (Barrile et al., 2018). Similar to the PA-on-a-Chip design (Ainscough et al., 2022), the Vessel-Chip consisted of two parallel microfluidic channels separated by a porous membrane. However, here the EnCs were cultured on all four inner surfaces of the cross-sectionally rectangular vascular channel to create a hollow endothelium-lined vessel (Fig. 1d). To reproduce embolism, the endothelial cells were treated with pro-inflammatory cytokine tumor necrosis factor alpha (TNFα). Then, whole human blood with full coagulation potential was continuously perfused through the vascular channel to induce clot formation. The authors showed ability of this model to reveal emergence of thrombosis in response to an experimental biologic (a CD40L-targeting monoclonal antibody called Hu5c8 for treatment of autoimmune disorders, which its clinical development was terminated due to unexpected thrombotic complications).

2.4. Disease-associated pulmonary vascular remodeling

Pulmonary vascular remodeling (PVR) occurs in multiple conditions including IPF (Barratt and Millar, 2014), COPD (Wick et al., 2011), and pH (Tuder, 2017). For example, clinical and in vivo reports show that in IPF, the lung vessel density is reduced (Renzoni et al., 2003) and capillary irregularity and dilatation increases (Kwon et al., 1991). Moreover, in many cases PVR triggered by PVDs leads to secondary pH which in turn, worsens the underlying PVD condition (Neubert et al., 2020). Therefore, it is important to further our understanding of PVR, especially when it is associated with a primary PVD.

One attempt to model PVR associated with PVD conditions using PV-Chip approach has been the work of Zeinali and colleagues (Zeinali et al., 2018). The authors developed a microvessel-on-a-chip that contained five consecutive channels and chambers: two perfusable rectangular cross-sectional channels alternatively arranged between three non-perfusable chambers (Fig. 1e). The chambers and channels were separated by arrays of micropillars which allowed exchanges of media and nutrients (Bichsel et al., 2015; Kim et al., 2013), a similar concept of design to that of Al-Hilal and colleagues (Al-Hilal et al., 2020). The two channels were used for perfusing culture media, while the chambers were filled with cell-laden hydrogels imitating the vessel’s walls and the connective tissues. Primary human umbilical vascular endothelial cells (HUVECs) were mixed fibrin hydrogels and cultured in the central chamber that was designed to mimic vessel walls, and normal hLFs were embedded separately in additional fibrin hydrogels and filled the two side chambers, imitating the vascular interstitial connective tissues. The model was used to study antiangiogenic effects of Nintedanib at different dosages. The authors found that the tested drug inhibits pulmonary vasculogenesis and angiogenesis in the context of IPF, confirming similar previously reported in vivo observations (Wollin et al., 2015).

3. Limitations of the current PV-Chip platforms

While existing PV-Chips have been utilized to model vascular and associated pathologies (discussed above), they suffer a number of biological and technical drawbacks needed for more accurate reproduction of in vivo-observed pathophysiology and tissue architecture. These limitations can also hinder future applications of these devices. First, all the aforementioned models used cross-sectionally square or rectangular blood channels rather than fully circular or oval vascular lumens seen in our bodies (Table 1). For disease applications, such as PAH, where vascular shear stress plays a crucial role in the disease modeling, using channel geometries that are not physiologically relevant can lead to biased results and conclusions as the mechanical forces of the shear stress are not uniformly distributed across the endothelial cells. Second, in the platforms (PA-on-a-Chip and Vessel-Chip) (Ainscough et al., 2022; Barrile et al., 2018) adopted from the two-channel Lung Alveolus-on-a-Chip design, the porous membrane is usually coated with a thin layer (< 100 μm) of hydrogel prior to cell seeding. This matrix thickness neither is sufficient to provide a realistic ECM microenvironment for EnC adhesion and maintenance, nor allows inclusion of stromal cells such as fibroblasts and/or SMCs for recreation of disease-relevant tissue-tissue crosstalk. The absence of an in vivo-emulating microenvironment, multicellularity, and structural organization can impact the ability of the discussed models in recapitulating PVD. For example, matrix remodeling triggered by endothelial-stromal and stromal-matrix interactions is known to be important in the development of both pH (Thenappan et al., 2018) and IPF (Kulkarni et al., 2016). In other words, the two-channel design used in PA-on-a-Chip and Vessel-Chip is not suitable to model diseases involving ECM and stromal cells-endothelium interactions. Importantly, the thin layer of ECM coating is not always maintained throughout the cell culture, and as such, the EnCs often get exposed to supra-physiologically stiff non-natural materials (such as polydimethylsiloxane [PDMS] or PET) that are used in device or membrane fabrication. Moreover, even in PV-Chips that incorporated ECM with matrix-embedded cells (Akinbote et al., 2021; Al-Hilal et al., 2020), the models lacked endothelial monolayers that create an effective vascular barrier separating the blood-like flow from the perivascular cellular and ECM components. This is because EnCs were embedded inside the hydrogel and the barrier formation entirely relied on the self-organization, which has been shown to be ineffective in creating vascular barrier in vitro (Darland and D’Amore, 2001; Montesano et al., 1983). Finally, in all the existing PV-Chip models except the Vessel-Chip (Barrile et al., 2018), cellular components covered only one surface of the vascular channel. Thus, when flowing circulating cells through the vascular channels, the cells would contact cell-free channel walls which are made of very stiff materials (usually PDMS). This may affect cellular responses or even damage them upon flowing at physiological shear stress levels (Ainscough et al., 2022), which require very high flow rates using common cell culture media. Even in the case that EnCs covered four surfaces of the vascular channel (Barrile et al., 2018), the thin layers of cells and the coatings beneath do not guarantee accurate reproduction of in vivo responses of circulating cells or whole blood.

Table 1.

Summary of Key Characteristics of Existing PV-Chip Systems.

Disease modeled	Channel Cross-section	ECM-embedded	Cellular Components	Vascular Barrier on-chip	Assays	Refs. (s)
pH	Rectangular	No	Primary hPAEnCs and hPASMCs	Yes	Testing Ambrisentan and AZD5153 on PAH	(Ainscough et al., 2022)
pH	Rectangular	Collagen	Primary EnCs, SMCs, ADCs from both healthy and PAH donors	No	Testing efficacy of PAH therapies including sexual hormones and anti-PAH drug	(Al-Hilal et al., 2020; Sarkar et al., 2022)
IPF	Rectangular	Fibrinogen	iPSC-induced EnCs; primary hLFs	No	Testing the role of TGFβ in inducing IPF	(Akinbote et al., 2021)
PE	Rectangular	No	HUVECs from healthy donors	Yes	Testing thrombotic complications of Hu5c8	(Barrile et al., 2018)
PVR	Rectangular	Fibrinogen	HUVECs and hLFs from healthy donors	No	Testing antiangiogenic effects of Nintedanib	(Zeinali et al., 2018)

pH: pulmonary hypertension; PAH: pulmonary arterial hypertension; IPF: idiopathic pulmonary fibrosis; PE: pulmonary embolism; PVR: pulmonary vascular remodeling; hPAEnCs: human pulmonary arterial endothelial cells; hPASMCs: human pulmonary arterial smooth muscle cells; EnCs: endothelial cells; SMCs: smooth muscle cells; ADCs: adventitial cells; iPSCs: induced pluripotent stem cells; hLFs: human lung fibroblasts; HUVECs: human umbilical vein endothelial cells.

It is also worth mentioning that all the above PV-Chip models only were focused on large blood vessels such as arterioles and venules. There is not any reported work focusing on modeling pulmonary capillaries with PVD conditions. This might be partially due to technical difficulties in reproducing physiologically relevant pulmonary capillary networks on-chip. Nevertheless, since capillaries are an important part of the lung vasculature associating with PVD pathologies and development (Kwon et al., 1991), future efforts should also address development of pulmonary capillaries models on-chip.

4. Emergening organ-on-a-chip technologies for on-chip modeling of human pulmonary vascular pathophysiology

4.1. Three-dimensional PV-Chip

Given the limitations discussed above, there is a need for novel PV-Chip models that can more faithfully reproduce microenvironment, tissue architecture, and cellular composition and localization of huma lung vasculature at health and disease. To this end, 3D vascular models utilizing rounded blood vessels embedded in ECM are well-suited candidates. Biologically, a sufficiently thick ECM in PV-Chip can house multiple types of cells, such as lung fibroblasts and lung mesenchymal stromal cells, building the interstitial connective tissues surrounding the vessels. Such microenvironment can promotes intercellular interaction between EnCs and stromal cells that are important in the development of many PVDs such as lung cystic fibrosis (CF) (Gaggar et al., 2011), pH (Thenappan et al., 2018), or IPF (Kulkarni et al., 2016). Mechanically, rounded vasculatures ensure uniform distributions of shear stress on the channel wall, which is directly related to the development of pH or PE (Wu and Birukov, 2019). Stiffness of the ECM is also an important factor affecting PVD pathologies. For instance, matrix stiffening has been associated with progressive decline of lung functions in IPF (Upagupta et al., 2018). Moreover, mechanical forces generated by the periodic breathing motions of the lung are a very specific factor affecting the development of various PVDs (Wu and Birukov, 2019). For example, endothelial mechano-transduction processes happening under cyclic stretching of pulmonary vasculature by breathing motions are known to be linked with progression of acute lung vascular injury and pulmonary fibrosis (Murata et al., 2014) and increase in pulmonary endothelial permeability which potentially itself can cause pulmonary edema (Birukova et al., 2008). Modeling the effects of the mechanical stretch caused by cyclic breathing motions on-chip is only possible if an ECM with suitable stiffness and deformability is used to construct the interstitial tissue surrounding the vasculature (Ferrari et al., 2023; Shimizu et al., 2020; Zeinali et al., 2021).

3D vascular models with rounded blood vessels embedded in suitably-thick ECM have been developed for general vascular related studies using various fabrication methods including casting (Jiménez-Torres et al., 2016) or bioprinting (Kolesky et al., 2016) for vessels of ~ 1 mm, viscous fingering (Bischel et al., 2012; Tsai et al., 2022) or lithography (Zheng et al., 2012) for vessels of few hundred microns, and self-assembly (Wong et al., 2012) for vessels < 100 μm. However, only very recently these models were applied to PVD modeling in several studies such as COPD (Wang et al., 2020), or lung tumor microvasculature (Bichsel et al., 2017; Campisi et al., 2020). This further highlights the potential of 3D PV-Chip models for PVD modeling.

Recently, Vo et al., developed a robust and highly reproducible process for constructing user-controlled long rounded ECM-embedded vascular microlumens lined with primary hLMVEnCs in co-culture matrix-localized primary hLFs on-chip (Vo et al., 2023); this platform was called so-called ECM MV-Chip (Fig. 2). Using simulation, Vo et al., (Vo et al., 2023) provided a practical guideline for design PV-Chip channels especially for shear-sensing study by demonstrating the critical role of microchannel cross-sectional geometry and length in determination of shear stress distribution on the endothelial monolayer. Specifically, the authors showed that to achieve uniform distribution of shear stress on the channel wall, the channels must have rounded (or ideally circular) cross-section and their length must be larger than a certain value determined by the shear stress (Fig. 2a). Experimentally, following the design requirements, the microvascular channel of the ECM MV-Chip was fabricated to create a uniformly rounded cross-section with sufficient length of up to 40 mm, aiming to achieve uniform distribution of shear tress on the channel wall with shear stress up to 10 dyne.cm⁻², i.e., the in vivo comparable shear stress (Anton Sidawy, 2022). The channel was cast in a thick natural gelatin-fibrinogen hydrogel block playing as an ECM (Fig. 2b). The hydrogel’s stiffness could be readily tuned from 1 kPa to ~10 kPa, an appropriate range for recapitulation of various lung conditions encompassing normal to fibrotic matrix (Fig. 2c). Primary hLFs were embedded inside the ECM while hLMVEnCs covered all inner surfaces of the rounded microchannel forming a complete endothelial monolayer on-chip (Fig. 2d, e). The endothelial monolayer effectively prevented diffusion of small molecules such as 70 kDa dextran molecules from the vascular channel into the ECM, mimicking the vascular barrier function seen in vivo (Fig. 2f).

Fig. 2.

Enabling Features of the ECM MV-Chip to Advance PVD Modeling On-Chip. (a) Simulation results showing the importance of vascular geometry and length in determination of shear stress distribution on channel wall. To achieve uniform shear stress distribution, the channel must have a smoothly rounded cross-section and a sufficient length for flow development. (b) Schematic illustrating the cross-section of a fully cellularized ECM MV-Chip. Primary human EnCs (hLMVEnCs) form a packed monolayer on the inner surfaces of the circular microchannel while primary human stromal cells (hLFs) are embedded within the ECM block in the perivascular region. This setting creates a 3D ECM-embedded microvasculature on-chip housing fibroblasts and allowing culture of other stromal cells providing a more in vivo-like multicellular microenvironment. (c) The ability to tailor stiffness of the ECM used in the model. The ECM stiffness can be varied from ≈1 to ≈10 kPa (higher ranges are possible too), encompassing normal to fibrotic lung tissue rheology. (d) Actual cross-sectional image of the fully cellularized ECM MV-Chip showing the compact endothelial monolayer (green) and the stromal embedded ECM (blue) surrounding the vascular channel. (e) A confocal micrograph of z-stacked immunofluorescent images taken from the side of the channel showing endothelial monolayer lining in the vascular channel on-chip. In (d) and (e), the scalebars represent 200 μm (f) Demonstration of effective 3D endothelial barrier formation via measurement of small molecules diffusion from the vascular channel into the perivascular ECM niche. Reproduced from (Vo et al., 2023).

The ECM MV-Chip model of Vo et al., is particularly useful in modeling PVD on-chip. First of all, the uniform distribution of shear stress obtained by the appropriate channel geometries is critical for reproduction of shear stress-sensitive vascular diseases. One example is pH, and its sub-category PAH, in which high shear stress and its distribution on the vascular inner surface play crucial roles in the development and pathology (Schafer et al., 2016). Moreover, the thick ECM block entirely enclosing the vascular lumen provides a realistic microenvironment not only for supporting the vascular lumen but also for housing stromal cells such as fibroblasts and SMCs. For example, the ECM allows embedded hLFs to grow and promotes fibroblast-EnCs interactions as well as cell-matrix interactions, which is an important factor in modeling IPF (Akinbote et al., 2021). Furthermore, the ability to tune the ECM’s stiffness in a wide range in this model is also important in applications such as modeling pulmonary fibrosis (Sundarakrishnan et al., 2018). Also, as the ECM beneath the endothelial layer is thick and has a physiological stiffness, the setting creates an in vivo-like blood vessel preventing damaging of cells that would flow through the vascular lumen. Besides, the ECM MV-Chip model allows formation of a 3D endothelial monolayer fully covering the rounded vascular lumen creating an effective vascular barrier which was not possible in other PV-Chip models using ECM with embedded EnCs (Akinbote et al., 2021; Al-Hilal et al., 2020). It is also worth mentioning that the improved fabrication method in Vo et al., (Vo et al., 2023), adapting the casting method with major improvements in channel stabilization and optimized endothelial seeding protocol, achieved 100 % success rate. Such a high success rate is important for Organ-on-Chip applications, that require high reproducibility.

4.2. Personalized PV-Chips using induced pluripotent stem cells

Another emerging approach for future PV-Chip technology, as well as the Organs-on-Chips field in general, is utilization of cells derived from stem cells. Currently, most studies use either immortalized cells or primary cells to construct PV-Chips. While immortalized cell lines offer a low-cost and more feasible cell culture strategy, they do not fully represent in vivo characteristics of tissues or organs. Primary cells, on the other hand, carry many of the in vivo properties of the cells and have become the gold standard for preclinical modeling using Organs-on-Chips (Low et al., 2021); however, they have limited proliferation potential and it is difficult to obtain them in some diseased settings.

Cells derived from pluripotent stem cells such as iPSCs offer a new opportunity for cellularization of the Organs-on-Chips. iPSCs not only have unlimited renewable potential but also maintain donors’ genetic backgrounds allowing more accurate recapitulation of pathologies, when mutations and genetic abnormalities play a key role in disease biogenesis, on-chip. Moreover, unlike primary cells, which can only be obtained from donors by invasive methods or from explants, iPSCs can be derived by reprogramming skin fibroblasts or blood cells procured by non-invasive methods (Malik and Rao, 2013), enabling studying disease pathologies of any desired patient population. Furthermore, as more protocols are established for successful differentiation of iPSCs into a variety of cell types and tissues, including vascular endothelium, mucociliated airway epithelium, and mesenchymal stromal cells, the use of iPSC-derived cells enables personalization of Organs-on-Chips. That is all lineage committed cells and tissues on-chip can be obtained from a single donor. This offers a unique advantage for modeling rare genetic pulmonary disorders such as pulmonary surfactant metabolism dys-functions (Wert et al., 2009). The utilization of iPSC-derived cells in Organs-on-Chips is still at its infancy with only several works focusing on separated organs such as airway (Sone et al., 2021), blood vessels (Luo et al., 2021), spinal cord (Sances et al., 2018), cardiac tissues (Huebsch et al., 2022). To our knowledge, the work of Akinbote and colleagues (Akinbote et al., 2021) was the only report that used iPSC-derived cells for PVD modeling.

5. Conclusions

Here, we reviewed the latest advances in development of Organ-on-a-Chip models of the PVD. Despite the unmet nature and pressing therapeutic needs of PVDs, and the broad range of these disorders, to date only few of them have been studied using PV-Chip models. Moreover, while the existing PV-Chips have achieved some level of success in design, development and application, their future potential and further utilization are partially impeded by their biological and technical drawbacks such as non-in vivo-like channel vascular channel geometries and the lack of suitable microenvironments and multicellular components. To address these limitations and move the field of forward, emerging technologies such as 3D PV-Chip models like ECM MV-Chip and iPSCs-derived cells for seeding into the chips are needed.

Data availability

Data will be made available on request.