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An overview of vascularization techniques. (A) Vascular structures are bioprinted by extrusion or droplet deposition of cells suspended in biocompatible gel in a patterned manner. (B) Photo-induced gelation of cell-containing liquid precursors is performed in a layer-by-layer fashion with DMD patterning. (C) 2-photon photopolymerization is used to directly fabricate perfusable tubular networks of arbitrary geometry which can be embedded in a cell-containing hydrogel. (D) A cell-laden extracellular matrix is cast over a sacrificial filament lattice created by bioprinting or stereo-lithography. In an aqueous environment, the sacrificial filaments are dissolved and the resulting hollow perfusable network is perfused with endothelial cells which form a conformal layer around the inner diameter of the vessels. (E) Microchannels fabricated in a cell-laden hydrogel by laser ablation can be directly seeded with endothelial cells to generate functional blood vessels. (F) Organoid angiogenesis can be achieved by grafting in a highly vascularized animal tissue, with the host vasculature infiltrating the organoid. Alternatively, organoid vascularization is also achieved in vitro by endothelial cell co-culture in compartmentalized microfluidic chip. VEGF and hypoxia gradients established on-chip provide spatial guidance cues to direct angiogenic sprouting.
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The development of increasingly biomimetic human tissue analogs has been a long-standing goal in two important biomedical applications: drug discovery and regenerative medicine. In seeking to understand the safety and effectiveness of newly developed pharmacological therapies and replacement tissues for severely injured non-regenerating tissues and...
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The tumor microenvironment (TME) plays a significant role in cancer growth and metastasis. Bioengineered models of the TME will advance our understanding of cancer progression and facilitate identification of novel anti-cancer therapeutics that target TME components such as extracellular matrix (ECM) and stromal cells. However, most current in vitr...
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... Furthermore, in order to be similar to those in vivo and be sustained, organoids need vasculature that provides oxygen and nutrients; otherwise, they develop into a static model and necrotic center. This hinders the development of an age-related functional model [148]. It is noteworthy that NDDs are often diagnosed in later stages, whereas most organoid models depict the early markers of the diseases. ...
The microbiota–gut–brain axis (MGBA) has emerged as a key prospect in the bidirectional communication between two major organ systems: the brain and the gut. Homeostasis between the two organ systems allows the body to function without disease, whereas dysbiosis has long-standing evidence of etiopathological conditions. The most common communication paths are the microbial release of metabolites, soluble neurotransmitters, and immune cells. However, each pathway is intertwined with a complex one. With the emergence of in vitro models and the popularity of three-dimensional (3D) cultures and Transwells, engineering has become easier for the scientific understanding of neurodegenerative diseases. This paper briefly retraces the possible communication pathways between the gut microbiome and the brain. It further elaborates on three major diseases: autism spectrum disorder, Parkinson’s disease, and Alzheimer’s disease, which are prevalent in children and the elderly. These diseases also decrease patients’ quality of life. Hence, understanding them more deeply with respect to current advances in in vitro modeling is crucial for understanding the diseases. Remodeling of MGBA in the laboratory uses many molecular technologies and biomaterial advances. Spheroids and organoids provide a more realistic picture of the cell and tissue structure than monolayers. Combining them with the Transwell system offers the advantage of compartmentalizing the two systems (apical and basal) while allowing physical and chemical cues between them. Cutting-edge technologies, such as bioprinting and microfluidic chips, might be the future of in vitro modeling, as they provide dynamicity.
... Hence, the majority of organoid culture systems lack stromal and immunological components that are key determinants of tumor biology. PDAC organotypic cultures lack TME components (e.g., fibroblasts, immune cells, endothelial cells) and several efforts have been made to better shape recapitulate the complexity of the disease by establishing co-cultures of organoids with other cell types (152,153). Moreover, the selection of the proper culture condition is necessary to control the expression of the more appropriate transcriptional profile. ...
Despite the efforts, pancreatic ductal adenocarcinoma (PDAC) is still highly lethal. Therapeutic challenges reside in late diagnosis and establishment of peculiar tumor microenvironment (TME) supporting tumor outgrowth. This stromal landscape is highly heterogeneous between patients and even in the same patient. The organization of functional sub-TME with different cellular compositions provides evolutive advantages and sustains therapeutic resistance. Tumor progressively establishes a TME that can suit its own needs, including proliferation, stemness and invasion. Cancer-associated fibroblasts and immune cells, the main non-neoplastic cellular TME components, follow soluble factors-mediated neoplastic instructions and synergize to promote chemoresistance and immune surveillance destruction. Unveiling heterotypic stromal-neoplastic interactions is thus pivotal to breaking this synergism and promoting the reprogramming of the TME toward an anti-tumor milieu, improving thus the efficacy of conventional and immune-based therapies. We underscore recent advances in the characterization of immune and fibroblast stromal components supporting or dampening pancreatic cancer progression, as well as novel multi-omic technologies improving the current knowledge of PDAC biology. Finally, we put into context how the clinic will translate the acquired knowledge to design new-generation clinical trials with the final aim of improving the outcome of PDAC patients.
... In particular, the absence of an external vascular supply and adequate stromal support may hinder proper growth, shorten lifespan, and impair functionality by preventing adequate differentiation and maturation [15]. Therefore, the development of effective strategies for organoid vascularization remains a challenge in the field [7,16]. Here we show that the combination of VUs with IOs within the VOC offers a promising approach to promote the formation of a vascularized stroma around multiple organoids in one single microfluidic device. ...
The timely establishment of functional neo-vasculature is pivotal for successful tissue development and regeneration, remaining a central challenge in tissue engineering. In this study, we present a novel (micro)vascularization strategy that explores the use of specialized “vascular units” (VUs) as building blocks to initiate blood vessel formation and create perfusable, stroma-embedded 3D microvascular networks from the bottom-up. We demonstrate that VUs composed of endothelial progenitor cells and organ-specific fibroblasts exhibit high angiogenic potential when embedded in fibrin hydrogels. This leads to the formation of VUs-derived capillaries, which fuse with adjacent capillaries to form stable microvascular beds within a supportive, extracellular matrix-rich fibroblastic microenvironment. Using a custom-designed biomimetic fibrin-based vessel-on-chip (VoC), we show that VUs-derived capillaries can inosculate with endothelialized microfluidic channels in the VoC and become perfused. Moreover, VUs can establish capillary bridges between channels, extending the microvascular network throughout the entire device. When VUs and intestinal organoids (IOs) are combined within the VoC, the VUs-derived capillaries and the intestinal fibroblasts progressively reach and envelop the IOs. This promotes the formation of a supportive vascularized stroma around multiple IOs in a single device. These findings underscore the remarkable potential of VUs as building blocks for engineering microvascular networks, with versatile applications spanning from regenerative medicine to advanced in vitro models.
... Achieving such vascularization is an iScience Article advancement in tissue engineering, since vascularization is important for growth, prevents necrosis as the aggregates increase in size, and prevents premature differentiation. 81 Until recently, the study of embryo and organ development has been restricted to animal embryos and fetal tissues. Although organogenesis can be observed in the embryo, these animal models and material sources are limited, and their use presents ethical concerns. ...
The developing mouse pancreas is surrounded by mesoderm compartments providing signals that induce pancreas formation. Most pancreatic organoid protocols lack this mesoderm niche and only partially capture the pancreatic cell repertoire. This work aims to generate pancreatic aggregates by differentiating mouse embryonic stem cells (mESCs) into mesoderm progenitors (MPs) and pancreas progenitors (PPs), without using Matrigel. First, mESCs were differentiated into epiblast stem cells (EpiSCs) to enhance the PP differentiation rate. Next, PPs and MPs aggregated together giving rise to various pancreatic cell types, including endocrine, acinar, and ductal cells, and to endothelial cells. Single-cell RNA sequencing analysis revealed a larger endocrine population within the PP + MP aggregates, as compared to PPs alone or PPs in Matrigel aggregates. The PP + MP aggregate gene expression signatures and its endocrine population percentage closely resembled those of the endocrine population found in the mouse embryonic pancreas, which holds promise for studying pancreas development.
... A typical tumor normally contains populations of stromal cells, fibroblasts, endothelium, as well as immune cells. Although more complex organoid systems are being explored, such as organoid co-cultures with immune [42,43] and endothelial [44] cells, none have established a complex organoid system that successfully incorporates all the cell types observed within the TME in vivo. ...
The recurrence of colorectal liver metastasis (CRLM) following liver resection is common; approximately 40% of patients will experience tumor recurrence post-surgery. Renin–angiotensin inhibitors (RASis) have been shown to attenuate the growth and progression of CRLM in pre-clinical models following liver resection. This study examined the efficacy of the RASi captopril on patient-derived colorectal liver metastasis organoids. Patient-derived organoids (PDOs) were established using fresh samples of colorectal liver metastasis from appropriately consented patients undergoing liver resection. To mimic the regenerating liver post-CRLM liver resection, PDOs were cultured under hepatocyte regeneration conditions in vitro. CRLM PDOs were established from three patients’ parent tissue. CRLM PDOs and parent tissue expressed markers of colorectal cancer, CDX2 and CK20, consistently. Furthermore, CRLM PDOs treated with captopril showed a dose dependent reduction in their expansion in vitro. In conclusion, CRLM PDOs recapitulate in vivo disease and displayed a dose-dependent response to treatment with captopril. RASis may be an additional viable treatment for patients with CRLM.
... The vascularization of lung organoids is critically important in enhancing the development and maturation of organoids. 56,57 Alveolar capillaries are composed of two distinct cell types: lung-specific aerocyte capillary (aCap) cells responsible for gas exchange and general capillary (gCap) cells that serve as the stem cells for capillaries. 32 Alveolar cells are closely aligned with microvasculature, forming an air-blood barrier that facilitates efficient gas exchange. ...
Amidst the recent coronavirus disease 2019 (COVID-19) pandemic, respiratory system research has made remarkable progress, particularly focusing on infectious diseases. Lung organoid, a miniaturized structure recapitulating lung tissue, has gained global attention because of its advantages over other conventional models such as two-dimensional (2D) cell models and animal models. Nevertheless, lung organoids still face limitations concerning heterogeneity, complexity, and maturity compared to the native lung tissue. To address these limitations, researchers have employed co-culture methods with various cell types including endothelial cells, mesenchymal cells, and immune cells, and incorporated bioengineering platforms such as air-liquid interfaces, microfluidic chips, and functional hydrogels. These advancements have facilitated applications of lung organoids to studies of pulmonary diseases, providing insights into disease mechanisms and potential treatments. This review introduces recent progress in the production methods of lung organoids, strategies for improving maturity, functionality, and complexity of organoids, and their application in disease modeling, including respiratory infection and pulmonary fibrosis.
... Recently, many studies have provided additional implementations on modeling blood vessel networks through synthetic devices [83][84][85][86]. Salmon and colleagues showed that cortical organoids (COs) can be vascularized through an avascular hydrogel matrix placed between two perfusing microfluidic channels [59]. ...
Brain organoids, three-dimensional cell structures derived from pluripotent stem cells, closely mimic key aspects of the human brain in vitro, providing a powerful tool for studying neurodevelopment and disease. The neuroectodermal induction protocol employed for brain organoid generation primarily gives rise to the neural cellular component but lacks the vital vascular system, which is crucial for the brain functions by regulating differentiation, migration, and circuit formation, as well as delivering oxygen and nutrients. Many neurological diseases are caused by dysfunctions of cerebral microcirculation, making vascularization of human brain organoids an important tool for pathogenetic and translational research. Experimentally, the creation of vascularized brain organoids has primarily focused on the fusion of vascular and brain organoids, on organoid transplantation in vivo, and on the use of microfluidic devices to replicate the intricate microenvironment of the human brain in vitro. This review summarizes these efforts and highlights the importance of studying the neurovascular unit in a forward-looking perspective of leveraging their use for understanding and treating neurological disorders.
Graphical Abstract
... The most important limitation in the use of scaffold-free techniques is the lack of vascularization. Although existing brain organoid systems simulate the successful formation of the brain in the embryonic period, they are far from mimicking the mature brain structure [13]. Vascularization is notably required for oxygen penetration, nutrient supply, and effective neural differentiation [14,15]. ...
Neurodegeneration is a catastrophic process that develops progressive damage leading to functional and structural loss of the cells of the nervous system and is among the biggest unavoidable problems of our age. Animal models do not reflect the pathophysiology observed in humans due to distinct differences between the neural pathways, gene expression patterns, neuronal plasticity, and other disease-related mechanisms in animals and humans. Classical in vitro cell culture models are also not sufficient for pre-clinical drug testing in reflecting the complex pathophysiology of neurodegenerative diseases. Today, modern, engineered techniques are applied to develop multicellular, intricate in vitro models and to create the closest microenvironment simulating biological, biochemical, and mechanical characteristics of the in vivo degenerating tissue. In THIS review, the capabilities and shortcomings of scaffold-based and scaffold-free techniques, organoids, and microfluidic models that best reflect neurodegeneration in vitro in the biomimetic framework are discussed.
... The ToC platform is created by co-culturing tumor and stromal cells in a continuously perfused chamber within a 3D biomimetic matrix on a microfluidic device [53]. ECMmimicking culture scaffolds provide structural and functional support to promote cell survival, proliferation, and differentiation [54], allowing ToC models to recapitulate key mechanobiological features of the TME for studying the collective migration and invasion of cancer cells [55]. The microfluidic device can also generate chemokine gradients for studying the chemotaxis of immune cells toward the tumor nest and distal metastasis of tumor cells [53,56]. ...
... Vascular co-culture can be added in vitro through layerby-layer deposition of endothelial cells or by selectively removing material to form tubular voids seeded with endothelial cells connected to the perfusion network. More complex organoids containing vascular-like structures can also be created [54,64]. Alternatively, angiogenesis can be induced and guided by hypoxia gradients in vascular endothelial growth factor using a separate microfluidic chip for organoid-endothelial cell co-cultures [54]. ...
... More complex organoids containing vascular-like structures can also be created [54,64]. Alternatively, angiogenesis can be induced and guided by hypoxia gradients in vascular endothelial growth factor using a separate microfluidic chip for organoid-endothelial cell co-cultures [54]. Organoid and vascular co-cultures have been extensively investigated across various cancer types in academic research. ...
In recent years, the three-dimensional (3D) culture system has emerged as a promising preclinical model for tumor research owing to its ability to replicate the tissue structure and molecular characteristics of solid tumors in vivo. This system offers several advantages, including high throughput, efficiency, and retention of tumor heterogeneity. Traditional Matrigel-submerged organoid cultures primarily support the long-term proliferation of epithelial cells. One solution for the exploration of the tumor microenvironment is a reconstitution approach involving the introduction of exogenous cell types, either in dual, triple or even multiple combinations. Another solution is a holistic approach including patient-derived tumor fragments, air-liquid interface, suspension 3D culture, and microfluidic tumor-on-chip models. Organoid co-culture models have also gained popularity for studying the tumor microenvironment, evaluating tumor immunotherapy, identifying predictive biomarkers, Cancer Innovation. 2023;1-25. wileyonlinelibrary.com/journal/cai2 | 1 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
... A multimode fiber (MMF) with a 50-μm core diameter collected the light pattern from the DMD [199,200]. In DMD patterning, a liquid gel precursor can be repeatedly exposed to projected sequential light patterns to produce desired 3D tissue structures [201]. For vascularized organoids, sacrificial perfusion networks can be created using DMD-based tools. ...
... The network was disintegrated to generate a co-culture model system in a NaOH-containing solution, leaving behind a vascularized scaffold that may be seeded with endothelial cells (HUVECs). This method integrates materials and fabrication technologies to attain the required features in intricate co-culture platforms [201]. Vascularized breast organoids can be produced using a Decellularized Macroporous Device (DMD). ...
In vitro models are necessary to study the pathophysiology of the disease and the development of efective, tailored
treatment methods owing to the complexity and heterogeneity of breast cancer and the large population afected
by it. The cellular connections and tumor microenvironments observed in vivo are often not recapitulated in conventional two-dimensional (2D) cell cultures. Therefore, developing 3D in vitro models that mimic the complex architecture and physiological circumstances of breast tumors is crucial for advancing our understanding of the illness.
A 3D scafold-free in vitro disease model mimics breast cancer pathophysiology by allowing cells to self-assemble/
pattern into 3D structures, in contrast with other 3D models that rely on artifcial scafolds. It is possible that this
model, whether applied to breast tumors using patient-derived primary cells (fbroblasts, endothelial cells, and cancer
cells), can accurately replicate the observed heterogeneity. The complicated interactions between diferent cell types
are modelled by integrating critical components of the tumor microenvironment, such as the extracellular matrix,
vascular endothelial cells, and tumor growth factors. Tissue interactions, immune cell infltration, and the efects
of the milieu on drug resistance can be studied using this scafold-free 3D model. The scafold-free 3D in vitro disease model for mimicking tumor pathophysiology in breast cancer is a useful tool for studying the molecular basis
of the disease, identifying new therapeutic targets, and evaluating treatment modalities. It provides a more physiologically appropriate high-throughput platform for screening large compound library in a 96–384 well format. We critically discussed the rapid development of personalized treatment strategies and accelerated drug screening platforms
to close the gap between traditional 2D cell culture and in vivo investigations.
