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Treatment of vascularized tumor organoids with antiangiogenic drugs. (A) Production of vascularized tumor organoids was repeated several times yielding similar organoid sizes and vascular network distribution. Six randomly picked organoids from two independent experiments are depicted. (B-D) The single treatment of tumor organoids with 100 nM 17-AAG (C) or 100 nM Sorafenib (D) at culture day 4 results in a disturbed formation of endothelial networks compared to DMSO treated controls (B). Pictures were taken at day 8. Tumor cells express GFP. (E-F) Quantification of branching points (E) and total vessel length (F). For quantification randomly picked maximum intensity projections from 6 individual whole-mount stained organoids per condition were analyzed. 17-AAG and Sorafenib treated aggregates showed significantly fewer branching points and reduced vessel length (p < 0.0125 determined by Wilcoxon-Mann-Whitney test).
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Organoids derived from human pluripotent stem cells are interesting models to study mechanisms of morphogenesis and promising platforms for disease modeling and drug screening. However, they mostly remain incomplete as they lack stroma, tissue resident immune cells and in particular vasculature, which create important niches during development and...
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... and fusion (Fig. 2L) within some cells of the tumor organoid suggesting lumen formation in parts of the capillary-like network 21 (Fig. 2K-L). Production www.nature.com/scientificreports www.nature.com/scientificreports/ of vascularized tumor organoids was repeated several times yielding similar organoid sizes and vascular network distribution (Fig. 3A). To further address the aspect of reproducibility, we repeated the experiment with an additional iPS cell line (Sendai NHDF iPSC) yielding similar results ( Figs S3, S4). The new iPS cell line was generated from commercially available dermal fibroblasts using a Sendai virus-based integration-free reprogramming method (Fig. S3). In ...
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... of vascularized tumor organoids was repeated several times yielding similar organoid sizes and vascular network distribution (Fig. 3A). To further address the aspect of reproducibility, we repeated the experiment with an additional iPS cell line (Sendai NHDF iPSC) yielding similar results ( Figs S3, S4). The new iPS cell line was generated from commercially available dermal fibroblasts using a Sendai virus-based integration-free reprogramming method (Fig. S3). ...
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... distribution (Fig. 3A). To further address the aspect of reproducibility, we repeated the experiment with an additional iPS cell line (Sendai NHDF iPSC) yielding similar results ( Figs S3, S4). The new iPS cell line was generated from commercially available dermal fibroblasts using a Sendai virus-based integration-free reprogramming method (Fig. S3). In addition, we randomly collected 20 organoids from one experiment and carefully quantified the organoid size and the surface area covered by CD31 + endothelial cells (Fig. S5A,B). We found that all tumor organoids display a similar CD31 + surface area (Fig. S5B). Regarding aggregate size, we observed a certain level of variation ...
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... we treated the tumor organoids with the tyrosine kinase inhibitor Sorafenib and the HSP90 inhibitor 17-AAG that had been shown to interfere with angiogenesis [22][23][24] . In both cases, we observe a disturbed formation of endothelial networks ( Fig. 3B-F) demonstrating the suitability of the model for drug testing applications and responsiveness to anti-angiogenic ...
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... Recent studies have demonstrated the successful development of vascularized organoids derived from iPSCs. In a specific study, the integration of stromal elements such as vasculature, fibroblasts, and immune cells was achieved by utilizing mesoderm progenitor cells induced from iPSCs [12]. Organoid culture offers the potential for long-term in vitro culture expansion while maintaining stable genetic characteristics, thus providing a solution to the challenges of liver disease models [13]. ...
... Furthermore, the blood vessel model produced by organoid systems can generate not only the endothelial cell (EC) vessel network but also the surrounding tissues and supportive cells, so that it can resemble in vivo tissue reactions properly [21]. They have provided invaluable observations in fundamental [18][19][20]. BEC brain endothelial cell, BPC brain pericyte, NPC neural progenitor cell, CTC circulating tumor cell, TC tumor cell, FB fibroblast biological investigations and potential pharmaceutical evaluations ( Fig. 1A) [8]. Practically, organoids have been known to be challenging to vascularize due to various reasons, including their size, the difficult spatial regulation of tissue, and the challenge of co-inducing endothelial cell (EC) supportive cells and organ-specific cells. ...
The vascular system, essential for human physiology, is vital for transporting nutrients, oxygen, and waste. Since vascular structures are involved in various disease pathogeneses and exhibit different morphologies depending on the organ, researchers have endeavored to develop organ-specific vascular models. While animal models possess sophisticated vascular morphologies, they exhibit significant discrepancies from human tissues due to species differences, which limits their applicability. To overcome the limitations arising from these discrepancies and the oversimplification of 2D dish cultures, microphysiological systems (MPS) have emerged as a promising alternative. These systems more accurately mimic the human microenvironment by incorporating cell interactions, physical stimuli, and extracellular matrix components, thus facilitating enhanced tissue differentiation and functionality. Importantly, MPS often utilize human-derived cells, greatly reducing disparities between model and patient responses. This review focuses on recent advancements in MPS, particularly in modeling the human organ-specific vascular system, and discusses their potential in biological adaptation.
... In recent decades, the CAM model has become increasingly important for the testing of angiogenic potential of biomaterials [17]. Recent studies have shown that the CAM can be used for the cultivation of vascularized brain and kidney organoids [18,19] and pre-vascularized cardiac organoids [20]. In 2022, Ribatti et al. [21] proposed the CAM model as a tool for cultivating organoids due to its extensive vascular network. ...
Osteosarcomas are the most common primary malignant bone tumors and mostly affect children, adolescents, and young adults. Despite current treatment options such as surgery and polychemotherapy, the survival of patients with metastatic disease remains poor. In recent studies, punicalagin has reduced the cell viability, angiogenesis, and invasion in cell culture trials. The aim of this study was to examine the effects of punicalagin on osteosarcomas in a 3D in vivo tumor model. Human osteosarcoma biopsies and SaOs-2 and MG-63 cells, were grown in a 3D in vivo chorioallantoic membrane (CAM) model. After a cultivation period of up to 72 h, the tumors received daily treatment with punicalagin for 4 days. Weight measurements of the CAM tumors were performed, and laser speckle contrast imaging (LSCI) and a deep learning-based image analysis software (CAM Assay Application v.3.1.0) were used to measure angiogenesis. HE, Ki-67, and Caspase-3 staining was performed after explantation. The osteosarcoma cell lines SaOs-2 and MG-63 and osteosarcoma patient tissue displayed satisfactory growth patterns on the CAM. Treatment with punicalagin decreased tumor weight, proliferation, and tumor-induced angiogenesis, and the tumor tissue showed pro-apoptotic characteristics. These results provide a robust foundation for the implementation of further studies and show that punicalagin offers a promising supplementary treatment option for osteosarcoma patients. The 3D in vivo tumor model represents a beneficial model for the testing of anti-cancer therapies.
... Since microglia are of mesodermal origin, a few labs have investigated the idea of incorporating mesodermal progenitors into organoids that would first differentiate into PMPs, migrate to the developing brain, and subsequently differentiate into microglia. In this regard, Wörsdörfer et al. were the first to develop a method that co-cultured Brachyury + hiPSCs-derived mesodermal progenitors with neural spheroids to generate vascularized neural organoids containing CD31 + vessel-like structures (Wörsdörfer et al., 2019). While the study objective was to generate vascularized organoids, Iba1 + microglia-like cells were surprisingly noted. ...
Microglia, resident immune cells of the brain that originate from the yolk sac, play a critical role in maintaining brain homeostasis by monitoring and phagocytosing pathogens and cellular debris in the central nervous system (CNS). While they share characteristics with myeloid cells, they are distinct from macrophages. In response to injury, microglia release pro-inflammatory factors and contribute to brain homeostasis through activities such as synapse pruning and neurogenesis. To better understand their role in neurological disorders, the generation of in vitro models of human microglia has become essential. These models, derived from patient-specific induced pluripotent stem cells (iPSCs), provide a controlled environment to study the molecular and cellular mechanisms underlying microglia-mediated neuroinflammation and neurodegeneration. The incorporation or generation of microglia into three-dimensional (3D) organoid cultures provides a more physiologically relevant environment that offers further opportunities to study microglial dynamics and disease modeling. This review describes several protocols that have been recently developed for the generation of human-induced microglia. Importantly, it highlights the promise of these in vitro models in advancing our understanding of brain disorders and facilitating personalized drug screening.
... A number of these approaches have been used, including the use of umbilical endothelial cells, mesodermal progenitors, and stem cells expressing a transcription factor for vascular lineage specification. With these experiments, organoids with tube-like vascular structures were generated [53,61,62]. Although, depending on the applied protocol, those multi-lineage approaches were variably able to originate perfusable vessels, they provided a model of human neurovascular interactions during development. ...
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
... As 3D structures, they enable more precise monitoring of processes in a more physiologically relevant environment and therefore open up new possibilities compared with previously used monolayer cultures [40]. Vascularized kidney, brain, and cardiac pre-vascularized organoids have already been successfully grown on the CAM [41,42]. The further establishment of the cultivation of organoids on the CAM could replace several of the in vivo models used to date, including various species of experimental animals, and enable research in tumor biology without the use of additional laboratory animals. ...
... Varzideh et al. engrafted three-dimensional cardiac organoids containing human ESCs-derived progenitor cells, endothelial cells, as well as mesenchymal stem cells onto the CAM, whereupon they induced a strong angiogenic response [61]. Wörsdörfer et al. demonstrated the functional connection of organoids obtained by co-culturing human mesodermal progenitors with progenitor cell types to the host blood circulation [42]. A connection to the chick´s circulating system was also observed by Schmidt et al. while cultivating blood vessel organoids in the CAM model [62]. ...
Although significant improvements have been made in the treatment of pancreatic cancer, its prognosis remains poor with an overall 5-year survival rate of less than 10%. New experimental approaches are necessary to develop novel therapeutics. In this study, the investigation of pancreatic cancer tissue growth in the chorioallantoic membrane (CAM) model and the subsequent use of indocyanine green (ICG) injections for the verification of intratumoral perfusion was conducted. ICG was injected into the CAM vasculature to visualize the perfusion of the tumor tissue. The presence of metastasis was investigated through PCR for the human-specific ALU element in the liver of the chicken embryo. Additionally, the usage of cryopreserved pancreatic tumors was established. Intratumoral perfusion of tumor tissue on the CAM was observed in recently obtained and cryopreserved tumors. ALU-PCR detected metastasis in the chick embryos’ livers. After cryopreservation, the tissue was still vital, and the xenografts generated from these tumors resembled the histological features of the primary tumor. This methodology represents the proof of principle for intravenous drug testing of pancreatic cancer in the CAM model. The cryopreserved tumors can be used for testing novel therapeutics and can be integrated into the molecular tumor board, facilitating personalized tumor treatment.
... Theoretically, by integrating a vascularized organoid into a perfusable system, the overall size and lifespan of the organoid can be significantly increased [25]. Early work on this took advantage of in vivo implantation of pre-vascularized or vascular organoids, whereby the implanted organoid was eventually anastomosed with the host vasculature, resulting in organoid survival [25][26][27][28]. The establishment of perfusion systems in vitro [29][30][31], mostly by incorporating organoids into microfluidic bioreactor systems, provides perfusion to increase the delivery of nutrients [32]. ...
... Wörsdörfer et al. initially incorporated iPSC-derived human mesodermal progenitors specifically into organoids. Since endothelial cells were differentiated from mesodermal progenitor cells treated with VEGF, the co-culture of mesodermal progenitor cells with spheroids resulted in the in vitro generation of vascularized organoids [27]. Likewise, hepatic endoderm cells can be prepared from human iPSCs, to construct three-dimensional vascularized liver buds. ...
Organoids have emerged as a powerful tool for studying organ development, disease modeling, and drug discovery due to their ability to mimic the in vivo structure and function of organs in a three-dimensional in vitro model. During in vivo organ maturation, the process of vascularization is crucial for the provision of nutrients and oxygen to cells and the removal of waste products as the organ increases in size. Similarly, organoids can grow to sizes greater than the millimeter scale, yet transport of oxygen and nutrients to the center becomes increasingly difficult, often resulting in the formation of a necrotic core. Herein, we provide a concise summary of the recent development of methods to initiate and maintain vascularization of organoids. Broadly, vascularization of organoids has been achieved primarily by two means: generating organoids that contain endothelial cells or employing the secretion of vascular growth factors to promote vascularization. Growth factors play a fundamental role in regulating blood vessel formation through chemical signals that cause changes in the cell–cell adhesions and ultimately the migration of endothelial cells. Furthermore, models with perfusable systems demonstrate that through the application of growth factors and cells, the vascular network in vascularization-based organoids can administer biological substances to the interior of the organoid, opening up new possibilities for long-term organoid culture in vitro. This goal is being realized through the development of bioengineering tools, such as vascularized organoids on a chip, which are currently tested for various organ systems, including the lung, brain, kidney, and tumors, with applications in cancer angiogenesis and metastasis research. Taken together, our review underlines the vast potential of vascularized organoids to improve the understanding of organ development, while also proposing exciting avenues of organoid-on-a-chip and disease modeling.
... THP-1s have been previously used in 3D-culture and can be polarized into both an M1 or M2 phenotype 7,28,29 . Although having successfully utilized iPS-derived mesenchymal progenitor cells as a source for stromal and endothelial cells in microorganoids previously 30 , we decided on employing mature primary cells for the drug screening platform. Cultivation of matured cells is less cost-intensive than iPS handling, especially if induction of pluripotency must be factored in. ...
Targeting the supportive tumor microenvironment (TME) is an approach of high interest in cancer drug development. However, assessing TME-targeted drug candidates presents a unique set of challenges: Adequate assays need to recreate the TME at least in part and provide intricate information about drug-induced changes in the TME’s interactions.
We have developed a comprehensive screening platform that allows to monitor, quantify, and rank drug-induced effects in self-organizing, vascularized tumor microorganoids (TMOs). Fully humanized, the confrontation of four different cell populations makes it possible to study complex changes in composition and cell-cell interaction. The platform is highly modular, allowing for adjustments regarding tumor entity, TME composition, or for genetic manipulation of individual cell populations. Treatment effects are recorded by light sheet fluorescence microscopy and translated by an advanced image analysis routine in processable multi-parametric datasets. The detailed data output allows for handling a wide range of potential inquiries. Nevertheless, the system proved to be robust, with strong interassay reliability.
We demonstrate the platform's utility for the side-by-side evaluation of TME-targeted antifibrotic and antiangiogenic drugs. The platform's output delivered a broad scope of information about treatment effects, enabling clear distinction of even closely related drug candidates according to projected therapeutic needs. Moreover, the modular character allowed for the differential evaluation of genetically targeting different cellular components, adding new possibilities for tailoring selective drugs.
... As a result, CNS organoids have been observed to lack the comprehensive array of cells originating from various germ layers found in the brain in vivo, including microglia [144,145]. However, recent research has introduced innovative approaches for generating organoids containing cells from all germinal layers, including the microglia [146][147][148][149][150][151][152]. This combination offers enhanced insights into the mechanisms underlying neurodegenerative disorders, including epilepsy. ...
Neuroinflammation represents a dynamic process of defense and protection against the harmful action of infectious agents or other detrimental stimuli in the central nervous system (CNS). However, the uncontrolled regulation of this physiological process is strongly associated with serious dysfunctional neuronal issues linked to the progression of CNS disorders. Moreover, it has been widely demonstrated that neuroinflammation is linked to epilepsy, one of the most prevalent and serious brain disorders worldwide. Indeed, NLRP3, one of the most well-studied inflammasomes, is involved in the generation of epileptic seizures, events that characterize this pathological condition. In this context, several pieces of evidence have shown that the NLRP3 inflammasome plays a central role in the pathophysiology of mesial temporal lobe epilepsy (mTLE). Based on an extensive review of the literature on the role of NLRP3-dependent inflammation in epilepsy, in this review we discuss our current understanding of the connection between NLRP3 inflammasome activation and progressive neurodegeneration in epilepsy. The goal of the review is to cover as many of the various known epilepsy models as possible, providing a broad overview of the current literature. Lastly, we also propose some of the present therapeutic strategies targeting NLRP3, aiming to provide potential insights for future studies.
... To circumvent these drawbacks, researchers increasingly employ MoA to produce complicated vascularized organotypic tissues. For example, Wörsdörfer et al suggested the guided insertion of mesodermal progenitor cell organoids into parenchymal organoids to form vascularized tumor or brain tissue models (figure 7) [134]. The generated blood arteries showed a characteristic ultrastructure and a hierarchical architecture. ...
Adequate vascularization is a critical determinant for the successful construction and clinical implementation of complex organotypic tissue models. Currently, low cell and vessel density and insufficient vascular maturation make vascularized organotypic tissue construction difficult, greatly limiting its use in tissue engineering and regenerative medicine. To address these limitations, recent studies have adopted pre-vascularized microtissue assembly for the rapid generation of functional tissue analogs with dense vascular networks and high cell density. In this article, we summarize the development of module assembly-based vascularized organotypic tissue construction and its application in tissue repair and regeneration, organ-scale tissue biomanufacturing, and advanced tissue modeling.
