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Recent Advances and Future Perspectives in Vascular Organoids and Vessel-on-Chip

William Harvey Research Institute, Faculty of Medicine and Dentistry, Queen Mary University of London, London EC1M 6BQ, UK

Author to whom correspondence should be addressed.

^†

These authors contributed equally to this work.

Organoids 2024, 3(3), 203-246; https://doi.org/10.3390/organoids3030014

Submission received: 11 July 2024 / Revised: 10 August 2024 / Accepted: 31 August 2024 / Published: 4 September 2024

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Figure 1
Diagram showing the main methods to generate vascular organoids (VOs), Created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>. The basic protocol for VO generation from hPSCs to hiPSCs is based on a stepwise differentiation of hPSC (or hiPSC) aggregates with sequential changing of culture media and further differentiation into vascular networks in a 3D matrix, with a variety of modifications as reported by Wimmer et al. [<a href="#B15-organoids-03-00014" class="html-bibr">15</a>], Schmidt et al. [<a href="#B16-organoids-03-00014" class="html-bibr">16</a>], and Dailamy et al. [<a href="#B17-organoids-03-00014" class="html-bibr">17</a>], respectively. It is worth noting that Penninger’s group [<a href="#B15-organoids-03-00014" class="html-bibr">15</a>,<a href="#B18-organoids-03-00014" class="html-bibr">18</a>,<a href="#B19-organoids-03-00014" class="html-bibr">19</a>] reported the first generation of VOs from hPSCs, which was quickly adapted by other laboratories such as Romeo et al. [<a href="#B20-organoids-03-00014" class="html-bibr">20</a>] and Nikolova et al. [<a href="#B21-organoids-03-00014" class="html-bibr">21</a>]. MIM, mesoderm induction medium (DMEM-F12, 2 mM, Ascorbic acid, 355 μM CHIR 99021, 10 μM BMP4); VGM, vascular growth medium (Neurobasal medium/DMEM-F12, N2B27, 2 mM Ascorbic acid, 100 ng/mL VEGF-A); OMM, organoid maintenance medium (Neurobasal medium/DMEM-F12, N2B27, 2 mM Ascorbic acid); VIM, vascular induction media (N2/B27 + VEGF + FSK); VMM, vascular maturation media (StemPro + 15%FBS + VEGF + bFGF). "> Figure 2
Schematic illustration of the key applications for vascular organoids (VOs). Increasing numbers of studies demonstrate a variety of promising applications for hPSC-derived VOs, including infectious disease pathogenesis (such as SARS-CoV2 and pathogenic bacteria), in vitro vascular disease modeling, high-throughput drug screening and drug toxicity testing, creating human VO biobanks for regenerative medicine, multi-omics analysis to probe novel insights into signaling pathways underlying vascular development and disease etiology, exploring key developmental events in human vascular systems, genetic engineering and editing for inherited disorders, and personalized and precision medicine. Diagram is created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>. "> Figure 3
Schematic diagram illustrating the main strategies to generate vessel-on-chip (VoC) and potential applications of VoCs. Different functional vascular cells can be derived from human-induced pluripotent stem cells (hiPSCs), which are incorporated into various 3D microporous scaffolds to create functional VoCs using a variety of fabrication techniques and microfluidic strategies. These human VoCs offer a wide range of potential applications, encompassing various fields such as studying vascular physiology, pathology, and potential therapeutic interventions, exploring infectious disease pathogenesis, using for in vitro vascular disease modeling as well as high-throughput drug screening and drug toxicity testing, providing novel insights into vascular aging and angiogenesis, and creating human VoC biobanks for regenerative medicine. EC, endothelial cell; SMC, smooth muscle cells; SMART, substrate modification and replication by thermoforming. Diagram is created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>. "> Figure 4
Schematic diagram illustrating the main strategies to generate vascularized organoids. Potentially, multiple methods could be applied to generate vascularized tissue organoids such as in vivo transplantation of tissues-specific organoids into mice which allows for host vasculature integration, co-culturing either hiPSCs/hPSCs with mature endothelial cells or hPSC-derived tissues-specific cells with hPSC-derived endothelial cells, genetic engineering vascular-inducing transcription factors (TFs, such as ETV2) into tissue-specific organoids, including vascular induction factors (such as VEGF-A, WNTs, BMPs) into tissue-specific organoids, and different fusion strategies (co-incubating tissue-specific spheroids/EBs (embryonic body) with vascular spheroids/EBs with our without ECM scaffolds, or co-culturing tissue-specific organoids with vascular organoids (Vos)). Diagram is created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>. "> Figure 5
Schematic diagram illustrating human multiple organs-on-chip (MOoC). HiPSCs/hPSCs-derived tissue-specific cells or organoids could be integrated into a human MOoC system by using sophisticated microfluidic devices or systems. These MOoC systems recapitulate the complex physiological environments and interactions of multiple organ systems and better mimic humans, allowing for the study of complex physiological system-system interactions and diseases in a high-throughput controlled manner. BOs, brain organoids; COs, cardiac organoids; IOs, intestinal organoids; KOs, kidney organoids; LOs, lung organoids; POs, pancreatic organoids; VOs, vascular organoids. Diagram is created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>. ">

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Abstract

Recent advancements in vascular organoid (VO) and vessel-on-chip (VoC) technologies have revolutionized our approach to studying human diseases, offering unprecedented insights through more physiologically relevant models. VOs generated from human pluripotent stem cells exhibit remarkable self-organization capabilities, forming complex three-dimensional structures that closely mimic human blood vessel architecture and function, while VoCs are engineered with microfluidic systems that meticulously recreate the physical and functional attributes of blood vessels. These innovative constructs serve as powerful tools for investigating vascular development, disease progression, and therapeutic efficacy. By enabling the creation of patient-specific VOs and VoCs, they pave the way for personalized medicine approaches, allowing researchers to delve into genetic variations, intricate cellular interactions, and dynamic processes with exceptional resolution. The synergy between VOs and VoCs with newly developed cutting-edge technologies has further amplified their potential, unveiling novel mechanisms underlying human pathologies and identifying promising therapeutic targets. Herein, we summarize different types of VOs and VoCs and present an extensive overview on the generation and applications of VOs and VoCs. We will also highlight clinical and translational challenges and future perspectives around VOs and VoCs.

Keywords:

vascular organoid; vessel-on-chip; pluripotent stem cell; induced pluripotent stem cell; cardiovascular disease; vascular disease

1. Introduction

Despite significant advances in understanding human diseases, traditional research models have shown limitations in fully capturing the intricacies of human tissue pathophysiology. Animal models, while valuable, often fail to translate findings to clinical outcomes due to inherent physiological differences between species. Similarly, conventional two-dimensional (2D) cell cultures lack the structural and functional complexity of native tissues [1], failing to replicate the dynamic microenvironment and multicellular interactions crucial for disease modeling. To address these challenges, recent years have witnessed remarkable progress in the development of advanced in vitro models that more closely mimic the human cardiovascular system. Among these innovations, vascular organoids (VOs) and vessel-on-chip (VoC) platforms have emerged as powerful tools for studying human diseases, including cardiovascular diseases (CVDs) [2,3]. These three-dimensional (3D) models offer remarkable opportunities to investigate disease mechanisms, test therapeutic interventions, and conduct drug screening in a physiologically relevant context [4]. VOs are self-organizing 3D structures derived from human pluripotent stem cells (hPSCs) that recapitulate key aspects of vascular development and function [4]. By leveraging the intrinsic capacity of these cells to differentiate and organize into complex tissues, researchers can now generate miniature blood vessels that exhibit remarkable similarity to their in vivo counterparts. These organoids provide insights into vascular development, disease progression, and regeneration, offering a unique platform for personalized medicine approaches. Complementing organoid technology, VoC devices integrate microfluidic systems with human vascular cells to create functional blood vessel models [5]. These platforms enable precise control over the cellular microenvironment, allowing to study the effects of hemodynamic forces, endothelial-blood cell interactions, and barrier function under both physiological and pathological conditions [6]. The ability to manipulate individual parameters while maintaining overall system complexity makes VoC models invaluable for dissecting the molecular and cellular events underlying human diseases. Researchers can now probe genetic variations [7,8,9,10,11], cellular heterogeneity, and dynamic processes at unprecedented resolution, leading to new insights into disease mechanisms and potential therapeutic targets. Moreover, VO and VoC platforms are beginning to address the limitations of traditional drug discovery pipelines. By providing more accurate predictions of drug efficacy and toxicity in humans, these models have the potential to significantly reduce reliance on animal testing, accelerate the development of novel therapies, and improve the success rate of clinical trials [2]. As we continue to unravel the complexities of human diseases, VO and VoC technologies stand at the forefront of a new era in cardiovascular research. Their ability to bridge the gap between simplified in vitro systems and the intricacies of human physiology offers hope for developing more effective strategies to combat human diseases. By espousing these innovative approaches, we move closer to realizing the goal of personalized, precise, and preventative medicine.

2. Vascular Organoids

VOs display intricate 3D structures that faithfully replicate the form and function of natural blood vessels, making them invaluable models for studying vascular biology and diseases. These organoids typically consist of various cellular elements, each playing a role in their growth and performance.

Endothelial cells (ECs) play a crucial role in the formation of the innermost layer of blood vessels, serving to establish a barrier between the blood and surrounding tissues. ECs could be derived from hPSCs, including human embryonic stem cells (hESCs) and human-induced pluripotent stem cells (hiPSCs), through a highly intricate and carefully regulated differentiation process. In the course of development, vascular progenitor cells with the capacity to transform into endothelial and mural cells originate from the lateral and posterior mesoderm [12]. Typically, protocols for inducing mesoderm from hPSCs entail activating the Wnt signaling pathway and stimulating BMP-4. Subsequent differentiation into ECs is accomplished by exposing the cells to VEGF-A, which promotes angiogenesis and the creation of new blood vessels [13,14].

Pericytes play a crucial role in stabilizing ECs, providing structural support, and regulating blood flow. These specialized cells originate from the same progenitor cells as ECs but differentiate in response to factors such as PDGF-BB and TGF-β [12]. The interaction between ECs and pericytes is essential for the maturation and proper functioning of the vascular network within VOs. In 3D cultures, these cells come together to form intricate endothelial/pericyte networks, closely resembling the in vivo environment. Moreover, vascular smooth muscle cells (SMCs) play an important role in maintaining the structural integrity and contractility of blood vessels. These cells can be generated from hPSCs through pathways involving mesoderm induction and exposure to specific growth factors. SMCs envelop the endothelial tubes, adding layers of support and contractile function that are essential for maintaining vessel tone and regulating blood pressure.

Fibroblasts play a key role as producers of the extracellular matrix (ECM), generating essential proteins such as collagen and fibronectin. These proteins form a scaffold that supports cell attachment, migration, and differentiation. The ECM is crucial for maintaining the structural integrity of VOs and for facilitating cell signaling pathways that impact VO development. Understanding the cellular components and their interactions within VOs is crucial for optimizing these models for research and therapeutic purposes, including drug testing and disease modeling. The ongoing refinement of these vascular models shows promise for substantial advancements in regenerative therapies.

2.1. Vascular Organoid Generation

Several methods have been applied to successfully generate VOs from stem cells (Figure 1). Among them, two primary methods are commonly utilized. One method involves differentiating and purifying various cell types and then merging them together, producing a multicellular organoid. A common, second method involves co-differentiation, which involves the simultaneous differentiation of various cell types from hPSCs.

The first protocol described for the development of human VOs was established by Wimmer et al. in 2019 [15]. This protocol leverages the differentiation of multiple vascular cell types from a pool of mesodermal progenitors, coupled with the innate self-organization capabilities of these cells throughout development. The protocol begins with hPSCs aggregating into embryoid bodies, followed by mesodermal fate induction by activating Wnt signaling and BMP-4 stimulation. After then, vascular cells will be differentiated from these mesodermal progenitors through supplementation with VEGF-A and Forskolin. The developed aggregates of approximately 100 to 200 microns will be embedded within 3D, bi-layered collagen I-Matrigel gels to encourage vasculature sprouting. Importantly, collagen and Matrigel are temperature-sensitive and have the potential to undergo early polymerization, resulting in irregular densities and uneven stiffness, which may inhibit sprouting [22]. Regardless, the ECM serves as a critical buffer between the organoids and the plastic surface of the culture dish. Without this assembly, the increase in density of the organoids compared to the non-polymerized matrix would cause them to sink and come into contact with the bottom of the plastic dish, impeding uniform sprouting [22]. Within a few hours of embedding, initial cellular infiltration into the ECM will occur, and the organoids will be cultured for an additional four days before being released from the Matrigel using fine syringe needles [15]. Timing is a vital part of this process; releasing the organoids too late can cause fusion of the organoids, while removing them too early may result in insufficient differentiation, evidenced by dense cores. Despite being successful, when optimizing protocols for VO development in the future, alternative approaches to this labor-intensive step, which also increases contamination risk, should be considered. This methodology has, for the first time, enabled the in vitro assembly of a self-organizing human capillary network.

More recent protocols for VOs heavily rely on Matrigel as an ECM to facilitate vessel sprouting. Matrigel is widely used due to its richness in ECM proteins such as collagen IV, laminin, and entactin, along with multiple growth factors. Despite this, the composition of Matrigel tends to fluctuate significantly between batches, with Matrigel lacking growth factors bearing inconsistency. In order to overcome this issue, Schmidt et al. [16] introduced a novel protocol for the development of VOs that eliminates the need for Matrigel. This approach utilized a conical agarose coating in 96-well plates to aggregate hiPSCs and support subsequent organoid culture. In a similar manner to earlier protocols, mesoderm induction was achieved using CHIR99021 and BMP4 over three days. However, vascular induction using this method entailed a single 48 h dose of 100 ng/mL of VEGF, following which the organoids were maintained in N2B27 medium without additional vascular-specific growth factors [16].

The lack of an ECM alternate and the modified culture conditions in the later protocol used by Schmidt et al. [16] have a considerable impact on organoid morphology. Early-stage organoids normally consist of loosely connected mesenchymal cells, with a vasculogenic region appearing by day seven. Cells within this region express CD31, which gradually infiltrates into other regions within the organoid. One limitation of the Schmidt et al. protocol is the absence of mural cells within in vitro cultures. Although these cells, along with perfusion and the combining of SMA⁺ mural cells with the vascular network, were observed when the organoids were transplanted into a chick chorioallantois membrane, lacking mural cells within these VOs may impact the stability and functionality of the vascular networks. Despite this, Schmidt et al. demonstrated that 3D VOs can form without depending on a matrix and sustained exposure to pro-angiogenic factors, exploiting the cells’ innate self-organizing capabilities within a suitable tissue context.

Both protocols for generating VOs from stem cells have contributed significantly to the field, each offering unique advantages and facing specific challenges. The protocol used by Wimmer et al. [15] was pioneering in its approach to creating VOs. This method’s reliance on a 3D culture system using collagen I-Matrigel gels was innovative but also introduced complexities related to the temperature sensitivity and batch variability of Matrigel. The manual release of organoids from Matrigel, while effective, is a significant bottleneck and poses a risk of contamination, highlighting the need for either automation or alternative techniques in future optimizations. Conversely, the protocol reported by Schmidt et al. [16] addresses some of these challenges by eliminating the need for Matrigel. Utilizing a conical agarose coating in 96-well plates for cell aggregation, this protocol simplifies the culture conditions and reduces variability. The mesoderm induction using CHIR99021 and BMP4 is followed by a single dose of VEGF for vascular induction, streamlining the process and minimizing the need for long-term exposure of angiogenic factors. This approach demonstrates that VOs can indeed form through the innate self-organizing capabilities of the cells; however, the absence of mural cells within in vitro cultures suggests that further refinement is required to ensure the stability and functionality of the vascular networks.

2.2. Functional Characteristics and Physiological Relevance of VOs

2.2.1. 3D Architecture

The benefits of 3D organoid systems surpass those of traditional 2D models in terms of architecture and physiology. Organoids developed from established differentiation protocols possess an inherent capacity for self-organization, leading to the formation of intricate 3D structures that closely mimic human organ morphology [23,24,25]. Unlike 2D monotypic cellular models, 3D organoids undergo multilineage differentiation, resulting in a diverse cell population that forms complex, tissue-like structures. This self-organization is facilitated by fundamental processes such as cellular migration, segregation, and spatially constrained lineage commitment, which are critical during organogenesis.

The use of 3D cell culture models has significantly advanced vascular biology research by providing more physiologically relevant representations of vessels and tissues. Traditional 2D models have been inadequate for accurately reproducing the spatial organization of blood vessels, cell-cell adhesion, and cell-extracellular matrix interactions in vascular diseases. The emergence of 3D VOs has overcome these limitations by offering a model that closely simulates the intricate architecture of vascular tissues [26].

In addition, as researchers look for options to replace animal models, hPSC-derived 3D VOs not only offer a more precise portrayal of human diseases but also allow for precise control of the microenvironment, including signaling pathways and transcriptional and translational regulators, due to their cultivated nature [27]. This ability is rather valuable for studying the spatial organization and interactions within vascular tissues, furthering our understanding of vascular development and pathology. Moreover, the 3D architecture of VOs plays a crucial role in mimicking the spatial arrangement of natural blood vessels and adjacent tissues, providing a more precise and physiologically relevant model for conducting research and developing therapeutics.

2.2.2. Transplantation Studies

The integration of vascularized organoids into a suitable system holds promise for significant enhancement in organoid size and lifespan. Previous research using single-cell RNA sequencing has shown that in vivo implantation of hPSC-derived VOs results in their integration with the host’s vasculature and improved organoid survival and maturation, as evidenced by matured endothelium with clear arteriovenous specification within transplanted VOs [21]. Moreover, VOs can also be transplanted into diabetic mice to generate chimeric humanized mouse models, enabling in vivo modeling of diabetic vasculopathy [15]. These models effectively mimic key diabetic vascular characteristics, including lumen narrowing and vessel regression, and facilitate the assessment of functional vessel parameters such as permeability, blood flow, and preclinical toxicology analysis of vasculopathy. In a similar vein, other pre-vascularized organoids, such as liver [28], pancreatic islet [29], and cortical [30] organoids, have also been transplanted into mice, which allows them to connect to the host vessels. The formation of functional vasculatures within transplanted organoids further promotes their maturation into organs and tissues that closely resemble their in vivo counterparts, enhancing their therapeutic potential against diseases.

2.3. VO Applications

The emergence of VOs has provided physiologically relevant models for studying vascular development and diseases with multiple applications, as illustrated in Figure 2. In recent years, these organoids have significantly advanced tissue engineering techniques, largely due to their intrinsic capacity for self-organization under in vitro conditions. This self-organization is critical for replicating the complex architecture and functionality of native vascular systems, which is essential for accurate disease modeling and therapeutic testing [15,31], as well as drug-induced cardiotoxicity [32]. In addition, the development of patient-derived, disease-specific VOs could enable researchers to investigate the underlying mechanisms of disease progression at a personalized level [26]. This approach allows for the identification of disease-related genes and pathways, which can vary significantly between individuals. Additionally, these organoids provide a platform for assessing drug efficacy and toxicity in a patient-specific context, thereby facilitating the development of more targeted and effective treatments. A notable example is the study by Dang et al. [33], which demonstrated the relationship between infectious disease and microcephaly using brain organoids, highlighting the potential of organoids in modeling complex disease interactions.

2.3.1. Infectious Disease Pathogenesis

In the realm of regenerative medicine, VOs have emerged as a pivotal tool, providing valuable insights into the complex interplay among blood vessels, pathogens, and immune cells [34]. The use of VOs in various culture systems has significantly contributed to our understanding of these interactions. Recently, studies have leveraged the use of VOs to explore the mechanisms of SARS-CoV-2 infection and potential therapeutic interventions. Specifically, researchers have demonstrated the direct infection of blood vessels by SARS-CoV-2 and the subsequent use of soluble human ACE2 to hinder viral entry. This approach was particularly valuable given the multiple expression patterns of ACE2, the primary receptor for SARS-CoV-2, in several tissues.

In a study conducted by Monteil et al. [35], human VOs were utilized to model the vascular aspects of SARS-CoV-2 infection. The choice of VOs was driven by the need to understand the mechanisms by which the virus interacts with ECs, which are crucial components of the vascular system. This enabled the group to directly observe how SARS-CoV-2 affects these cells, contributing to the severe vascular complications seen in COVID-19 patients. It was observed that these VOs could be readily infected by SARS-CoV-2, with it also being demonstrated that infection could be significantly inhibited by the application of clinical-grade human recombinant soluble ACE2 during the initial stages of the infection [35]. This ability to infect VOs and subsequently inhibit this infection with ACE2 provides a powerful platform for testing the efficacy of antiviral compounds in a controlled, physiologically relevant environment. Understanding the role of ACE2 in different tissues, as demonstrated by the infection patterns in the VOs, aids in explaining the multi-organ impact of SARS-CoV-2 [36,37]. The use of VOs has provided pivotal insights into the vascular aspects of SARS-CoV-2 infection and highlighted the therapeutic potential of ACE2. These advancements highlight the importance of VO models in biomedical research, particularly for investigating intricate infectious diseases and developing targeted treatments.

Another similar, notable discovery involves the stimulation of VOs with SARS-CoV-2 antigens. In this study, Khan et al. [38] aimed to determine the precise mechanisms by which SARS-CoV-2 induced endotheliitis, which remains unknown. The group investigated vascular permeability in the context of SARS-CoV-2-mediated endotheliitis using both patient samples and human 3D VOs composed of vascular endothelium, pericytes, and fibroblasts. By employing these VOs, it was revealed that ACE2 is predominantly expressed in pericytes adjacent to vascular networks. Upon VOs being exposed to SARS-CoV-2 or its antigens, there was a significant reduction in the CD144 expression, which is essential for maintaining EC junctions of blood vessels.

The utilization of VOs in these studies is crucial for multiple reasons. Firstly, these VOs replicate the intricate cellular architecture and function of the human vasculature, offering a more relevant model for investigating the interactions between SARS-CoV-2 and ECs than conventional 2D cell cultures. The VO model also enabled a detailed, comprehensive observation of ACE2 expression in pericytes and the subsequent effects of SARS-CoV-2 infection on endothelial permeability. This insight is crucial for understanding how the virus-induced vasculopathy and thrombotic complications. Additionally, the findings of these studies support the development of biomarker-guided therapies to mitigate thrombotic risks in COVID-19 patients by highlighting potential targets for therapeutic intervention.

2.3.2. Disease Modeling

The VOs developed by Wimmer et al. [19] have also been employed to model diabetic vasculopathy. Analysis of dermal vasculature in patients with type 2 diabetes reveals a thick, multi-layered basement membrane. Similarly, VOs exposed to elevated levels of glucose, TNF (tumor necrosis factor), and IL-6 (interleukin 6) in vitro exhibited an expanded basement membrane, mirroring the patient phenotype. Excessive ECM synthesis is predominantly mediated by pericytes, supporting previous findings that mural cells are responsible for basement membrane production and maintenance. Treatment with the γ-secretase inhibitor DAPT prevented basement membrane thickening and restored EC proliferation. In addition to this, pharmacological inhibition and genetic ablation studies identified DLL4 and NOTCH3 as key mediators and potential therapeutic targets in diabetic vasculopathy [19]. Other micro-engineered perfusable 3D VOs, including collagen-patterned, endothelialized microchannels, biomimetic 3D EC-SMC vascular model, and collagen-patterned constricted vascular microchannels, were created to study barrier permeability and neutrophil transendothelial migration, vascular inflammation, and stenosis in atherosclerosis, respectively [39]. Interestingly, 3D VOs have also been adapted for creating a blood-brain barrier that could accurately replicate the in vivo neurovascular organization, offering an innovative and valuable platform for drug screening as well as studying pathological neurovascular functions in neurodegenerative disease [40].

2.3.3. Drug Testing and Development

VOs derived from hiPSCs present significant promise as platforms for personalized drug testing. These organoids maintain the epigenetic characteristics of the donor patient, thus offering a highly physiologically relevant model for investigating vascular cell dysfunction that can result in CVDs. The retention of patient-specific epigenetic information enables a tailored approach to studying disease mechanisms and testing therapeutic interventions, enhancing the relevance and applicability of the findings to the specific patient condition [31]. This advantageous characteristic makes iPSC-derived VOs particularly valuable for precision medicine, where understanding the nuances of each patient’s disease at the cellular level is crucial for developing effective treatments.

Although the current body of research utilizing VOs for drug testing remains relatively limited, the promising results from studies by Monteil et al. [35] and Wimmer et al. [15] highlight their significant potential. These studies demonstrate that VOs can serve as robust platforms for personalized drug testing, offering a high degree of physiological relevance. Monteil et al. showcased how VOs could model SARS-CoV-2 infection and evaluate antiviral interventions, while Wimmer et al. utilized these organoids to replicate diabetic vasculopathy and test therapeutic compounds. Emerging studies have also confirmed that VOs are a nice model system to study vascular development and underlying signal pathways such as B-cell CLL/lymphoma 6 member B [41] and connective tissue growth factor [20]. The abovementioned findings indicate that VOs hold great promise for advancing precision medicine by enabling the tailored testing of drugs on patient-specific models and unraveling key signaling pathways underlying vascular development.

2.4. Challenges and Limitations of Vascular Organoids

While vascular organoids mark a major breakthrough in regenerative medicine, they face several challenges and limitations that impede their full potential. These difficulties arise from their relatively small size and functional inconsistencies when compared to normal tissues, largely because they lack a fully mature vascular system. Additionally, the absence of certain microenvironmental cells, specifically immune cells and stromal cells [42,43], further restricts the potential of VOs.

A notable limitation of the protocol developed by Wimmer et al. [15] is the inability to generate hierarchical vascular tree structures in vitro. Throughout the culture stage, the in vitro VOs fail to develop the complexity required to form arteriole- or venule-like structures. As a result, these organoids lack the tunica media and adventitia, which complicates studies of atherosclerosis that affect these specific layers. While it remains possible to investigate certain disease characteristics, such as endothelial gene expression changes and angiogenic sprouting, the current organoid models are insufficient for investigating the excessive proliferation of VSMCs, macrophage infiltration, or plaque formation.

One strategy to overcome these limitations involves transplanting the organoids into the renal capsule of immunodeficient NOD-SCID (non-obese diabetic/severe combined immunodeficiency) mice. These organoids subsequently demonstrated integration with the host vasculature, forming a fully human endothelial and mural cell vasculature, which remained stable for over six months without the need for co-transplantation of mouse mesenchymal cells [15]. This approach allows for the maturation and integration of the VOs within a living organism, perhaps enabling the study of more complex vascular structures and processes.

The lack of a consistent cellular microenvironment might compromise the physiological relevance and functional complexity of VOs. A well-defined cellular microenvironment is crucial for the normal functioning and development of organoids. The presence of immune cells is particularly important, as they play a crucial role in maintaining homeostasis, facilitating repair, and responding to pathogens. Similarly, stromal cells provide structural support and secrete essential growth factors that influence cellular behavior. The absence of these cells leads to an inadequate representation of in vivo conditions, thus limiting the usefulness of organoids in modeling complex biological processes and diseases. Despite this, Kim et al. [44] have suggested that the lack of a microenvironment in human VOs could also present certain advantages. However, it remains unclear whether this benefit extends specifically to VOs.

It has been suggested that a potential solution to circumvent such a limitation may be to coculture VOs with mesenchymal and immune cell populations [27,43,45,46]. This approach aims to enhance the cellular complexity and functionality of VOs, thereby making them more akin to mature organs. For example, integrating ECs promotes vascularization, effectively addressing the traditional challenge of nutrient and oxygen diffusion in larger tissue constructs. Moreover, the absence of a cellular microenvironment also provides certain benefits, as it allows for the focused study of specific cell types in isolation, ultimately simplifying the analysis of cell-specific behaviors and interactions. The development of VOs often faces limitations in generating larger organoids, which results in higher variability and complicates comparability between research studies. To mitigate these issues, it is advised to adhere to standardized design protocols [47,48].

Another major issue associated with VOs is their frequent irreproducibility, which arises from inconsistencies in differentiating hPSCs into a specific type of organoid using organ-specific inducers. This often results in cellular composition that is heterogeneous in nature with an unidentified number of cells [49]. When attempting to improve reproducibility, it has been proposed to use methods that determine organoid patterning, managing both the spatial and temporal aspects of organoid formation rather than relying on more speculative approaches [50]. Additionally, optimizing differentiation protocols for region-specific organoids and increasing standardization using microwell-based techniques have also been suggested. This includes compartmentalization to facilitate organoid-organoid communication while preventing uncontrolled fusion [51,52]. Moreover, it has also been demonstrated that using Matrigel or collagen to generate human VOs, both of which are widely available and frequently used, may inadvertently introduce heterogeneity and irreproducibility. To overcome this, researchers have started creating both mechanically and chemically defined synthetic ECMs for organoid culture [53,54].

Incorporating an ECM is crucial for the development of a fully mature and stable organoid structure. Despite this, the inclusion of an ECM is not without complications, particularly regarding cryopreservation. In this case, the ECM may hinder effective and rapid infiltration of the cryopreservation media, which introduces difficulties when attempting to recover and achieve optimal cell viability post freeze-thaw cycles. Therefore, developing, refining, and implementing cryopreservation protocols tailored for VOs with ECMs is essential to ensuring the widespread application of VOs in research and clinical settings.

3. Vessel-on-Chip

VoC technology, an advanced subset of organ-on-chip (OoC) technology, replicates the intricate structure and function of the human vascular system within a controlled, miniaturized, and biomimetic in vitro environment, bridging the gap between traditional in vitro models and in vivo studies. By offering a more physiologically relevant and ethically sound platform, VoC technology provides unprecedented opportunities to understand vascular biology and disease mechanisms (Figure 3).

One key aspect of VoC technology is the ability to fabricate biomimetic vascular structures. Marder et al. [55] developed stem cell-derived VoC for CVD modeling, highlighting the potential of using patient-specific cells to create personalized vascular models. Additionally, Yan et al. [56] introduced a rapid-patterning 3D VoC platform, enabling imaging and quantitative analysis of cell-cell junction phenotypes, which are crucial for understanding vascular processes.

Integrating physiologically relevant features into VoC models is another area of focus. Furthermore, de Graaf et al. [57] presented a multiplexed fluidic circuit board for controlled perfusion of 3D VoC, enabling precise control over the microenvironment and fluid dynamics. Advanced microfabrication techniques are also being employed to create intricate vascular structures. Wu et al. [58] demonstrated the use of acoustofluidic engineering to create functional VoC models, showcasing the potential of novel fabrication methods. Incorporating patient-derived cells is another key aspect of VoC technology. Bulut et al. [59] developed a 3D VoC platform based on hiPSC-derived vascular ECs and SMCs, enabling the study of patient-specific vascular biology and disease mechanisms. Vascularized OoC models have also gained attention, as highlighted by Yin et al. [60], who discussed advances in the model structure of in vitro vascularized OoC systems. These models incorporate functional vascular networks, allowing for the investigation of vascular-tissue interactions and the role of the vascular system in various physiological and pathological processes. Imaging and monitoring capabilities are essential for studying dynamic processes in VoC models. Cuartas-Vélez et al. [61] employed visible-light optical coherence tomography to track the dynamics of thrombus formation in a blood-VoC system, demonstrating the potential of advanced imaging techniques for investigating vascular pathologies.

3.1. Microfluidic System in VoC Technology

Vascular cell biology is fundamental for understanding the mechanisms underlying major diseases such as atherosclerosis, diabetes, and cancer [62]. The primary challenge in vascular research is replicating the dynamic, 3D microenvironment of blood vessels in vitro. Traditional cell culture methods, which often involve static, two-dimensional conditions, fail to accurately mimic the complex interactions and mechanical forces experienced by vascular cells in vivo [63]. This gap in modeling physiological conditions has driven the adoption of microfluidic technology, which offers unparalleled precision in simulating the microenvironment of blood vessels.

As illustrated in Figure 3, the conventional microfluidic architecture has been widely adapted to generate VoC, more often referred to as “Microvascular Networks On-Chip” (MVN) in the literature [64,65,66,67]. Briefly, a central gelified chamber (shown as blue in VoC, Figure 3) is created to support the development of VoCs that self-organize from a single cell suspension. Nutrients and oxygen are provided via two lateral channels (shown in red in VoC, Figure 3) where the growth media flows.

Microfluidic devices manipulate small volumes of fluids within microscale channels, enabling precise control over the physical and chemical conditions to which cells are exposed. These devices are commonly fabricated from materials such as glass, polymers, and polydimethylsiloxane (PDMS), with PDMS being particularly favored due to its biocompatibility, optical transparency, and ease of fabrication [62]. The fabrication process of microfluidic devices typically involves soft lithography, where a master mold is created using photolithography techniques. PDMS is then cast onto this mold to replicate the desired microstructures. The resulting PDMS chips can be bonded to glass or other PDMS layers to form enclosed microfluidic channels through which fluids can be precisely directed.

One of the significant advantages of microfluidic technology in vascular research is its ability to replicate the hemodynamic conditions of blood flow. ECs, which line the interior surface of blood vessels, are highly responsive to shear stress generated by blood flow. Microfluidic devices can generate controlled shear stress, enabling the study of EC responses under physiologically relevant conditions [62]. This capability is crucial for understanding how mechanical forces influence vascular function and pathology.

Additionally, microfluidic platforms can establish gradients of growth factors, cytokines, and other signaling molecules, which are essential for studying processes such as angiogenesis and inflammation. Microfluidic devices also facilitate the coculture of multiple cell types, which is critical for mimicking the complex cellular interactions within the vascular system. For instance, co-culturing ECs with SMCs or pericytes within a 3D ECM allows researchers to study the intricate signaling networks and structural organization of blood vessels [58]. These multi-cellular models provide insights into the cellular dynamics and tissue architecture that are difficult to capture with traditional two-dimensional cultures.

Furthermore, the small scale of microfluidic devices reduces the consumption of cells and reagents, making experiments more efficient and cost-effective. The integration of microfluidic technology with OoC systems has further expanded the potential for vascular research. These advanced platforms aim to recreate the functional units of organs, incorporating features such as fluid flow, mechanical stretch, and complex cellular arrangements. VoC models, for instance, can simulate the permeability of blood vessels, the interaction of ECs with circulating immune cells, and the impact of pharmaceutical compounds on vascular integrity [58,59].

3.2. Main Materials for Fabricating VoC

3.2.1. Elastomers and Thermoplastics

As shown in Table 1 and illustrated in Figure 3, a variety of materials (elastomers and thermoplastics) and gel components (alginate, collagen, gelatin, fibrin, polyethylene glycol (PEG), polyvinyl alcohol (PVA), and hybrid hydrogels) have been used for fabricating VoC; each offers distinct advantages and applications.

Elastomers and thermoplastics are two fundamental materials in VoC platforms, each offering unique advantages. PDMS, an elastomer, has emerged as a cornerstone in biomedical and microfluidic research due to its excellent optical, electrical, and mechanical properties. Its chemical inertness, optical transparency, gas permeability, and thermal stability make it particularly attractive for microfluidic platforms and VoC devices [58]. PDMS also exhibits low interfacial free energy, high physical toughness, and biocompatibility, making it suitable for cell culture and biomedical applications [59]. In microfluidics, PDMS enables the fabrication of intricate microstructures with high precision and cost-effectiveness through soft lithography techniques. Replica molding, microcontact printing, and micro-molding in microcapillaries are common methods, with replica molding being the most prevalent [60]. This method involves creating a master mold, usually from silicon, followed by replicating the desired structure in PDMS.

PDMS is the most widely used elastomer in VoC applications [58]. Its exceptional elasticity, transparency, and biocompatibility make it ideal for mimicking blood vessel mechanical properties. Importantly, unlike thermoplastics, PDMS could allow high gas permeability due to its flexible polymer chains. This flexibility enables PDMS to maintain good permeability even at low temperatures and transmembrane pressures. Its permeability can be further modified by adjusting the ratio of polymer to crosslinking agent during membrane preparation. Soft lithography techniques have significantly advanced the rapid prototyping of PDMS-based microfluidic devices, allowing the creation of customized vascular models with high fidelity [62].

However, PDMS has limitations, particularly in terms of small hydrophobic molecule absorption, leaching of uncrosslinked oligomers, and mechanical stiffness, which can impact cellular behavior and experimental outcomes [63]. Its hydrophobic nature can lead to nonspecific adsorption of hydrophobic molecules, affecting drug screening assays and pharmacokinetic studies. Additionally, PDMS’s Young’s modulus may not accurately replicate native blood vessel mechanical properties.

To address these limitations, alternative elastomers with enhanced mechanical properties and reduced small-molecule absorption are being explored. Surface modifications and incorporation of alternative materials with PDMS are being investigated. Polyurethane-based elastomers and UV-curable polymers offer promising alternatives, providing greater control over material stiffness and surface properties [64].

Thermoplastic polymers, such as polycarbonate and cyclic olefin copolymers, have emerged as viable alternatives to elastomers in VoC applications [65]. These materials offer structural stability, compatibility with high-throughput manufacturing processes, and reduced small-molecule absorption. Thermoplastics enable the fabrication of robust microfluidic systems with precise control over channel dimensions and geometries. They exhibit excellent biocompatibility and chemical inertness, minimizing the risk of adverse reactions with cultured cells or biological samples [65].

Thermoplastics can withstand a wide range of temperatures and mechanical forces, making them suitable for long-term cell culture experiments. However, they lack the elasticity of elastomers, which is essential for accurately replicating dynamic vascular environments. The significant difference in Young’s modulus between thermoplastics and native ECM materials can affect cell adhesion, migration, and mechanotransduction processes [59].

To address this limitation, composite approaches that combine thermoplastics with hydrogels or elastomers to create hybrid devices with improved mechanical properties and cellular compatibility are being explored. By incorporating elastomeric components into thermoplastic-based microfluidic platforms, the physiological relevance and functionality of VoC systems can be enhanced [59].

By combining the unique properties of both materials, individual limitations can be overcome, creating hybrid devices with enhanced functionality and performance. For instance, combining PDMS membranes with thermoplastic substrates allows capitalizing on PDMS’s elasticity for mimicking dynamic vascular environments while utilizing the structural stability and reduced small-molecule absorption of thermoplastics [59].

This hybrid approach enables the fabrication of microfluidic devices that accurately replicate physiological flow conditions while minimizing experimental artifacts associated with small molecule adsorption. Moreover, the development of composite materials that combine elastomeric and thermoplastic properties opens up new possibilities for creating advanced VoC platforms with tailored mechanical and biochemical properties [59]. By engineering materials with controlled stiffness, porosity, and surface chemistry, microfluidic devices that better mimic the complex microenvironment of native blood vessels can be designed, leading to more physiologically relevant experimental results.

In conclusion, elastomers and thermoplastics represent two distinct yet complementary classes of materials in VoC platforms. While elastomers offer exceptional elasticity and biocompatibility, thermoplastics provide structural stability and reduced small-molecule absorption.

3.2.2. Key Gel Components in VoC Technology

Hydrogels are crucial in VoC technology due to their biocompatibility and ability to mimic the ECM of blood vessels. Various types of hydrogels are used, each with unique properties and applications. Alginate, a natural polysaccharide from brown seaweed, is biocompatible, non-toxic, and forms hydrogels through ionic crosslinking. Alginate-based hydrogels are widely used to create perfusable vascular channels and networks [68,69]. Due to its limited mechanical properties and degradation rates, alginate is often combined with gelatin or fibrin for improved functionality.

Collagen, the most abundant ECM protein, is highly biocompatible and capable of promoting cell adhesion, proliferation, and differentiation. Collagen hydrogels are frequently used in studying EC behavior and vascular permeability [70]. However, its low mechanical strength and rapid degradation necessitate crosslinking or blending for stability [71]. Gelatin, a hydrolyzed form of collagen, offers greater versatility but shares similar limitations to collagen. Therefore, a modified version of gelatin, gelatin methacrylate, which allows photo-crosslinking for improved stability, has been used to study endothelialization and cell interactions under flow conditions [72].

Fibrin forms hydrogels through fibrinogen polymerization, supporting cell migration, proliferation, and differentiation. It mimics dynamic ECM remodeling but is weak and degrades quickly. To address these issues, fibrin is often combined with other hydrogels or synthetic polymers in VoC systems [73]. Synthetic polymers such as PEG and PVA offer tunable properties and stability. PEG hydrogels are hydrophilic, biocompatible, and protein-resistant, allowing the incorporation of bioactive molecules and cell adhesion peptides. They enable the study of cellular responses to various mechanical and biochemical cues [74].

PVA hydrogels, known for their excellent mechanical properties, form stable and flexible gels useful for creating robust vascular channels and studying fluid dynamics and EC behavior [75]. Hybrid hydrogels in VoC technology combine natural and synthetic materials for enhanced biological functionality and mechanical strength, which could enable precise adjustment of hydrogel properties, thereby creating novel VoC systems [76].

Table 1. Main materials used for fabricating VoC.

	Key Functional Traits	Advantages	Disadvantages	Potential Solutions
Materials
PDMS (polydimethylsiloxane)	Elasticity, optical transparency, and gas permeability [56,77]	Biocompatible, enables intricate microstructure, and cost-effective fabrication [56,77]	Absorbs small hydrophobic molecules, leaching of uncrosslinked oligomers, and low Young’s modulus [78,79]	Surface modifications, and incorporation of alternative materials [79,80]
Thermoplastics (e.g., PC, COCs)	Structural stability and chemical inertness [81,82]	Reduced small molecule absorption, compatibility with high-throughput manufacturing, and wide temperature tolerance [81,82]	Lack of elasticity and high Young’s modulus compared to native ECM [77,81]	Combining with hydrogels or elastomers to create hybrid devices [80,83]
Key gel components
Alginate	Biocompatibility and ease of gelation [84]	Non-toxic and provides 3D matrix for cell growth [84]	Suboptimal mechanical properties and non-ideal degradation rates [84,85]	Combining with other biopolymers (e.g., gelatin, fibrin) [84,85]
Collagen	High biocompatibility and promotes cellular activities [85,86]	Easily moldable and natural presence in human body [85,86]	Low mechanical strength and rapid degradation [85,86]	Crosslinking with agents such as glutaraldehyde, and blending with more robust materials [85,86]
Gelatin	Biocompatibility and low immunogenicity [87]	Retains collagen’s biological properties and forms hydrogels at physiological temperatures [87]	Weak mechanical properties and rapid degradation at body temperature [87]	Chemical modifications (e.g., methacrylation) and photo-crosslinking [87]
Fibrin	Supports cell migration and proliferation, and mimics dynamic ECM remodeling [85]	-Highly biocompatible, and natural role in angiogenesis [85,88]	Relatively weak, and degrades quickly [85,88]	Combining with other hydrogels, and reinforcing with synthetic polymers [85,88]
Polyethylene Glycol (PEG)	Hydrophilicity, and tunable mechanical properties [89]	-Resistant to protein adsorption, and customizable with bioactive molecules [89]	Lacks inherent bioactivity [89]	Incorporating cell-adhesion peptides and bioactive molecules [89]
Polyvinyl Alcohol (PVA)	Highly hydrophilic, and excellent mechanical properties [89]	Stable, flexible, and durable [89]	Limited cell adhesion properties [89]	Modification with bioactive molecules [89]
Hybrid Hydrogels	Combines properties of natural and synthetic materials [80]	Tailored mechanical and biochemical properties, and enhanced functionality [80]	Complexity in fabrication and characterization [80]	Optimizing composition and crosslinking methods [80]

3.3. Key Fabrication Techniques for VoC

As shown in Table 2 and illustrated in Figure 3, multiple fabrication techniques have been extensively explored for fabricating VoC; each comes with unique advantages and limitations. Fabrication techniques play a crucial role in developing VoC systems. This section explores three key methods: soft lithography, photolithography, and non-lithographic approaches, along with various microfluidic strategies for recreating vasculature in vitro.

Soft lithography is essential for creating microfluidic structures in VoC systems. The process typically begins with making a master mold using photolithography or micromilling. A PDMS pre-polymer mixture is then poured over the mold, degassed, and cured to form an elastomeric replica. PDMS’s gas permeability allows for controlled microenvironments within channels, crucial for cell culture. Soft lithography enables the fabrication of multilayered devices with complex channel networks, valves, and pumps, simulating physiological conditions such as pulsatile blood flow. However, PDMS has limitations, such as high hydrophobicity and potential degradation when exposed to certain solvents [90].

Photolithography is another technique for fabricating VoC systems. Li et al. developed a PDMS-based device with multi-height structures using a single-step photolithography process [91]. Revzin et al. created poly(ethylene glycol) hydrogel microstructures [92], while Cokelet et al. pioneered in vitro microvascular blood flow systems [93]. Mathur et al. developed a VoC platform using endothelial progenitor cells to model vascular thromboinflammation [94]. De Graaf et al. reported on a scalable microphysiological system for 3D blood vessels [95]. Photolithography offers precise control over geometrical features and compatibility with various materials but requires cleanroom facilities and has limitations in feature resolution.

Non-lithographic methods have been explored to overcome some limitations of photolithography. Kappings et al. proposed the substrate modification and replication by thermoforming (SMART) technology [96]. This technique involves irradiating a polycarbonate film with heavy ions and using micro-thermoforming to create a semicircular form, which is then bonded to form a porous microchannel. While promising, SMART technology has not yet been extensively demonstrated for fabricating multiscale VoCs.

Microfluidic strategies offer precise fluid control, low sample consumption, and high throughput [65,97,98]. The integration of nanomaterials into microfluidic platforms has further expanded their capabilities. Three main approaches for recreating vasculature in VoC systems are the wall trapping method, the microencapsulation method, and 3D bioprinting.

The wall trapping method involves seeding ECs onto microfluidic channel sidewalls to form an endothelial barrier [99,100,101]. This can be achieved using porous membranes or hydrogel matrices. Porous membranes allow for coculture of multiple cell types [102], while hydrogels enable the creation of lumenized channels without an intermediate membrane. The hydrogel-based approach allows for full interaction between trapped ECs and surrounding cells, mimicking the tubular architecture of blood vessels.

The microencapsulation method, also known as self-assembling or self-morphogenesis, involves encapsulating ECs within microfluidic chambers under controlled conditions [40]. This approach induces vasculogenesis and angiogenesis without subjecting cells to high shear stress. Vascular endothelial growth factor (VEGF) plays a crucial role in promoting vascular sprouting [103], while other factors such as fibroblast growth factor (FGF) and angiopoietin (ANG) modulate vascular formation and stabilization [104]. Campisi et al. created a vascularized network using hiPSC-derived ECs in a microfluidic device supplemented with VEGF [40].

3D bioprinting is used for creating complex biomimetic structures, including blood vessels and vascular networks. This technology combines biomaterials, cells, and computer-aided design to fabricate 3D constructs with high precision. Various approaches have been developed, including the use of microfluidic chips, continuously perfusable platforms, and direct bioprinting of vessel-like structures. Salmon et al. demonstrated the engineering of neurovascular organoids using 3D-printed microfluidic chips [97]. Gao et al. developed a method for 3D bioprinting vessel-like structures with multilevel fluidic channels using a coaxial nozzle system and sacrificial material [105]. Xu et al. proposed a strategy for creating biomimetic blood vessels using a coaxial nozzle system to print a multi-layered construct mimicking blood vessel structure [106].

Several bioprinting methods have been explored for fabricating in vitro tubular blood vessel models, including extrusion-based, inkjet-based, and laser-assisted bioprinting. These approaches consider factors such as cell types, biomaterials, and printing parameters to achieve physiologically relevant and functional vascular constructs.

Injection molding is a fabrication technique for producing VoC devices, offering precision, repeatability, and scalability. The process involves injecting molten polymer into a designed mold cavity to create intricate microvascular networks. PDMS is commonly used due to its biocompatibility and flexibility, but alternative materials such as cyclic olefin copolymer or polystyrene may be used for chemical resistance or reduced absorption. While injection molding provides high precision and consistency, challenges including high initial costs for mold creation and potential polymer shrinkage during cooling remain to be circumvented [99,107].

Laser-assisted bioprinting uses laser technology to deposit biomaterials and living cells in predetermined patterns. This approach allows for the creation of on-demand patterns with high spatial resolution, enabling the fabrication of complex microvascular networks with varying diameters and branching patterns. Laser-assisted bioprinting facilitates the integration of inorganic materials, such as hydroxyapatite, enhancing the biomimetic properties of engineered vasculature [108]. However, it faces some challenges, such as optimizing laser parameters to minimize damage to deposited biomaterials and cells [109].

Micro-extrusion bioprinting involves extruding bioinks through microscale nozzles to generate intricate 3D constructs. This technique offers high spatial control and the ability to create heterogeneous structures. Various natural and synthetic biomaterials have been explored as bioink components, providing a supportive environment for encapsulated cells [110,111,112]. Micro-extrusion bioprinting enables the creation of multi-material constructs, facilitating the integration of different cell types and biomaterials, but it still comes with some remaining challenges, such as maintaining cell viability during the printing process and achieving precise control over bioink deposition [113,114].

Stereolithography bioprinting utilizes photopolymerization to create intricate 3D objects layer by layer. This technology has enabled the creation of complex biological constructs and VoC systems. Stereolithographic 3D bioprinting allows for the fabrication of highly detailed and biofunctionalized hydrogel constructs, mimicking the microarchitecture of native tissues. Applications include the development of 3D culture chips with integrated perfusion networks and the creation of multivascular networks within biocompatible hydrogels [115,116].

Sacrificial bioprinting involves fabricating sacrificial structures that are subsequently removed to create perfusable channels mimicking vasculature. This approach has been used to generate multi-scale vascular networks within 3D hydrogels, incorporating complex channel geometry and multiple fluidic channels. Sacrificial bioprinting has been applied in cancer research to create vascularized tumor-on-chip models. Advanced fabrication techniques, such as two-photon 3D printing and scaffold molding, have been explored to improve the fidelity and resolution of sacrificial bioprinting processes [117].

In conclusion, each fabricating method has its strengths and limitations, and ongoing research aims to overcome challenges and enhance the physiological relevance of engineered vascular models.

Table 2. Key strategies for creating VoC.

Fabrication Technique	Characteristics of Vessels	Relative Cost	Key Advantages	Disadvantages	Potential Solutions to Limitations
Soft-lithography	unknown	Low cost	Precise control over microstructures, biocompatible (PDMS), gas permeable, and enables multilayered devices	PDMS absorbs small hydrophobic molecules and PDMS may swell or degrade with organic solvents	Surface modifications, use of alternative materials, and careful selection of experimental conditions
Photolithography	Blood vessel diameter (BVD)—50 µm to several hundred micrometers (100, 200, and 500 µm)	Low-cost	High precision for microfluidic structures, compatible with various materials, and enables complex channel designs	Requires cleanroom facilities, limited feature resolution, and material compatibility issues	Development of single-step processes, exploration of alternative photo-sensitive materials, and integration with other fabrication techniques [118,119]
Non-lithographic methods (e.g., SMART)	unknown	unknown	Creates rounded cross-sections and simplifies biomimetic scaffold creation [96]	Limited demonstration for multiscale structures and less established than other methods [96]	Further research and development and integration with existing fabrication strategies [96]
3D bioprinting	BVD: 300–500 µm	Moderate to high: Requires specialized equipment and bioinks, involves multiple cell types and hydrogels, and is limited to feature sizes of hundreds of microns.	Creates complex 3D structures, enables vascularized tissues, and high precision and customization [97,105,120,121]	Technical complexity, limited by printable materials, and challenges in maintaining cell viability [120,121]	Development of advanced bioinks, optimization of printing parameters, and integration with other fabrication methods [97,105,120,121]
Injection molding	unknown	Cost-efficient	High precision and repeatability, scalable for mass production, and efficient for complex microchannels [122]	High initial mold cost, polymer shrinkage and deformation, and limited material choices [122]	Careful mold design to account for shrinkage, development of new moldable biomaterials, and optimization of cooling processes [122]
Laser-assisted bioprinting	BVD: 50–200 µm; Number of vessel branching: High (5–10+ branches); changes in BVD: gradual and sudden; other vessel characteristics: high precision, suitable for complex branching structures	High	High spatial resolution, on-demand patterning, and enables multi-material constructs [123,124]	Potential cell damage from laser and challenges in integrating with other components [123,124]	Optimization of laser parameters, development of protective bioinks, and integration of real-time monitoring systems [123,124]
Micro-extrusion bioprinting	BVD: 100–1000 µm; Number of vessels branching: limited (1–3 branches); changes in BVD: gradual; other vessel characteristics: suitable for larger vessels, good for creating straight segments	unknown	Creates complex, heterogeneous structures, enables multi-material constructs, and provides a wide range of printable materials [122,125]	Shear stress may damage cells and challenges in precise bioink deposition [122,125]	Development of shear-thinning bioinks, optimization of extrusion parameters, and integration with other fabrication techniques [122,125]
Stereolithography bioprinting	BVD: 100–500 µm; Number of vessels branching: moderate (3–7 branches); changes in BVD: gradual; other vessel characteristics: high structural integrity, suitable for complex geometries	Moderate to High	High resolution and precision, enables complex 3D structures, and rapid fabrication [126,127,128]	Limited by photo-crosslinkable materials and potential cytotoxicity of photoinitiators [127,128]	Development of biocompatible photopolymers, optimization of light exposure parameters, and integration with other bioprinting techniques [127,128]
Sacrificial bioprinting	Unknown	unknown	Creates perfusable channels, enables complex vascular networks, and suitable for multi-scale structures	Challenges in removing sacrificial material and limited by properties of sacrificial materials	Development of easily removable sacrificial materials, integration with other fabrication techniques, and optimization of removal processes
Microfluidic strategies
Wall trapping method	not applicable	not applicable	Enables coculture of multiple cell types and creates lumenized channels [129,130]	Planar membrane structure (for porous membranes), and high shear stress during cell seeding [129,130]	Use of hydrogels instead of membranes, optimization of cell seeding techniques, and integration with advanced bioprinting [129,130]
Microencapsulation method	not applicable	not applicable	Induces spontaneous vascular formation, low shear stress on cells, and mimics natural vasculogenesis [40,131,132]	Unpredictable sprouting patterns and challenges in controlling precise structures [40,131,132]	Application of microfluidic forces to guide growth, and optimization of growth factor combinations, and integration with other fabrication methods [40,131,132]

3.4. System Integration in Microfluidic Devices and VoC Technology

The design and integration of microchannels, oxygen supply systems, pumps, and valves are pivotal in the development of advanced microfluidic devices. These components collectively enhance the functionality, precision, and application range of microfluidic platforms, particularly in tissue engineering and disease modeling.

Microchannels serve as the foundational architecture of microfluidic devices. Their design and material composition significantly impact the overall performance and application scope of the system [133]. For instance, cyclic olefin copolymer is frequently chosen for fabricating microchannels due to its robustness, suitability for mass production, favorable optical properties for imaging, and low chemical absorption. The typical configuration involves multiple microchannels motored via a multi-channel syringe pump, enabling precise control over fluid dynamics within the device. The serpentine-shaped microchannels are particularly advantageous for hydrodynamic trapping of organoids, ensuring accurate positioning within predefined locations. These channels can be adjusted in dimensions to accommodate different sizes of organoids, which is critical for experiments involving various cell types and sizes. This adaptability allows for the encapsulation of organoids within a fibrin hydrogel matrix without morphological alterations, maintaining their structural integrity and functionality [134].

Oxygen supply is a crucial consideration in microfluidic systems, especially for applications involving cell culture and tissue engineering [88]. The microcirculation within these devices must mimic physiological conditions to ensure cell viability and function. In natural systems, oxygen is delivered to tissues primarily through diffusion, facilitated by the vascular architecture that ensures proximity between cells and capillaries. In microfluidic devices, oxygen delivery can be managed through the integration of gas-permeable materials or by embedding oxygen-generating elements within the system. Hemoglobin’s role in oxygen transport in vivo highlights the necessity for efficient diffusion and convection mechanisms in microfluidic models. Computational models and experimental setups often aim to replicate these conditions, ensuring that oxygen diffusion aligns with physiological parameters to support cellular metabolism and tissue health [134].

Pumps and valves are integral to the regulation of fluid flow within microfluidic devices [135]. Syringe pumps, hydrostatic pressure systems, and pressure controllers are commonly employed to achieve precise flow control. Syringe pumps offer high precision and ease of use but are limited by the volume they can dispense. Hydrostatic pressure systems provide continuous flow and are straightforward to implement but may lack the precision needed for certain applications. Pressure controllers, on the other hand, offer stable and responsive flow control, making them ideal for experiments requiring fine adjustments. They can be integrated with feedback loops and sensors to maintain constant flow rates and pressures, essential for replicating physiological conditions within the microchannels.

Valves in microfluidic systems serve to control the direction and rate of fluid flow, enabling complex fluid manipulation and isolation of specific regions within the device. Various types of valves, including solenoid, pneumatic, and piezoelectric valves, can be integrated into microfluidic platforms. Solenoid valves are electrically controlled and provide rapid response times, making them suitable for high-throughput applications. Pneumatic valves use air pressure to act and are favored for their simplicity and reliability. Piezoelectric valves, which rely on piezoelectric materials that change shape under an electric field, offer precise control and are advantageous for applications requiring minimal mechanical disturbance.

Integration of sensors within microfluidic systems further enhances their capabilities [136]. Pressure sensors, for instance, can monitor changes in fluid dynamics in real-time, providing critical data for assessing the system’s performance and making necessary adjustments. In one approach, pressure sensors are integrated into the microfluidic chip using capillaries to track pressure changes by observing the movement of the liquid-gas boundary. This method allows for continuous monitoring and can be used to determine occlusion times and other dynamic parameters within the system. Additionally, advanced detection tools such as surface acoustic wave lysis devices and multiplexed sensors can be incorporated to measure biochemical markers such as miRNAs, offering rapid and quantitative analysis essential for applications such as ischemia-reperfusion injury assessment.

The integration of microchannels, oxygen supply systems, pumps, and valves into a cohesive microfluidic platform is fundamental to creating a reliable and versatile system for biological and medical research. The choice of materials and design configurations directly impacts the functionality and application range of these devices. For instance, the use of fibrin hydrogels in microchannels supports the encapsulation and growth of organoids, mimicking the ECM and providing a conducive environment for cellular interactions. The ability to adjust microchannel dimensions to fit specific organoid sizes ensures precise positioning and minimizes mechanical stress on the cells, maintaining their physiological relevance.

Oxygen delivery within these systems is managed through careful design and material selection, ensuring that the diffusion and convection of oxygen match physiological conditions. Computational models play a crucial role in optimizing these parameters, allowing researchers to simulate and adjust the microenvironment within the device. The integration of pumps and valves with feedback systems ensures stable and controlled fluid flow, replicating the dynamic conditions found in vivo. This is particularly important for applications involving the culture of delicate cell types or the formation of vascular networks, where precise control over flow rates and pressures is essential. The use of sensors within microfluidic devices provides real-time data on system performance, enabling dynamic adjustments and enhancing the reliability of the platform. This is particularly valuable in high-throughput screening applications, where rapid and accurate measurements are crucial. By integrating advanced detection tools, microfluidic platforms can perform complex biochemical analyses, broadening their application scope to include areas such as drug testing and disease modeling.

3.5. Key Advancements in VoC Technology

VoC technology has revolutionized the field of biomedical research by providing physiologically relevant in vitro models for studying vascular biology, disease mechanisms, and therapeutic interventions. Recent advancements in this domain have been driven by interdisciplinary contributions from bioengineering, microfluidics, stem cell research, and beyond. Herein, we explore the latest developments in VoC technology, highlighting their potential applications and future directions.

One of the most significant breakthroughs in this field has been the utilization of stem cell-derived vessels for CVD modeling. Researchers have successfully engineered VoC that mimic the physiological environment, enabling the study of disease mechanisms and drug responses in a more accurate and personalized manner. By leveraging stem cell technology, these platforms offer insights into individual-specific disease pathophysiology and therapeutic responses, paving the way for personalized medicine and drug testing [137]. The development of continuously perfusable and customizable VoC platforms has addressed the limitations of traditional static culture systems [138]. These innovative platforms allow precise control over vascular architecture and flow dynamics, incorporating matrix-free environments and microfluidic technologies. As a result, researchers can create biomimetic vascular networks with intricate geometries and physiologically relevant flow conditions, opening up new avenues for studying vascular development, angiogenesis, and disease progression in a more realistic setting [139].

Bioprinting techniques have also made significant contributions to the advancement of VoC technology [140]. Researchers have demonstrated the 3D bioprinting of vessel-like structures with multilevel fluidic channels, achieving biomimetic vascular networks [140]. This approach offers spatial control over vascular architecture, enabling the creation of complex and intricate vascular structures that closely resemble their in vivo counterparts. In bioprinting, vascularization is crucial for maintaining tissue viability and function. The integration of vascularized tumor-on-a-chip models has advanced cancer research by providing a more accurate representation of the tumor microenvironment [141,142]. These models incorporate vascularization to recapitulate the complex interplay between tumor cells, ECs, and the surrounding ECM. By incorporating vascularization, researchers can study tumor angiogenesis, drug efficacy, and metastasis in a physiologically relevant context, facilitating the development of personalized cancer therapies. Infectious disease research has also benefited from the advent of OoC models, including VoC platforms [143]. These platforms offer a controlled environment to investigate host-pathogen interactions and evaluate antimicrobial agents. By recapitulating the vascular environment, researchers can gain insights into disease dynamics, such as pathogen dissemination, immune cell recruitment, and the effectiveness of therapeutic interventions.

Microfluidic technologies have played a pivotal role in advancing VoC platforms [87,88]. These microscale systems provide precise control over cellular microenvironments, enabling detailed mechanistic studies of vascular development, cell migration, and disease progression. Researchers have leveraged microfluidic technologies to create intricate vascular networks, manipulate flow conditions, and observe cellular responses in real-time. Advancements in stem cell differentiation have also contributed significantly to the field of VoC technology [137]. Researchers have made progress in differentiating hPSCs into vascular cells, offering a scalable and reproducible approach for generating functional vascular networks.

Furthermore, the integration of VoC platforms with other OoC systems has enabled the creation of more complex and physiologically relevant multi-organ models [73,88]. These integrated systems allow researchers to study the interconnected processes of various organ systems, including the vascular system’s role in nutrient and oxygen delivery, waste removal, and immune cell trafficking. Recent advancements in VoC technology include using collagen culture vessels to fabricate 3D cardiac tissue. This approach yields tissues with 20 times greater contractile ability compared to conventional plastic substrates. The collagen vessels suppress tissue detachment, allow for thicker constructs, and improve drug response sensitivity. This innovation enhances the potential for more accurate drug screening and cardiotoxicity evaluations [144].

VoC models also show promise for evaluating tumor activities in a drug screening platform [67] and studying cancer immunotherapies such as CAR-T cells [145]. A breast cancer-on-chip model successfully captured T cell infiltration, tumor growth, and cytokine responses over eight days. It demonstrated differences between CAR-T and control T cells and revealed how ECs impact CAR-T cell recruitment. The model also integrated patient-derived organoids, allowing for patient-specific evaluations. This approach enables faster testing of CAR-T cell products and intervention strategies compared to animal models [145].

Looking ahead, the field of VoC technology is poised for further advancements as researchers continue to push the boundaries of biomimicry and physiological relevance. The incorporation of advanced materials, such as hydrogels and scaffolds, will enable the creation of more intricate and biologically accurate vascular networks [73]. Additionally, the integration of emerging technologies, such as machine learning and artificial intelligence, will facilitate the analysis of complex data sets generated by these platforms, providing insights into vascular biology and disease mechanisms [146]. Moreover, the development of automated and high-throughput VoC platforms will enable more efficient and scalable drug screening and toxicity testing, accelerating the drug development process [141]. These platforms will also facilitate personalized medicine approaches by allowing researchers to study patient-specific vascular responses and tailor therapies accordingly.

Additional key advancements in VoC technology have emerged from recent efforts to combine organoids with microfluidic systems. These studies have demonstrated significant improvements in organoid vascularization and maturation when cultured under controlled fluid flow conditions [134,147]. In kidney organoids, the application of fluid shear stress (FSS) during development has led to a fivefold increase in vessel percent area and a tenfold increase in junctional density compared to static cultures. High FSS conditions resulted in the formation of perfusable vascular networks with varying diameters, reflecting multiscale vessel formation. Notably, glomerular vascularization was enhanced under high FSS, with over 60% of podocyte clusters invaded by vascular structures compared to only 10–20% in static controls. This process appeared to be regulated by endogenous VEGF gradients, as both VEGF inhibition and exogenous addition disrupted proper glomerular vascularization. The maturation of tubular epithelia was also improved under flow conditions, with enhanced polarity, brush border formation, and ciliary assembly. Gene expression profiles showed upregulation of tubular epithelial transporters and mature proximal tubule markers [147].

In conclusion, these innovations have paved the way for more physiologically relevant in vitro models, enabling detailed studies of vascular biology, disease mechanisms, and drug responses.

3.6. Applications of VoC Technology

VoC technology has emerged as a promising approach in various biomedical research areas, offering unique opportunities to study vascular physiology, pathology, and potential therapeutic interventions (Figure 3). This technology leverages microfluidic systems and advanced biomaterials to create miniaturized models that mimic the intricate structure and function of blood vessels [148]. One significant application of VoC technology is the fabrication of biomimetic vascular structures for in vitro modeling and investigation. In a study by Jia et al., microfluidic techniques were employed to fabricate helical hydrogel microfibers that mimic the natural helical structure of blood vessels. These biomimetic hydrogel microfibers can serve as valuable platforms for studying vascular physiology, pathology, and potential therapeutic interventions in a controlled and physiologically relevant environment [72]. Another promising application lies in the realm of infectious disease research. Alonso-Roman et al. highlighted the potential of VoC for investigating infectious diseases. These models can provide insights into the interactions between pathogens and vascular cells, as well as the mechanisms underlying vascular dysfunction during infectious processes [149]. Vascularized OoC models have also gained attention for their potential in studying various aspects of vascular biology and disease. Yin et al. discussed advances in the model structure of in vitro vascularized OoC systems. These models incorporate functional vascular networks, enabling the investigation of vascular-tissue interactions, angiogenesis, and the role of the vascular system in various physiological and pathological processes [60]. VoC technology has also shown promise in the fields of tissue engineering and regenerative medicine. Gao et al. explored the 3D bioprinting of vessel-like structures with multilevel fluidic channels, paving the way for the development of personalized vascular grafts [73]. The study of vascular aging has also benefited from the advent of VoC technology. Jiao et al. discussed advances in the differentiation of hPSCs into vascular cells [150], which can be utilized in VoC models to investigate the mechanisms underlying vascular aging. Furthermore, VoC systems have been employed to study the effects of fluid flow and shear stress on vascular cells and structures. Catros et al. utilized laser-assisted bioprinting to create on-demand patterns of human osteoprogenitor cells and nano-hydroxyapatite, demonstrating the potential of integrating bioprinting techniques with VoC technology [151]. Bioprinting techniques have also been integrated with VoC technology, enabling the creation of complex vascular structures and the study of vascular-tissue interactions. Chliara et al. discussed the development and applications of bioprinting on OoC platforms, including VoC systems [152].

3.7. Limitations and Challenges in VoC Technology

VoC models have emerged as powerful tools for studying vascular physiology, disease modeling, and drug testing. However, like any technology, they face several challenges and limitations that need to be addressed [87]. One of the key challenges in VoC models is the accurate recapitulation of the complex microenvironmental cues present in vivo. Factors such as the dynamic interplay between different cell types, the presence of biochemical gradients, and the influence of mechanical forces need to be carefully considered and integrated into these models [97]. Another challenge lies in the fabrication and integration of functional vascular networks within these microfluidic devices. Rafiee et al. highlight the advances in coaxial additive manufacturing and their applications in creating physiologically relevant vascular structures [153]. The integration of multiple tissue types and their interactions with the vascular component is also a significant challenge. Rothbauer et al. discuss the importance of recapitulating the biomimetic epithelium/endothelium interface in OoC models for studying physiological processes and disease mechanisms involving the crosstalk between these tissue types [154]. Another limitation of VoC models is the difficulty in accurately modeling the dynamic processes involved in vascular pathologies. Dasgupta et al. highlight how microfluidic OoC technology is revolutionizing the study of mucosal tissues and vasculature, emphasizing the need for advanced imaging techniques to track dynamic processes [155]. The incorporation of patient-specific cells and the ability to recapitulate individual variability is another challenge faced by VoC models. Marder et al. discuss the use of stem cell-derived VoC for CVD modeling, emphasizing the need for robust protocols to generate patient-specific vascular cells and integrate them into these platforms [156]. Furthermore, the validation and correlation of data obtained from VoC models with in vivo observations remain a critical step. Cuartas-Vélez et al. utilized a VoC platform to investigate endothelial COVID-19 fingerprints, highlighting the need for thorough validation and comparison with clinical data to ensure the relevance and translational potential of these models [61]. Despite these challenges and limitations, VoC technology continues to evolve, with ongoing research efforts focused on addressing these issues. Multidisciplinary collaborations involving bioengineers, material scientists, biologists, and clinicians are crucial for overcoming these obstacles and advancing the development of more physiologically relevant and predictive VoC models [157].

4. Vascularized Organoids

Unsurprisingly, the central cells localized in larger tissue organoids often struggle with acquiring nutrients and removing waste, which limits their growth and functionality. In recent years, both in vitro and in vivo vascularization approaches have been explored to address these issues (Figure 4). The process of in vivo vascularization involves the transplantation of organoids into suitable animal models, thereby encouraging organoid integration with the host’s vascular system. This approach has shown potential in generating functional VOs. For instance, W. Van den Berg et al. successfully transplanted hPSC-derived renal organoids into the renal capsule of immunodeficient mice, leading to the formation of a vascular network between the transplanted organoid and host blood vessels [158]. Microscopy imaging confirmed the presence of a vascular system within the organoids. Functional study also demonstrated the functionality of these vascularized renal organoids, showing their ability for selective permeability and re-uptake, similar to human kidney function.

Various studies have employed similar strategies for different types of organoids. For instance, it has been demonstrated that brain organoids transplanted into the mouse brain established blood circulation, showing potential for modeling neurological diseases [159]. However, challenges persist in in vivo vascularization. The main concern is the integration of human organoids with animal vasculature, which could result in the gradual replacement of organoid cells, extracellular matrix, and structure by host animal cells, compromising the human-specific properties of the organoids. This approach is inherently limited by the use of conventional animal models and thus requires further research to fully comprehend and address these issues.

The concept of in vitro vascularization involves creating a vascular system within organoids using gene editing, mixed cell cultures, and microfluidic platforms. The goal is to establish an independent vascular network capable of sustaining the growth, development, and function of the organoid without relying on animal models. This has been achieved by Cakir et al. [30], who employed genetic engineering techniques to induce the expression of EVT2, a gene involved in regulating angiogenesis, in human cortical organs, ultimately generating vascular brain organoids with characteristics akin to the blood-brain barrier. Another technique often proposed is the introduction of pro-angiogenic factors that promote the co-culturing of ECs with other cell types to facilitate the natural development of vascular structures. This was achieved by Shi et al. [160], who cocultured hESCs with human umbilical vein endothelial cells (HUVECs) in vitro, ultimately giving rise to cerebral organoids that had an optimal, well-developed tube-like vascular system. In contrast to the co-culturing technique, gene editing techniques will depend on the overexpression of various cytokines, and as a result, these techniques are accompanied by some shortcomings, namely an increase in the occurrence of gene mutations, the requirement for technical expertise, and the relatively high cost.

Although it is acceptable to develop these organoids with intricate vascular characteristics using these co-culturing and gene editing techniques, the organoids themselves will therefore be selective with regard to the delivery of oxygen and nutrients, an attribute that conflicts with innate perfusion vessels. In the following section, we will discuss different types of vascularized tissue organoids (Figure 4).

4.1. Vascularized Cardiac Organoids (COs)

Vascularized COs are advanced in vitro models that integrate hPSC-derived COs with microfluidic systems [161,162]. These sophisticated platforms mimic the structural and functional properties of the human heart by incorporating a functional vascular network within the 3D COs [163,164]. The development of these models involves several key steps. First, hPSCs are differentiated into cardiomyocytes, ECs, and other supporting cell types using specific signaling molecules and growth factors [150,165]. These differentiated cells are then assembled into 3D COs using techniques such as self-organization or bioprinting [166,167]. To achieve vascularization, ECs are cocultured with the cardiac cells, and angiogenic factors are introduced to promote the formation of a functional vascular network [168,169]. The vascularized COs are subsequently integrated into microfluidic devices that can precisely recreate physiological conditions of the heart, including fluid flow, shear stress, and nutrient gradients [170,171]. Vascularized COs on a chip offer significant advantages over traditional in vitro models and animal models for studying cardiac development, disease modeling, and drug testing [79,172]. By recapitulating the complex cellular interactions and physiological processes of the human heart, these organoids provide a more accurate representation of cardiac physiology and pathophysiology compared to 2D cell cultures [164,173]. Additionally, they enable personalized medicine by utilizing patient-derived iPSCs, allowing for the study of individual genetic variations and their impact on disease progression [55]. Furthermore, these advanced systems reduce the usage of animals for drug screening and toxicity testing, facilitating the evaluation of potential therapeutic compounds on human-relevant tissue models [161,174]. Despite the significant progress made in the development of vascularized COs on a chip, several challenges remain. One major challenge is achieving long-term organoid viability and maturation, which is hindered by the limited diffusion of nutrients and oxygen within COs [175]. Strategies to address this challenge include improving the efficiency of vascular network formation and integrating perfusion systems to facilitate nutrient and waste exchange [161,162]. Another challenge is the accurate recapitulation of the complex architecture and physiological functions of the human heart, including the formation of well-organized ventricular and atrial chambers, as well as the integration of various cell types, such as cardiac conduction system cells [79]. Future directions in this field may involve the incorporation of advanced bioengineering techniques, such as 3D bioprinting, to precisely control the spatial organization of cells and the integration of vascular networks. Additionally, the development of multi-organ systems, combining vascularized COs with other organ models, could enable the study of inter-organ interactions and systemic effects [176].

4.2. Vascularized Brain Organoids (BOs)

BOs derived from hPSCs have emerged as powerful in vitro models for studying brain development, function, and disease mechanisms. However, a major limitation of these BOs is the lack of a functional vascular system, which hinders their long-term viability, maturation, and physiological relevance. To address this challenge, researchers have developed vascularized BOs, which incorporate a functional vascular network within the 3D brain-like structures [177,178,179]. Several strategies have been explored to generate vascularized BOs. First, hPSCs are differentiated into neural progenitor cells, which are then guided to form organized brain-like structures through techniques such as spinning bioreactors or microfluidic devices. These BOs are subsequently cocultured with ECs, either derived from hPSCs or obtained from other sources, to facilitate the formation of a vascular network within the organoid [180]. Second, genetic engineering techniques were employed by Cakir et al. [30] to induce the expression of proangiogenic genes, which increase angiogenesis in BOs, ultimately generating vascularized BOs with characteristics akin to the blood-brain barrier. Third, as a good alternative to the approach described by Cakir et al. [30], Wörsdörfer et al. [181,182] were the first to coculture human BOs with hiPSC-derived VOs to generate vascularized BOs, observing a typical blood vessel ultrastructure including EC-EC junctions, basement membrane, luminal caveolae, and microvesicles. This VO-BO fusion method was further reproduced and refined by others to study human cortical development and neuro-vascular crosstalk [183,184,185,186].

Vascularized BOs offer several advantages over traditional in vitro models and animal models for studying brain development, disease modeling, and drug testing [61]. By recapitulating the complex cellular interactions, tissue architecture, and physiological processes present in the human brain, these BOs provide a more accurate representation of brain physiology and pathophysiology compared to 2D cell cultures or non-vascularized BOs. Additionally, vascularized BOs enable the study of neurovascular interactions, which play crucial roles in various neurological disorders, such as stroke, Alzheimer’s disease, and brain tumors [187]. Despite the significant progress in the development of vascularized BOs, several challenges remain. One major challenge is achieving long-term organoid viability and maturation, as the limited diffusion of nutrients and oxygen within the organoid can hinder proper development and functionality [124]. Strategies to address this challenge include improving the efficiency of vascular network formation and integrating perfusion systems to facilitate nutrient and waste exchange [157]. Another challenge is the accurate recapitulation of the complex architecture and physiological functions of the human brain, including the formation of distinct brain regions, the integration of various cell types (e.g., neurons, astrocytes, oligodendrocytes), and the establishment of proper connectivity and neural circuitry [61,97]. Future directions in the field of vascularized BOs may involve the incorporation of advanced bioengineering techniques, such as 3D bioprinting and microfluidic devices, to precisely control the spatial organization of cells and the integration of vascular networks. Additionally, the development of more sophisticated OoC systems that can mimic the dynamic microenvironment of the brain, including mechanical and electrical stimuli, could further enhance the physiological relevance of these organoids.

4.3. Vascularized Kidney Organoids (KOs)

KOs are 3D cellular structures that mimic the architecture, physiology, and functionality of the human kidney. However, one of the major challenges in developing functional KOs is the lack of a functional vascular system, which is crucial for nutrient and oxygen supply as well as waste removal [188]. Vascularized KOs aim to address this challenge by incorporating a vascular network, enhancing the organoids’ maturation, survival, and physiological relevance [188,189]. One widely adopted approach is the coculture of KOs with ECs, which can self-organize and form primitive vascular structures within the organoid [190,191]. Various types of ECs, such as HUVECs or hPSC-derived ECs, have been employed in these coculture systems. Another strategy involves the incorporation of decellularized ECM derived from kidney tissue as a scaffold for vascular network formation and organoid maturation [191,192]. The ECM provides a natural, biocompatible environment that retains the structural and biochemical cues necessary for cellular organization and vascularization. Bioprinting techniques, which involve the precise deposition of cells, biomaterials, and growth factors, have emerged as a powerful tool for engineering vascularized KOs [188,193,194]. These techniques allow for the creation of intricate vascular architectures within the organoid, mimicking the complex hierarchical structure of the kidney vasculature.

Microfluidic OoC systems have also been employed to provide a controlled microenvironment and perfusion of nutrients, enabling the development and maintenance of vascularized KOs [195]. These systems consist of micrometer-scale channels and chambers that mimic the physiological conditions of the body, including fluid flow, shear stress, and nutrient gradients. Researchers have combined multiple strategies, such as coculture systems, ECM scaffolds, and microfluidic platforms, to enhance the vascularization and maturation of KOs. Additionally, the incorporation of other cell types, such as pericytes and SMCs, has been explored to further stabilize and mature the vascular networks within the organoids. Despite significant progress, challenges remain in achieving long-term stability, functional maturation, and adequate perfusion throughout the organoid [196,197].

Ongoing research efforts focus on optimizing engineering strategies, exploring novel biomaterials, and integrating advanced technologies to create more physiologically relevant and predictive vascularized KOs. Vascularized KOs offer numerous applications in biomedical research and clinical settings. They can serve as models for studying kidney development, disease modeling, and drug testing, providing a more physiologically relevant and predictive platform compared to traditional cell culture models [198,199]. Additionally, together with hiPSCs, vascularized KOs may pave the way for personalized medicine by enabling the development of patient-specific disease models and the testing of potential therapeutic interventions. Vascularized KOs offer several advantages over traditional in vitro models, such as 2D cell cultures and animal models. They more closely recapitulate the complex 3D architecture, cellular composition, and microenvironment of the human kidney, providing a more accurate representation of physiological processes and disease mechanisms. Furthermore, vascularized KOs reduce the need for animal experiments, aligning with the principles of ethical and humane research practices [189].

4.4. Vascularized Lung Organoids (LOs)

LOs are 3D biomimetic models derived from hPSCs, or adult stem cells, that recapitulate key structural and functional features of the human lung [200,201]. These self-organized, multicellular constructs mimic the cellular complexity, architecture, and physiological processes of the native lung, providing a powerful in vitro platform for studying lung development, modeling respiratory diseases, and testing potential therapeutic interventions [199,202]. The generation of LOs typically involves co-culturing lung epithelial cells with mesenchymal cells and, in some cases, ECs to mimic the complex cellular interactions found in the native lung. This coculture system facilitates the self-organization and differentiation of the various cell types into organoid structures that resemble the intricate branching patterns and cellular composition of the lung [200]. Advanced techniques, such as microfluidic systems and bioprinting, have been employed to create preformed vascular networks within LOs, enhancing their physiological relevance and longevity [188].

Recently, significant progress has been made in improving the complexity and maturity of LOs. One notable advancement is the incorporation of immune cells, enabling the study of host-pathogen interactions and the modeling of viral infections, such as SARS-CoV-2 [157,201]. This has facilitated the development of potential therapeutic strategies and a deeper understanding of disease mechanisms. Additionally, the integration of biomaterials and scaffolds has facilitated the creation of organoid constructs with enhanced structural integrity and vascularization, further improving their physiological relevance [189,202] Furthermore, advances in bioengineering and stem cell technology have led to the generation of more mature and functional LOs. For instance, researchers have developed bioengineered niches that promote the in vivo engraftment and maturation of hPSC-derived LOs, enabling better recapitulation of lung development and function [189].

Despite the remarkable progress, several challenges remain in the development and application of LOs. One of the major limitations is the incomplete recapitulation of the complex cellular heterogeneity and architectural features of the human lung. While LOs can capture key aspects of lung biology, they may not fully represent the diverse cell types and intricate organization found in the native organ. Additionally, achieving long-term viability, functional maturation, and proper vascularization of LOs remains a significant challenge. Maintaining the organoids in a physiologically relevant state over extended periods is crucial for their use in disease modeling, drug testing, and regenerative medicine applications [203,204]. Another key challenge is the lack of standardized protocols and scalable manufacturing processes, which can hinder reproducibility and high-throughput applications. Variations in LOs generation techniques and culture conditions can lead to inconsistencies in LOs quality and behavior, hampering their widespread adoption in research and clinical settings. Furthermore, the ethical considerations surrounding the use of hPSCs need to be carefully addressed, particularly in the context of potential clinical applications [199].

LOs hold significant potential for various applications in biomedical research and clinical translation. They can serve as valuable tools for studying lung development, elucidating disease mechanisms, and testing the efficacy of potential therapeutic interventions in a physiologically relevant context. For instance, LOs have been employed to investigate respiratory diseases such as lung cancer, chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis [188]. Moreover, LOs offer a promising platform for personalized medicine approaches. Patient-derived LOs can be used to develop personalized treatment strategies and test the efficacy of potential therapies, paving the way for precision medicine. This is particularly valuable for conditions with high inter-individual variability, where tailored treatments may be more effective. Additionally, LOs have been explored for their potential in regenerative medicine applications, such as tissue engineering and organ transplantation. By providing a physiologically relevant microenvironment and supporting vascular networks, these organoid constructs may facilitate the integration and functionality of implanted tissues or organs [189]. Importantly, LOs have proven valuable in the study of viral infections, particularly in the context of the COVID-19 pandemic. Researchers have utilized LOs to model SARS-CoV-2 infection, investigate host-pathogen interactions, and develop potential therapeutic strategies [201,205]. This has contributed to a better understanding of the disease pathogenesis and the identification of potential treatment targets.

4.5. Vascularized Pancreatic Organoids (POs)

POs are 3D in vitro models that recapitulate the cellular composition, architecture, and functionality of the pancreas [206,207]. The generation of POs typically involves culturing pancreatic stem cells or progenitor cells in a specialized medium that promotes their self-organization into 3D structures [208]. This process aims to mimic the intricate cellular interactions and signaling pathways involved in pancreatic organogenesis [207,209]. Advanced techniques, such as co-culturing with supporting cell types such as ECs or mesenchymal cells, have been employed to enhance the maturity and functionality of these organoids [210,211].

One of the significant advancements in the field of POs is their ability to model pancreatic diseases, including pancreatic cancer and diabetes [212,213]. POs derived from patient samples can recapitulate the genetic and molecular features of the disease, providing a valuable platform for studying disease mechanisms and testing potential therapeutic interventions [212]. Additionally, pancreatic cancer organoids have been used for personalized drug screening, enabling the identification of effective treatment strategies tailored to individual patients [212]. Furthermore, recent studies have demonstrated the potential of POs for regenerative medicine applications, particularly in the context of diabetes. By providing a physiologically relevant microenvironment and supporting cellular interactions, these POs can facilitate the differentiation and maturation of insulin-producing beta cells, paving the way for the generation of functional islet cells for transplantation. Furthermore, Rambol et al. [214] developed a microfluidic platform to study pancreatic islet vascularization. They created perfusable microvascular networks using HUVECs supported by mesenchymal stem cells in fibrin gels. When integrated with rat islets, the microvasculature was actively recruited to the islets, though it rarely invaded them. The platform allowed real-time monitoring of islet-network interactions and offered flexibility to investigate parameters affecting microvascular development and islet integration.

Despite the remarkable progress, several challenges remain in the development and application of POs. One of the major hurdles is achieving the complete recapitulation of the complex cellular heterogeneity and spatial organization found in the native pancreas. While current techniques can generate organoids with multiple pancreatic cell types, ensuring proper ratios, distribution, and functional integration of these cells remains a significant challenge. Another challenge is the limited vascularization and nutrient supply within POs, which can impact their long-term viability and functionality [211]. Strategies such as incorporating vascular networks or implementing microfluidic systems are being explored to address this issue [190,211]. Furthermore, the scalability and reproducibility of POs production are essential for their widespread adoption in research and clinical applications. Efforts are underway to develop standardized protocols and automated manufacturing processes to ensure consistent and scalable POs generation.

POs have demonstrated their potential in various applications, including disease modeling, drug discovery, and regenerative medicine. In the realm of disease modeling, POs have been employed to study host-pathogen interactions, such as viral infections or the interplay between pancreatic cells and the gut microbiome. Additionally, they have facilitated the investigation of developmental processes, including pancreatic organogenesis and the differentiation of endocrine and exocrine cell types [207,209]. In the context of drug discovery, pancreatic cancer organoids have shown promise for personalized drug screening [212]. By recapitulating the genetic and molecular features of a patient’s tumor, these organoids can be used to test the efficacy of various therapeutic agents, enabling the identification of tailored treatment strategies [212]. Moreover, POs hold significant promise for regenerative medicine applications, particularly in the treatment of diabetes. By mimicking the complex cellular interactions and microenvironmental cues found in the native pancreas, these POs may facilitate the generation of functional islet cells for transplantation, potentially offering a more effective and long-lasting treatment for diabetic patients. Furthermore, POs have emerged as valuable tools for studying pancreatic cancer biology and developing novel therapeutic approaches [206,213]. They provide a physiologically relevant platform for investigating the molecular mechanisms underlying pancreatic cancer progression, metastasis, and drug resistance, ultimately contributing to the development of more effective treatments [213]. As research in this field progresses, interdisciplinary collaborations among bioengineers, cell biologists, clinicians, and regulatory bodies will be crucial in addressing the remaining challenges and translating the promising potential of POs into tangible benefits for patients and the biomedical community.

Taken together, with their ability to recapitulate the cellular composition and functionality of the pancreas, these organoids provide a physiologically relevant platform for investigating various aspects of pancreatic biology. However, further research is needed to overcome the challenges related to vascularization, scalability, and reproducibility to fully harness the potential of POs in clinical settings. Interdisciplinary collaborations and continued efforts in developing standardized protocols and advanced techniques will be essential to drive the field forward and ultimately translate the promising findings into patient care.

4.6. Multiorgans-on-Chip (MOoC) Systems with Vascular Components

MOoC systems are advanced in vitro models that aim to recapitulate the complex physiological environments and interactions of multiple organ systems within a microfluidic device [215]. These systems have emerged as powerful tools for studying human physiology, disease mechanisms, and drug testing, offering a more accurate representation of in vivo conditions compared to traditional cell culture models [5,216]. By integrating multiple organ models within a single platform, researchers can investigate the intricate crosstalk and systemic responses that occur between different organ systems, enabling a more comprehensive understanding of physiological processes and disease pathways [217] (Figure 5). The development of MOoC systems has its roots in the convergence of various disciplines, including microfluidics, tissue engineering, and microfabrication techniques [215]. Early efforts focused on creating single OoC models, such as lung-on-a-chip [5], which paved the way for the integration of multiple organ models within a single platform [215,216]. These initial developments demonstrated the potential of microfluidic systems to recreate organ-specific microenvironments and physiological functions, providing valuable insights into disease mechanisms and drug responses [216]. As the field progressed, researchers recognized the importance of incorporating vascular components to better simulate the interconnectivity and crosstalk between different organ systems [218]. The inclusion of vascular networks not only enables the transport of nutrients, oxygen, and waste products but also facilitates communication between different organ models through signaling molecules and circulating factors [215]. This realization led to the development of more sophisticated MOoC systems that integrate vascular components, allowing for a more accurate representation of in vivo conditions [217].

The incorporation of vascular components is a crucial aspect of MOoC systems, as it enables the simulation of blood flow, nutrient transport, and communication between different organ models [217]. These vascular components can take various forms, ranging from simple microfluidic channels lined with ECs to mimic blood vessels to more complex vascular networks created using advanced techniques such as bioprinting or microfabrication [215]. One approach to creating vascular components involves patterning microfluidic channels and seeding them with ECs, which can form a continuous endothelial layer mimicking the structure and function of blood vessels [216]. These endothelialized channels can be integrated with organ compartments, allowing for the exchange of nutrients, signaling molecules, and waste products between the organ models and the vascular network [215].

Another strategy involves the use of bioprinting techniques to create more intricate vascular networks within the MOoC system [218]. By using biocompatible hydrogels and specialized bioprinting methods, researchers can fabricate complex 3D vascular structures that better mimic the intricate architecture of in vivo vasculature [217]. These bioprinted vascular networks can be perfused with culture media or blood-mimicking fluids, enabling the study of organ-specific vascular interactions and responses [217]. Specifically, the design and fabrication of MOoC systems with vascular components involve interdisciplinary approaches, leveraging expertise in fields such as microfluidics, biomaterials, and microfabrication [215].

Soft lithography, a widely used technique in microfluidics, involves the fabrication of microstructures using elastomeric materials such as PDMS [215]. This technique allows for the creation of precisely defined microchannels and chambers, which can be used to construct organ compartments and vascular networks within the MOoC system [218]. 3D printing technologies, such as stereolithography and two-photon polymerization, have also been employed in the fabrication of MOoC systems [215]. These techniques enable the creation of complex 3D structures with high resolution, facilitating the integration of intricate vascular networks and organ compartments within a single device [219].

MOoC systems with vascular components have numerous applications in biomedical research and drug development. They enable the study of organ-organ interactions, systemic responses to drugs or toxins, and the investigation of disease mechanisms involving multiple organ systems [216]. For instance, these systems can be used to evaluate the effect of a potential therapeutic compound on various organ models simultaneously, providing insights into its systemic effects and potential off-target toxicities [215]. Skardal et al. created a three-tissue OoC system, comprised of liver, heart, and lung tissue constructs, and observed drug responses that depend on inter-tissue interaction [220]. Importantly, such MOoC systems have also been successfully proven as an excellent platform for quantitative prediction of human pharmacokinetic responses to drugs [221] and the identification of early biomarkers of cardiotoxicity [222], respectively. Additionally, MOoC systems with vascular components can be utilized for personalized medicine applications by incorporating patient-derived cells or tissues [218]. This approach allows for the study of individual patient responses to drugs or treatments, potentially leading to more personalized and effective therapeutic strategies [219]. Furthermore, these systems can be employed in the study of vascular diseases, such as atherosclerosis, thrombosis, and vascular inflammation [215]. By incorporating vascular components and mimicking blood flow conditions, researchers can investigate the mechanisms underlying vascular pathologies and evaluate potential therapeutic interventions [216].

While MOoC systems with vascular components offer significant advantages, they also present several technological challenges. These challenges include achieving physiologically relevant flow rates, maintaining long-term culture conditions, integrating complex vascular networks, and ensuring scalability and reproducibility [215]. Achieving physiologically relevant flow rates within the vascular networks of the MOoC system stands out as one of the key challenges facing this system. Improper flow rates can lead to disturbances in nutrient transport, shear stress patterns, and overall organ functionality [217]. Researchers have explored various solutions, such as incorporating on-chip pumps or using external pumping systems to precisely control flow rates within the vascular channels [215]. Maintaining long-term culture conditions is another challenge, as the viability and functionality of the organ models and vascular components need to be sustained over extended periods to accurately represent physiological processes and disease progression [219]. Strategies such as incorporating perfusion systems, optimizing culture media formulations, and implementing advanced monitoring systems have been explored to address this challenge [218]. Integrating complex vascular networks that accurately mimic in vivo vasculature is also a significant hurdle. Advanced techniques such as bioprinting and microfabrication have been employed to create intricate 3D vascular structures, but challenges related to perfusion, cell seeding, and integration with organ compartments remain [215]. Ensuring scalability and reproducibility is crucial for the widespread adoption and translation of MOoC systems into clinical applications. Researchers have explored the use of automated fluidic handling systems and standardized protocols to improve the reproducibility and scalability of these systems [218]. Furthermore, the integration of vascularized BOs with other organ models, such as COs, KOs, LOs, POs, and/or other tissue organoids, could lead to the development of human systems on chip, enabling the study of inter-organ interactions and systemic effects of the whole human body [97].

5. Future Prospects and Innovations as Well as Potential Challenges

The field of VOs, VoC, OoC, and MOoC systems with vascular components is rapidly evolving, with ongoing efforts to enhance their complexity, functionality, and clinical relevance. Future prospects include the integration of advanced biosensing technologies, the development of self-organizing vascular networks, and the incorporation of immune system components [215]. Advanced biosensing technologies, such as electrochemical sensors, optical sensors, and microelectrode arrays, can be integrated into these systems to enable real-time monitoring of various physiological parameters, including pH, oxygen levels, and metabolite concentrations [219]. This continuous monitoring capability will provide valuable insights into organ-organ interactions and facilitate the study of dynamic physiological processes.

The development of self-organizing vascular networks within these systems is another exciting prospect. By leveraging principles from developmental biology and tissue engineering, researchers aim to create vascular networks that can self-assemble and remodel in response to environmental cues and organ-specific demands [218]. This approach could lead to more physiologically relevant models and enable the study of processes such as angiogenesis and vascular remodeling [215]. Additionally, the incorporation of immune system components, such as immune cells and lymphoid tissues, into VOs, VoC, OoC, or MOoC systems is an area of active research [216]. These advancements will enable the investigation of immune-related processes, such as inflammation, immune responses to pathogens, and immune-mediated diseases, providing a more comprehensive understanding of human physiology and disease mechanisms [219]. Furthermore, the use of machine learning and artificial intelligence for data analysis and predictive modeling holds promise for further optimizing these functional systems and accelerating biomedical discoveries [218]. By analyzing the vast amounts of data generated from these systems, machine learning algorithms can identify patterns, predict outcomes, and optimize experimental conditions, leading to more efficient and accurate research outcomes.

VOs and VoC systems have emerged as promising tools for studying vascular biology, disease modeling, and drug screening. However, these advanced in vitro models face several biological challenges that need to be addressed to improve their physiological relevance and reliability [43,223].

One of the primary challenges is achieving the full spectrum of cellular complexity found in native vasculature. While many models incorporate ECs and some supporting cell types such as pericytes or SMCs, they often lack the complete array of cells present in vivo. This includes various immune cells, progenitor cells, and specialized stromal cells that contribute to vascular function and homeostasis [224,225]. Another significant challenge is recreating the hierarchical structure of the vascular system. Native vasculature consists of a complex network of vessels ranging from large arteries and veins to small capillaries. Current VOs and VoC models often struggle to generate this hierarchical organization, which is crucial for proper blood flow dynamics and oxygen/nutrient distribution [226,227]. The complexities of ECM composition and structure found in native blood vessels present another challenge for VOs and VoC systems. The vascular ECM plays a critical role in regulating cell behavior, vessel stability, and mechanotransduction. Many current models rely on simplified ECM components or animal-derived materials such as Matrigel, which may not accurately represent the complex and tissue-specific ECM found in human vasculature [53,54].

Perfusion and fluid dynamics pose significant challenges in both VOs and VoC systems. While chip-based models often incorporate flow, achieving physiologically relevant shear stress levels and flow patterns throughout complex vascular networks remains difficult. For VOs, the lack of perfusion can lead to limitations in nutrient and oxygen availability, particularly in larger constructs. This can result in necrotic cores and altered cellular behavior, limiting the long-term viability and functionality of the models [228,229]. The maturation of vascular structures is another critical challenge. Many current models produce vessels that resemble early developmental stages rather than fully mature, functional vasculature. This includes limitations in barrier function, proper vessel wall organization, and functional specialization of different vessel types [230,231]. Innervation of vascular models presents an additional challenge. The autonomic nervous system plays a crucial role in regulating vascular tone and function, yet most current models lack this neural component. Incorporating functional innervation into VOs and VoC systems could significantly enhance their physiological relevance and ability to model neurovascular interactions [232].

The integration of vascular systems with other tissue types poses both opportunities and challenges. While vascularization can enhance the function and viability of other organoid types, achieving proper integration and cross-talk between vascular and parenchymal components remains difficult. This includes challenges in coordinating the developmental timing of different tissue types and ensuring appropriate interactions between vascular and tissue-specific cells [28,233]. Modeling disease states in VOs and VoC systems presents its own set of challenges. While these models offer the potential to recapitulate aspects of vascular pathologies, accurately representing complex, multifactorial diseases such as atherosclerosis or diabetic vasculopathy remains challenging. This includes difficulties in recreating the long-term progression of these diseases and incorporating systemic factors that influence vascular health. The immune component of vascular biology presents another significant challenge. The vasculature plays a crucial role in immune cell trafficking and inflammatory responses, yet many current models lack a robust immune component. Incorporating functional immune cells and modeling their interactions with the vascular system is crucial for studying inflammation-related vascular diseases and immune-mediated drug responses [234].

Standardization and reproducibility remain ongoing challenges in the field. The complexity of these models, combined with variations in cell sources, materials, and protocols, can lead to significant variability among research laboratories and even between different experiments. Developing standardized protocols and quality control measures is crucial for the widespread adoption and reliability of these models [235]. Long-term stability and functionality of vascular models is another area of concern. Many current models show decreased viability or altered function over extended culture periods, limiting their utility for studying chronic conditions or long-term drug effects. Improving the longevity of these models while maintaining their physiological relevance is an important goal [236]. Finally, scaling and high-throughput applications present challenges, particularly for more complex, perfused systems. While VOs and VoC systems offer some advantages in terms of scalability, integrating advanced vascular models into high-throughput screening platforms for drug discovery and toxicity testing remains difficult [237].

In conclusion, while VOs and VoC systems offer exciting opportunities for advancing vascular biology research and drug development, they face numerous biological challenges. Addressing these challenges will require interdisciplinary approaches, combining advances in stem cell biology, tissue engineering, microfluidics, and materials science. As these models continue to evolve, they hold the potential to provide increasingly accurate and physiologically relevant platforms for studying vascular health and disease [43,223].

In a similar way, OoC technology has emerged as a revolutionary approach to studying various physiological processes and disease mechanisms, aiming to recapitulate the complex microenvironment of living organs by combining microfluidics, bioengineering, and cell biology techniques [152,172]. OoCs are miniaturized devices that mimic the structural, functional, and biochemical characteristics of specific organs, enabling researchers to investigate cellular responses, drug interactions, and disease progression in a controlled and physiologically relevant setting [83,201]. While single OoCs have provided valuable insights into organ-specific processes, there is a growing interest in developing MOoC platforms. These platforms integrate multiple organ models on a single chip, allowing for the study of inter-organ communication, systemic responses, and the potential impact of one organ’s dysfunction on others [172,201]. MOoCs offer a more comprehensive and realistic representation of the human body, bridging the gap between traditional cell culture models and animal studies [202,238]. The development of OoCs and MOoCs has been facilitated by advancements in microfabrication techniques, such as 3D bioprinting [152,202]. These techniques enable the creation of intricate microfluidic channels, biomimetic structures, and the precise patterning of cells and biomaterials. Consequently, researchers can precisely control the cellular microenvironment, mimicking in vivo conditions more accurately [83,201].

Moreover, multi-organ chip systems with vascular components represent a transformative approach to studying human physiology, disease mechanisms, and drug responses. The integration of vascular networks enhances the physiological relevance of these systems, enabling more accurate modeling of organ-organ interactions and systemic responses [215]. Despite the technological challenges, ongoing advancements and interdisciplinary collaborations are driving the field forward, with promising prospects for future innovations and applications [219].

6. Conclusions

The advent of VOs and VoC models revolutionized the study of vascular biology and pathology. These advanced in vitro systems offer unprecedented opportunities for modeling human vascular diseases, drug testing, and personalized medicine, addressing significant gaps left by traditional 2D cell cultures and animal models. The development of VOs has provided a robust platform for mimicking the complex architecture and cellular interactions of human vasculature. Studies utilizing these organoids have shed light on critical mechanisms of diseases such as SARS-CoV-2-induced endotheliitis and diabetic vasculopathy. By offering a more physiologically relevant model, VOs enable detailed investigations into endothelial permeability, vessel maturation, and the interactions between ECs and other cells, including mural cells, SMCs, fibroblasts, and immune cells. The potential to use patient-derived iPSCs for creating personalized VOs further enhances their value in precision medicine, allowing for the study of individual-specific disease mechanisms and drug responses.

Complementing the utilization of VOs, VoC models excel in providing controlled microenvironments to study specific aspects of vascular physiology, such as shear stress effects and blood-brain barrier dynamics. These models have been instrumental in exploring the impact of mechanical forces on EC behavior and the functionality of tissue-tissue interfaces, such as the alveolar-capillary interface and the renal vascular-tubule unit. The precise control over experimental conditions in VoC systems makes them ideal for detailed mechanistic studies and for testing drug efficacy and toxicity in a patient-specific context.

However, the adoption of VoC technology is accompanied by several technical challenges that require innovative solutions. This may include optimizing physiological flow rates within vascular networks, maintaining long-term viability and functionality of integrated organotypic models, and ensuring scalability and reproducibility across various experimental setups.

Advances in microfluidic design, biomaterial development, and tissue engineering are actively addressing these challenges, aiming to enhance the reliability and applicability of VoC systems. Moving forward, the continued evolution of VoC technology holds promise for transformative advancements in vascular biology and therapeutic innovation. Future developments may focus on integrating advanced biosensing technologies for real-time monitoring of physiological parameters, enhancing the complexity of vascular networks through bioprinting and microfabrication techniques, and incorporating immune and stromal components to better mimic in vivo vascular microenvironments.

Ultimately, the complementary strengths of VOs and VoC models suggest that integrated approaches could offer even greater insights into vascular biology and disease. Hybrid models combining the detailed mechanistic control of VoC systems with the complex tissue-like environment of organoids could lead to the development of more physiologically relevant vascularized tissue constructs. Such advancements could significantly enhance our understanding of organ-specific vascular diseases and improve the predictive accuracy of therapeutic responses.

Despite their potential, both VOs and VoC models face challenges that need to be addressed. Issues such as the lack of a fully mature vascular system in organoids, functional inconsistencies, and the absence of certain microenvironmental cells limit their physiological relevance. The standardization of protocols and the incorporation of immune and stromal cells could enhance the functionality and reproducibility of these models. Additionally, advancements in cryopreservation techniques and the development of synthetic ECMs could further improve the stability and applicability of VOs. As these technologies mature and become more accessible, they are poised to drive significant progress in understanding and treating vascular diseases, ultimately benefiting patients and advancing human health.

Author Contributions

Conceptualization, G.R.C., C.V.W. and Q.X.; methodology, formal analysis, and investigation, G.R.C., C.V.W. and S.R.; resources, Q.X.; writing—original draft preparation, G.R.C., C.V.W. and S.R.; writing—review and editing, Q.X.; supervision, Q.X.; project administration, Q.X.; funding acquisition, Q.X. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the British Heart Foundation (PG/15/11/31279, PG/15/86/31723, PG/20/10458, and PG/23/11371 to Q.X). This work forms part of the research portfolio for the National Institute for Health Research Biomedical Research Centre at Barts.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable since no new data were created in this review article.

Conflicts of Interest

The authors declare no conflicts of interest. The funders had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.

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Figure 1. Diagram showing the main methods to generate vascular organoids (VOs), Created with BioRender.com. The basic protocol for VO generation from hPSCs to hiPSCs is based on a stepwise differentiation of hPSC (or hiPSC) aggregates with sequential changing of culture media and further differentiation into vascular networks in a 3D matrix, with a variety of modifications as reported by Wimmer et al. [15], Schmidt et al. [16], and Dailamy et al. [17], respectively. It is worth noting that Penninger’s group [15,18,19] reported the first generation of VOs from hPSCs, which was quickly adapted by other laboratories such as Romeo et al. [20] and Nikolova et al. [21]. MIM, mesoderm induction medium (DMEM-F12, 2 mM, Ascorbic acid, 355 μM CHIR 99021, 10 μM BMP4); VGM, vascular growth medium (Neurobasal medium/DMEM-F12, N2B27, 2 mM Ascorbic acid, 100 ng/mL VEGF-A); OMM, organoid maintenance medium (Neurobasal medium/DMEM-F12, N2B27, 2 mM Ascorbic acid); VIM, vascular induction media (N2/B27 + VEGF + FSK); VMM, vascular maturation media (StemPro + 15%FBS + VEGF + bFGF).

Figure 2. Schematic illustration of the key applications for vascular organoids (VOs). Increasing numbers of studies demonstrate a variety of promising applications for hPSC-derived VOs, including infectious disease pathogenesis (such as SARS-CoV2 and pathogenic bacteria), in vitro vascular disease modeling, high-throughput drug screening and drug toxicity testing, creating human VO biobanks for regenerative medicine, multi-omics analysis to probe novel insights into signaling pathways underlying vascular development and disease etiology, exploring key developmental events in human vascular systems, genetic engineering and editing for inherited disorders, and personalized and precision medicine. Diagram is created with BioRender.com.

Figure 3. Schematic diagram illustrating the main strategies to generate vessel-on-chip (VoC) and potential applications of VoCs. Different functional vascular cells can be derived from human-induced pluripotent stem cells (hiPSCs), which are incorporated into various 3D microporous scaffolds to create functional VoCs using a variety of fabrication techniques and microfluidic strategies. These human VoCs offer a wide range of potential applications, encompassing various fields such as studying vascular physiology, pathology, and potential therapeutic interventions, exploring infectious disease pathogenesis, using for in vitro vascular disease modeling as well as high-throughput drug screening and drug toxicity testing, providing novel insights into vascular aging and angiogenesis, and creating human VoC biobanks for regenerative medicine. EC, endothelial cell; SMC, smooth muscle cells; SMART, substrate modification and replication by thermoforming. Diagram is created with BioRender.com.

Figure 4. Schematic diagram illustrating the main strategies to generate vascularized organoids. Potentially, multiple methods could be applied to generate vascularized tissue organoids such as in vivo transplantation of tissues-specific organoids into mice which allows for host vasculature integration, co-culturing either hiPSCs/hPSCs with mature endothelial cells or hPSC-derived tissues-specific cells with hPSC-derived endothelial cells, genetic engineering vascular-inducing transcription factors (TFs, such as ETV2) into tissue-specific organoids, including vascular induction factors (such as VEGF-A, WNTs, BMPs) into tissue-specific organoids, and different fusion strategies (co-incubating tissue-specific spheroids/EBs (embryonic body) with vascular spheroids/EBs with our without ECM scaffolds, or co-culturing tissue-specific organoids with vascular organoids (Vos)). Diagram is created with BioRender.com.

Figure 5. Schematic diagram illustrating human multiple organs-on-chip (MOoC). HiPSCs/hPSCs-derived tissue-specific cells or organoids could be integrated into a human MOoC system by using sophisticated microfluidic devices or systems. These MOoC systems recapitulate the complex physiological environments and interactions of multiple organ systems and better mimic humans, allowing for the study of complex physiological system-system interactions and diseases in a high-throughput controlled manner. BOs, brain organoids; COs, cardiac organoids; IOs, intestinal organoids; KOs, kidney organoids; LOs, lung organoids; POs, pancreatic organoids; VOs, vascular organoids. Diagram is created with BioRender.com.

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Cheruku, G.R.; Wilson, C.V.; Raviendran, S.; Xiao, Q. Recent Advances and Future Perspectives in Vascular Organoids and Vessel-on-Chip. Organoids 2024, 3, 203-246. https://doi.org/10.3390/organoids3030014

AMA Style

Cheruku GR, Wilson CV, Raviendran S, Xiao Q. Recent Advances and Future Perspectives in Vascular Organoids and Vessel-on-Chip. Organoids. 2024; 3(3):203-246. https://doi.org/10.3390/organoids3030014

Chicago/Turabian Style

Cheruku, Gowtham Reddy, Chloe Veronica Wilson, Suriya Raviendran, and Qingzhong Xiao. 2024. "Recent Advances and Future Perspectives in Vascular Organoids and Vessel-on-Chip" Organoids 3, no. 3: 203-246. https://doi.org/10.3390/organoids3030014

APA Style

Cheruku, G. R., Wilson, C. V., Raviendran, S., & Xiao, Q. (2024). Recent Advances and Future Perspectives in Vascular Organoids and Vessel-on-Chip. Organoids, 3(3), 203-246. https://doi.org/10.3390/organoids3030014

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