Organoids

Studying human placental development and function presents significant challenges due to the inherent difficulties in obtaining and maintaining placental tissue throughout the course of an ongoing pregnancy. Here, we provide a detailed protocol for generating trophoblast organoids from chorionic villi obtained during ongoing pregnancy. Our method results in efficient generation of trophoblast organoids from chorionic villus sampling, does not require preselection of chorionic villi, and controls contamination of decidual gland organoids. The resulting trophoblast organoids spontaneously form syncytiotrophoblasts that start secreting hCG hormone amongst other placenta-specific factors. Our approach facilitates the generation of trophoblast organoids from a variety of genetic backgrounds, including trisomies and gene mutations, and can be aligned with prenatal diagnostic routines. The protocol requires up to 14 days and can be carried out by users with expertise in cell culture. Full article

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. Full article

Existing mRNA COVID-19 vaccines have shown efficacy in reducing severe cases and fatalities. However, their effectiveness against infection caused by emerging SARS-CoV-2 variants has waned considerably, necessitating the development of variant vaccines. Ideally, next-generation vaccines will be capable of eliciting broader and more sustained immune responses to effectively counteract new variants. Additionally, in vitro assays that more closely represent virus neutralization in humans would greatly assist in the analysis of protective vaccine-induced antibody responses. Here, we present findings from a SARS-CoV-2 VLP vaccine encompassing three key structural proteins: Spike (S), Envelope (E), and Membrane (M). The VLP vaccine effectively produced neutralizing antibodies as determined by surrogate virus neutralization test, and induced virus-specific T-cell responses: predominantly CD4⁺, although CD8⁺ T cell responses were detected. T cell responses were more prominent with vaccine delivered with AddaVax compared to vaccine alone. The adjuvanted vaccine was completely protective against live virus challenge in mice. Furthermore, we utilized air–liquid-interface (ALI)-differentiated human nasal epithelium (HNE) as an in vitro system, which authentically models human SARS-CoV-2 infection and neutralization. We show that immune sera from VLP-vaccinated mice completely neutralized SARS-CoV-2 virus infection, demonstrating the potential of ALI-HNE to assess vaccine induced Nab. Full article

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. Full article

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. Full article

While research on stem cell-derived tissues and organoids is rapidly expanding, the technically related creation of complex embryoids has recently excited a vivid discussion since it raises ethical questions about individuation and the possible gain of viability. The present study focuses on the onset of organismic development and the proposed biological and legal definitions for the terms embryo, embryoid, and organoid. It is concluded that such considerations have become important for investigators’ choices of the appropriate in vitro model systems, allowing the formation of organoids vs. complex embryoids. Full article

Meningiomas are the most common tumours of the central nervous system. According to the World Health Organization (WHO), this disease is classified into three different grades: 80% of meningioma patients present with benign grade I tumours, while less than 2% present with malignant grade III meningiomas. Despite affecting thousands of people worldwide, much remains unknown about this disease, and the development of systemic treatments is still far behind in comparison to other types of tumours. Therefore, forming 3D structures (spheroids and organoids) could facilitate research on the mechanisms of formation, proliferation, migration, and invasion of these, for the most part, benign tumours, while also helping in the process of drug development. To date, there are three published methods for the formation of meningioma organoids primarily derived from patient tissue samples. Organoids offer many advantages in the development of treatments because they recapitulate the cellular complexity within tumours. These new methodological advances could open a substantial number of possibilities for the further characterisation and treatment of meningiomas. This review includes an overview of the disease and a description and comparison of established protocols for meningioma organoid formation. Full article

A three-dimensional cell culture in hydrogel beads can support cell growth and differentiation into multi-cellular structures, and these gel beads could be used as building blocks for more complex three-dimensional assemblies. This requires hydrogel beads that are robust enough to sort via FACS yet can be degraded by cell-secreted enzymes. Collagen polymers form hydrogels that are excellent cell growth substrates; however, collagen-containing hydrogel beads typically include additional polymers that limit their degradation. Here, we introduce a simple microfluidic method to generate robust, sortable, cell-laden collagen hydrogel beads. We use on-device pH control to trigger collagen gelation without exposing cells to low pH, ensuring high cell viability. We fabricate microfluidic devices to generate droplets with a wide size range, as demonstrated by production of both small (~55 µm diameter) and large (~300 µm diameter) collagen gels. All hydrogels are sufficiently robust to allow for sorting using FACS. Moreover, high cell viability is maintained throughout the process. Full article

The generation of gastrointestinal tissues from human pluripotent stem cells has provided unprecedented insight into the molecular mechanisms that drive the patterning of the primitive gut tube. Previous work has identified bone-morphogenetic-protein (BMP) signaling as an important mediator of mid/hindgut versus foregut and hindgut versus midgut cell fate choice. Inhibition of BMP signaling during gut tube morphogenesis inhibits the expression of the pan-intestinal transcription factor CDX2. Treatment of CDX2+ mid/hindgut cultures with BMP patterns them into hindgut, which gives rise to colonic organoids (HCOs). While the role for BMP signaling is clear, the molecular mechanisms through which BMP signaling patterns the mid/hindgut and colon remain unclear. BMPs bind to BMP receptors, activating a signaling cascade that results in the activation of SMADs, which function as transcription factors. We hypothesized that one of these factors, SMAD1, would be necessary for establishing the CDX2 domain and the colon domain. Unexpectedly, endoderm derived from SMAD1-deficient induced pluripotent stem cells was capable of inducing CDX2 in response to WNT and FGF signaling. In addition, CDX2+ gut tube cultures could activate posterior HOX genes in response to BMP. However, examination of HCOs following cytodifferentiation revealed that SMAD1-deficient HCOs ectopically expressed small-intestinal markers despite expressing posterior HOX genes. These results indicate that there is redundancy of SMADs during early hindgut patterning but that SMAD1 is required for the inhibition of small-intestinal gene expression in HCOs. Full article

Ensuring drug safety for patients with specific neurological disorders is of paramount importance. For instance, certain antiepileptic drugs (AEDs) are contraindicated in Dravet Syndrome (DS), which is characterized by a deficiency in Na⁺ channel function. Constructing in vitro assessment methods capable of detecting contraindicated drug responses and medication effects on neurons derived from DS patients is highly anticipated for drug safety assessment and therapeutic innovation. This study used micro electrode array (MEA) measurements with low-frequency analysis on human iPSC-derived DS organoids to investigate AED responses. When exposed to the contraindicated drugs carbamazepine and phenytoin, the number of network oscillations increased in DS organoids while maintaining oscillation intensity. Furthermore, carbamazepine administration appeared to enhance activities beyond oscillations which is partially consistent with findings in the DS mouse model. Conversely, treatment with the therapeutic drug sodium valproate resulted in a similar decrease in activity both in healthy and DS organoids. The frequency characteristics of spontaneous firings and AEDs responsiveness in DS organoids demonstrated partial correlation with typical electroencephalography patterns observed in vivo. In conclusion, this study, employing MEA measurements with low-frequency analysis, revealed contraindicated drug responses and disease-specific functional characteristics in DS organoids, effective for DS patient safety assessment, precision medicine, and antiepileptic drug screening. Full article

Human cerebral organoids are readily generated from human embryonic stem cells and human induced pluripotent stem cells and are useful in studying human neurodevelopment. Recent work with human cerebral organoids have explored the creation of different brain regions and the impacts of soluble and mechanical cues. Matrigel is a gelatinous, heterogenous mixture of extracellular matrix proteins, morphogens, and growth factors secreted by Engelbreth-Holm-Swarm mouse sarcoma cells. It is a core component of almost all cerebral organoid protocols, generally supporting neuroepithelial budding and tissue polarization; yet, its roles and effects beyond its general requirement in organoid protocols are not well understood, and its mode of delivery is variable, including the embedding of organoids within it or its delivery in soluble form. Given its widespread usage, we asked how H9 stem cell-derived hCO development and composition are affected by Matrigel dosage and delivery method. We found Matrigel exposure influences organoid size, morphology, and cell type composition. We also showed that greater amounts of Matrigel promote an increase in the number of choroid plexus (ChP) cells, and this increase is regulated by the BMP4 pathway. These results illuminate the effects of Matrigel on human cerebral organoid development and the importance of delivery mode and amount on organoid phenotype and composition. Full article

We evaluated the redox status, precisely glutathione levels, which have a major impact in cellular detoxification and antioxidant defence in IBD-derived and healthy intestinal organoids. Therefore, we wanted to explore the differences in terms of their redox balance and mitochondrial fitness. To this end, we introduced a Grx1-roGFP2 construct into the organoids by lentiviral transduction before performing a stress assay by treating the organoids with hydrogen peroxide and examined the GSH/GSSG ratio using confocal imaging. Using ratio imaging, we could detect statistically significant differences between healthy and IBD-derived samples. To gain more insight, we also performed a GSH/GSSG assay, which directly measured glutathione levels. This analysis revealed that both organoid lines had higher levels of oxidized glutathione due to the stress treatment demonstrated by a lower GSH/GSSG ratio compared to the untreated control. Nevertheless, the results showed no significant difference between healthy and IBD-derived organoids. We further challenged organoids with hydrogen peroxide after incubation with MitoTracker^® to see if mitochondrial fitness might be different in IBD-derived organoids. However, these results were also very comparable. In summary, our preliminary findings indicate that both organoid lines demonstrate a well-functioning system in terms of analysis but show no clear difference between healthy and IBD-derived samples. Full article

Organoid-based research has made significant discoveries and contributions to our understanding of human organ function in both health and disease. To continue making progress, it is crucial to acknowledge the crucial role of the immune system in all organs. Various immune cells, such as macrophages, T cells, and neutrophils, are resident in almost all human tissues and play essential roles in organ homeostasis, function, and disease. Using diverse methods, researchers have begun integrating immune cells into organoid models, leading to more physiologically relevant models that better represent various aspects of human disease. These methods range from immune cell injection to co-culture and tissue expansion with existing immune cells. Immune cells can be sourced from mature patients or generated from stem cells as immature immune cells. The successful incorporation of immune cells into organoids will enhance our understanding of organ function and provide a more accurate approximation of human disease. Full article

Challenges to the musculoskeletal system negatively impact the quality of life of people suffering from them, leading to pain, a decline in mobility, genetic alterations, and potential disorders. The bone marrow (BM) forms an integral part of the musculoskeletal system responsible for erythropoiesis and optimal survival of the various immune and stem cells within the BM. However, due to its dynamic and complex three-dimensional (3D) structure, replicating the BM physiologically in traditional two-dimensional (2D) cell culture settings is often challenging, giving rise to the need for 3D in vitro models to better dissect the BM and its regeneration. Several researchers globally have been investigating various approaches to define an appropriate 3D model for their research. Organoids are novel preclinical models that provide a 3D platform for several tissues and have been analysed using next-generation sequencing (NGS) to identify new molecular pathways at the genetic level. The 3D in vitro models and organoids are increasingly considered important platforms for precision medicine. This review outlines the current knowledge of organoid and 3D in vitro models for the BM. We also discuss different types of 3D models which may be more adaptable for the BM. Finally, we critically review the NGS techniques used for such models and the future combination of these techniques. Full article

(1) Background: There are no high-throughput microtissue platforms for generating bone marrow micro-ossicles. Herein, we describe a method for the assembly of arrays of microtissues from bone marrow stromal cells (BMSC) in vitro and their maturation into bone marrow micro-ossicles in vivo. (2) Methods: Discs with arrays of 50 microwells were used to assemble microtissues from 3 × 10⁵ BMSCs each on a nylon mesh carrier. Microtissues were cultured in chondrogenic induction medium followed by hypertrophic medium in an attempt to drive endochondral ossification, and then they were implanted in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice, where they were remodeled into bone marrow micro-ossicles. Mice were transplanted with 10⁵ human umbilical cord blood CD34⁺ cells. (3) Results: Micro-ossicles contained more human CD45⁺ cells, but fewer human CD34⁺ progenitor cells than mouse marrow. Human hematopoietic progenitor cells cycle rapidly at non-physiological rates in mouse marrow, and reduced CD34⁺ cell content in micro-ossicles is consistent with the notion that the humanized niche better controls progenitor cell cycling. (4) Conclusions: Assembling microtissues in microwells, linked by a nylon membrane carrier, provides an elegant method to manufacture and handle arrays of microtissues with bone organ-like properties. More generally, this approach and platform could aid bridging the gap between in vitro microtissue manipulation and in vivo microtissue implantation. Full article

Bovine respiratory disease (BRD) is the leading cause of morbidity and mortality in feedlot cattle. Bovine herpesvirus-1 (BHV-1) is one of the main culprits of BRD; however, research on BHV-1 is hampered by the lack of suitable models for infection and drug testing. In this study, we established a novel bovine tracheal organoid culture grown in a basement membrane extract type 2 (BME2) matrix and compared it with the air–liquid interface (ALI) culture system. After differentiation, the matrix-embedded organoids developed beating cilia and demonstrated a transcriptomic profile similar to the ALI culture system. The matrix-embedded organoids were also highly susceptible to BHV-1 infection and immune stimulation by Pam₂Cys, an immunomodulator, which resulted in robust cytokine production and tracheal antimicrobial peptide mRNA upregulation. However, treatment of bovine tracheal organoid cultures with Pam₂Cys was not sufficient to inhibit viral infection or replication, suggesting a role of the non-epithelial cellular microenvironment in vivo. Full article