Organoids doi: 10.3390/organoids5020011

Authors: Gautami R. Kelkar Balaji M. Rao Albert J. Keung

Human cerebral organoids, derived from pluripotent stem cells, are powerful models for studying human brain development. The understanding of how morphogens can be used to guide patterning and differentiation has matured rapidly; however, the influence of basal media components on organoid development remains unclear. Standard organoid media frequently contain non-physiological concentrations of nutrients, including glucose, a central regulator of cellular metabolism and signaling. Here, we examine how glucose availability shapes cerebral organoid growth, morphology, and cell type composition by comparing conventional hyperglycemic media to media with glucose levels more closely resembling normoglycemic conditions. We find that organoids derived from multiple human pluripotent stem cell lines can grow in low glucose, but they exhibit altered growth rates, structural features, and lineage distributions. In H9 embryonic stem cell-derived organoids, inhibition of the mammalian target of rapamycin pathway under low glucose restores neurodevelopmental cell types otherwise diminished in these conditions. These findings highlight glucose as a key determinant of organoid lineage specification and cellular signaling. Importantly, however, glucose modulation does not reduce variability across organoids or cell lines, underscoring the need to better understand and control sources of heterogeneity to improve organoid models.

Organoids doi: 10.3390/organoids5020010

Authors: Mingyang Kong Sanjima Pal Shuyuan Wang Julie Bérubé Ruoyu Ma Yifei Yan Wotan Zeng France Bourdeau Betty Giannias Hong Zhao Nathan Osman Yehonatan Nevo Kulsum Tai Hellen Kuasne James Tankel Gertruda Evaristo Pierre O. Fiset Xin Su Swneke Bailey Morag Park Nicholas Bertos Veena Sangwan Lorenzo Ferri

A major limitation in studying gastroesophageal adenocarcinoma (GEA) has been the lack of reliable models that represent the disease’s complexity. We present lessons learned from a comprehensive large-scale biobanking effort combining traditional sample collection with several in vitro models, including 3-dimensional patient-derived organoids (PDOs), 2-dimensional cancer-associated fibroblasts (CAFs), tumor-infiltrating lymphocytes (TILs), and/or in vivo xenografts. This initiative started in 2018, integrating multiple advanced ex vivo models such as PDOs, patient-derived xenografts (PDXs), and organoids (PDXOs). This unique resource now includes tumor avatars from over 380 consented patients, making it the world’s largest living GEA biobank. We achieved a >90% success rate in creating per-patient models, including 227 tumor-derived and 203 neighboring normal PDOs. These organoids accurately mirror key features of the original tumors, such as their histology (e.g., microsatellite instability), mutations, and drug response across treatment points. Notably, PDOs can predict individual patient responses to chemotherapy within five weeks, underscoring their clinical relevance. Furthermore, high-throughput drug screening on PDO subsets with known genetic landscapes generates personalized chemosensitivity profiles for 22 drugs. Through a process of continued refinement of culture techniques and tumor sampling approach, our large-scale comprehensive collection of GEA avatars represents a unique and valuable preclinical experimental resource for precision oncology.

Organoids doi: 10.3390/organoids5010009

Authors: Zhifeng Xue Runze Yang Yaling Liu Han Luo

As a three-dimensional in vitro model, organoid technology represents a revolutionary breakthrough in precision medicine. By harnessing the self-organizing capabilities of stem cells within biomimetic extracellular matrices, it enables the generation of miniature tissues that recapitulate key structural and functional characteristics of their source organs. Conventional two-dimensional cell cultures lack tissue architecture and microenvironmental cues, whereas animal models are hindered by interspecies differences and inadequate representation of human pathological heterogeneity. By effectively addressing these limitations, organoids have emerged as powerful platforms that are highly representative of human physiology and disease processes in oncology, genetic disorders, and infectious diseases. They demonstrate significant potential for use in drug screening, toxicity assessment, and the development of personalized treatment strategies. Although challenges such as limited vascularization, lack of standardized culture protocols, and ethical considerations remain, the integration of multidisciplinary approaches such as AI-assisted analysis, organ-on-a-chip systems, and 3D bioprinting, together with increasing policy support and industrial advancement, is accelerating the clinical translation of organoid technology. In this review, the construction strategies for and applications of organoid models are systematically summarized, and their value and limitations in disease modeling, precision medicine, and preclinical research are highlighted. Finally, future development pathways driven by multidisciplinary collaboration and standardization are outlined.

Organoids doi: 10.3390/organoids5010008

Authors: Patricia Mateos-Martínez Deanira Patrone Milagros González-Flores Cristina Soriano-Amador Rosa González-Sastre Sabela Martín-Benito Andreea Rosca Raquel Coronel Victoria López-Alonso Isabel Liste

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder and the leading cause of dementia, for which there is currently no cure. The causes of AD are still not well understood, although 5% of cases are known to have a genetic origin, associated with pathogenic genetic variants of the APP and PSEN1/2 genes. There is growing evidence that both APP and PSEN1/2 are also essential for proper human brain development and neural/neuronal function. This implies that abnormalities in early brain development could increase neuronal vulnerability to AD later in life. Human cerebral organoids (hCOs), generated from induced pluripotent stem cells (iPSCs) from AD patients, provide an exceptional model for better understanding the cellular and molecular mechanisms involved in human brain development, as well as early neurological alterations in the evolution of AD. This review compiles the main studies in which hCOs are used as a model for studying AD and for the discovery of new biomarkers. We also discuss the advantages and applications of these hCOs for studying the early stages of AD from a neurodevelopmental perspective. Finally, we mention the main current challenges in the use of hCOs for future research into AD.

Organoids doi: 10.3390/organoids5010007

Authors: Vinothini Prabhakaran Jennifer Z. Paxton

The tendon-to-bone enthesis is a multiphasic structure with four structurally continuous and compositionally distinct regions: tendon, uncalcified fibrocartilage, calcified fibrocartilage and bone. Our study aimed to develop 3D scaffold-free in vitro spheroids and macro-tissues of the enthesis for applications as experimental tools to understand the development and repair of enthesis injury. This study hypothesises that integrating tendon and bone cell spheroids with bone marrow mesenchymal stem cell spheroids will facilitate the production of a fibrocartilaginous interface. 3D Spheroids: The biphasic (tendon–bone) and triphasic co-culture (tendon–stem cell–bone) of spheroids in growth media and chondrogenic media were investigated to establish fusion kinetics, and the cellular and ECM components produced via histology and immunohistochemistry. Complete fusion between spheroids occurred within 6-to-8 days in biphasic co-culture, and 15-to-20 days in triphasic co-culture. Compared to biphasic, the triphasic co-culture in chondrogenic media showed a continuous interface connecting the tendon and bone regions. The presence of collagen I, sulphated proteoglycans and collagen type II in the interface region of triphasic co-culture indicates fibrochondrogenic differentiation. 3D macro-tissues: The modular tissue engineering strategy was used in this study to produce enthesis macro-tissues using spheroids as building blocks. Spheroids were bio-assembled in the triphasic manner (12 tendon spheroids, 12 stem cell spheroids and 8 bone spheroids) in the custom-designed and 3D-printed temporary supports (Formlabs Clear Resin®) using a customised spheroid bio-assembly system. The fusion of spheroids occurred by day 8 after bio-assembly, and they were removed from temporary supports and cultured in scaffold-free conditions. Although the bio-assembly methodology was successful in producing fused scaffold-free macro-tissues, the histological analysis revealed the presence of an extensive necrotic core due to the large-sized constructs. To conclude, the findings support the hypothesis that a triphasic co-culture has the potential to produce a structurally continuous fibrocartilaginous interface but requires further optimisation to produce macro-tissues with anatomical morphologies and reduced necrotic cores.

Organoids doi: 10.3390/organoids5010006

Authors: Hiroyuki Uematsu Chieko Saito Jumpei Kondo Kunishige Onuma Roberto Coppo Hiroko Endo Takahiro Nakayama Katsuhide Yoshidome Taisei Nomura Arihiro Kohara Masahiro Inoue

Breast cancer progression and treatment responsiveness are significantly influenced by the tumor microenvironment. Therefore, transplantation into the mammary fat pad is widely employed to establish a mouse xenograft model of breast cancer. This study reports chimeric organoids derived from breast cancer xenografts composed of human and mouse cells. During passaging of an organoid line derived from breast cancer xenografts, characteristic cell clusters composed of smaller cells were observed. Immunostaining with a mouse-specific antibody revealed that the smaller cells were mouse cells composed of luminal- and basal-like cells. Chimeric organoids were observed in four of the six xenograft-derived organoid lines. Organoids composed solely of human cells rapidly diminished after passaging, with chimeric and mouse-cell-only organoids becoming predominant. When human breast cancer cells were co-cultured with mouse mammary epithelial cells, chimeras were frequently observed. The PCNA positivity rate in breast cancer cells within chimeras was higher than that in breast cancer cells within organoids composed solely of human cells. These findings indicate that xenograft-derived breast cancer organoids frequently contain mouse cells and that mouse mammary epithelial cells promote the proliferation of human breast cancer cells.

Organoids doi: 10.3390/organoids5010005

Authors: Christoph Grün Cordula Nies Magdalena Klesen Enja Schwarz Jonah ter Haseborg Cornelius Dettmer Christian Beyer Larissa Funk Eric Gottwald

Precise control and measurement of the cellular microenvironment, particularly oxygen concentration, are crucial for developing physiologically relevant in vitro models. However, current methods often lack the spatial resolution and throughput needed to investigate complex, oxygen-dependent biological mechanisms in 3D cell cultures. Here, we present an advanced platform based on microcavity arrays featuring integrated, ratiometric oxygen sensors, so-called SensoSpheres. A unique bevel design at the cavity entrance enables the non-invasive, real-time measurement of pericellular oxygen concentration and oxygen gradients. We established protocols for generating spheroids from various cell lines (e.g., HepG2, HeLa) and characterized their metabolic responses under precisely controlled hypoxic, normoxic, and hyperoxic conditions. Using a dose–response assay, we demonstrate the platform’s sensitivity in capturing distinct metabolic shifts in response to acetaminophen and cisplatin. Furthermore, we introduce the Oxygen Consumption Recovery Rate (OCRR) as a novel parameter to quantify cellular resilience after exposure to toxic compounds such as cisplatin and acetaminophen. This high-throughput-compatible platform represents a significant methodological advancement, enabling detailed studies of oxygen-dependent cellular processes, drug toxicity, and metabolic adaptation. Its potential for integration into microfluidic systems paves the way for more sophisticated organ-on-chip models, ultimately improving the predictive power of preclinical research.

Organoids doi: 10.3390/organoids5010004

Authors: Gijs J. F. van Son Femke C. A. S. Ringnalda Markus J. van Roosmalen Thomas A. Kluiver Quinty Hansen Evelien Duiker Marius C. van den Heuvel Vincent E. de Meijer Ruben H. de Kleine Ronald R. de Krijger József Zsiros Weng Chuan Peng Ruben van Boxtel Marc van de Wetering Karin Sanders Hans Clevers

Hepatoblastoma (HB) is a paediatric liver malignancy arising from hepatic precursor cells, with >90% of cases harbouring a mutation in exon 3 of CTNNB1. We present a fully genetically characterised HB tumour organoid (tumoroid) biobank, which allows for in vitro studies of disease progression and clonal dynamics in vitro. We established a biobank of 14 tumoroid lines from 9 different patients. Tumours and tumoroids were characterised by whole genome sequencing (WGS) and histology, revealing strong concordance in cell morphology and β-catenin staining. In tumour—tumoroid pairs, identical pathogenic CTNNB1 variants were found, alongside shared copy number alterations (CNAs) and mutations. Variant allele frequency (VAF) was consistently higher in tumoroids, indicating increased tumour purity in vitro. In addition to CTNNB1, we frequently observed ARID1A alterations (single-nucleotide variants [SNVs] or CNAs in 56% of patients), and MYC gains as described previously. In paired pre- and post-treatment samples, we observed a clear increase in mutational load, attributed to a chemotherapy signature. Notably, from one patient, we analysed 4 tumour samples (3 post-treatment) with 4 matching tumoroid lines, all carrying a novel BCL6 mutation and loss of ARID1A. Mutational profiles varied across samples from different locations, suggesting intratumoral heterogeneity and clonal selection during tumoroid derivation. Taken together, this biobank allows detailed analysis of HB tumour biology, including treatment-induced progression and clonal dynamics across temporally and spatially distinct samples.

Organoids doi: 10.3390/organoids5010003

Authors: Shumaila Khalid Sobia Ekram Faiza Ramzan Asmat Salim Irfan Khan

Human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) possess the potential for chondrogenic differentiation, offering a promising alternative source for cartilage regeneration. To address the limited availability and expansion capacity of autologous chondrocytes, we investigated the effect of co-overexpression of Sox9, TGFβ1, and type II collagen (Col II) on the chondrogenic differentiation of hUC-MSCs using both 2D and 3D pellet culture systems. Following transfection, the cells exhibited a chondrocyte-like morphology and a marked downregulation of the stemness marker Stro-1. After 21 days in a 3D pellet culture system, the cells formed cartilage-like tissue characterized by the strong expression of chondrocyte-specific genes (Sox9, TGFβ1, Col II, Aggrecan) along with the significant secretion of sulfated glycosaminoglycans (sGaGs). These effects were attributed to enhanced cell–cell contact and extracellular matrix interactions promoted by the 3D environment. Our findings suggest that genetically modified hUC-MSCs cultured in a 3D pellet system represent a robust in vitro model for cartilage regeneration, with potential applications in transplantation and drug toxicity screening.

Organoids doi: 10.3390/organoids5010002

Authors: Elena Gkantzou Alexandro Rodríguez-Rojas Aleksandra Chmielewska Barbara Pratscher Surina Surina Patricia Freund Iwan A. Burgener

Organoids are three-dimensional multicellular structures that mimic key aspects of native tissues consisting ideal tools to study organ development and pathophysiology when incorporated in customized bioscaffolds. In vivo, the extracellular matrix (ECM) maintains tissue integrity and regulates cell adhesion, migration, differentiation, and survival through biochemical and mechanical signals. Tissue-derived decellularized extracellular matrix (dECM) can preserve organ-specific biochemical signals and cell-adhesive motifs, creating a bioactive environment that supports physiologically relevant organoid growth. 3D bioprinting technology marks a transformative phase in organoid research by enhancing the structural and functional complexity of organoid models and expanding their application in pharmacology and regenerative medicine. These systems enhance tissue modeling and drug testing while adhering to the principles of animal replacement, reduction, and refining (3Rs) in research. Remaining challenges include donor variability, limited mechanical stability, and the lack of standardized decellularization protocols that can be addressed by adopting quality and safety metrics. The combination of dECM-based biomaterials and 3D bioprinting holds great potential for the development of human-relevant, customizable, and ethically sound in vitro models for regenerative medicine and personalized therapies. In this review, we discuss the latest (2021–2025) developments in applying extracellular matrix bioprinting techniques to organoid technology, presenting examples for the most commonly referenced organoid types.

Organoids doi: 10.3390/organoids5010001

Authors: Kathrin Kostka-Wirtz Nataniel Białas Ivanna Kostina Matthias Epple

Organoids consisting of primary human cells, i.e., astrocytes, pericytes, and endothelial cells, form a functional blood–brain barrier (BBB) in vitro. The ability of FITC-dextran (70 kDa), calcium phosphate nanoparticles (100 nm), Escherichia coli bacteria (2 µm), and MS2 coliphages (27 nm, a model for viruses) to penetrate the BBB under normoxic and hypoxic conditions (2.5% oxygen) for up to 12 days was assessed by fluorescence microscopy and confocal laser scanning microscopy. All agents were fluorescently labeled to trace them inside the organoids. Under normoxia, FITC-dextran, calcium phosphate nanoparticles, E. coli bacteria and MS2 coliphages did not penetrate the BBB. However, oxygen deficiency (hypoxia) triggered the penetration of the BBB by FITC-dextran and E. coli cells. This was underscored by a strong hypoxic center inside the organoids that developed in the presence of E. coli bacteria.

Organoids doi: 10.3390/organoids4040033

Authors: Süleyman Ergün Philipp Wörsdörfer

Over the past decade, organoids representing a wide range of tissues have been developed, with increasing efforts to enhance their complexity, maturity, and resemblance to the corresponding native organs [...]

Organoids doi: 10.3390/organoids4040032

Authors: Daniel Skubleny Hasnaien Ahmed Sebastiao N. Martins-Filho David Ross McLean Daniel E. Schiller Gina R. Rayat

Three-dimensional cell model systems such as tumour organoids allow for in vitro modelling of self-organized tissue with functional and histologic similarity to in vivo tissue. However, there is a need for standard protocols and techniques to confirm the presence of cancer within organoids derived from tumour tissue. The aim of this study was to assess the utility of a Nanostring gene expression-based machine learning classifier to determine the presence of cancer or normal organoids in cultures developed from both benign and cancerous stomach biopsies. A prospective cohort of normal and cancer stomach biopsies were collected from 2019 to 2022. Tissue specimens were processed for formalin-fixed paraffin-embedding (FFPE) and a subset of specimens were established in organoid cultures. Specimens were labelled as normal or cancer according to analysis of the FFPE tissue by two pathologists. The gene expression in FFPE and organoid tissue was measured using a 107 gene Nanostring codeset and normalized using the Removal of Unwanted Variation III algorithm. Our machine learning model was developed using five-fold nested cross-validation to classify normal or cancer gastric tissue from publicly available Asian Cancer Research Group (ACRG) gene expression data. The models were externally validated using the Cancer Genome Atlas (TCGA), as well as our own FFPE and organoid gene expression data. A total of 60 samples were collected, including 38 cancer FFPE specimens, 5 normal FFPE specimens, 12 cancer organoids, and 5 normal organoids. The optimal model design used a Least Absolute Shrinkage and Selection Operator model for feature selection and an ElasticNet model for classification, yielding area under the curve (AUC) values of 0.99 [95% CI: 0.99–1], 0.90 [95% CI: 0.87–0.93], and 0.79 [95% CI: 0.74–0.84] for ACRG (internal test), FFPE, and organoid (external test) data, respectively. The performance of our final model on external data achieved AUC values of 0.99 [95% CI: 0.98–1], 0.94 [95% CI: 0.86–1], and 0.85 [95% CI: 0.63–1] for TCGA, FFPE, and organoid specimens, respectively. Using a public database to create a machine learning model in combination with a Nanostring gene expression assay allows us to allocate organoids and their paired whole tissue samples. This platform yielded reasonable accuracy for FFPE and organoid specimens, with the former being more accurate. This study re-affirms that although organoids are a high-fidelity model, there are still limitations in validating the recapitulation of cancer in vitro.

Organoids doi: 10.3390/organoids4040031

Authors: Roberto Coppo Edoardo Bertone

Ensuring access to safe drinking water is a fundamental public health priority, yet the growing diversity of contaminants demands more human-relevant toxicity assessment frameworks. Conventional models based on immortalized cell lines or sentinel species, while informative, lack the tissue complexity and inter-individual variability required to capture realistic human responses. Organoids, three-dimensional epithelial structures derived from adult or pluripotent stem cells, retain the genomic, histological, and functional characteristics of their original tissue, enabling assessment of contaminant-induced toxicity, short-term peak exposures, and inter-donor variability within a single system. This study examined whether current international drinking water guidelines remain protective or if recent organoid-based findings reveal toxicity at differing concentrations. Comparative synthesis indicates that per- and polyfluoroalkyl substances (PFAS) often display organoid toxicity at concentrations above current thresholds, suggesting conservative guidelines, whereas most metals are properly regulated. However, some metals exhibit toxicity at concentrations that include levels below guideline values, highlighting the need for further investigation. Emerging contaminants, including pesticides, nanoparticles, microplastics, and endocrine disruptors, induce adverse effects at environmentally relevant concentrations, despite limited or absent regulatory limits. Integrating organoid-based toxicology with high-frequency monitoring and dynamic exposure modeling could refine water quality guidelines and support adaptive regulatory frameworks that better reflect real-world exposure patterns and human diversity.

Organoids doi: 10.3390/organoids4040030

Authors: Elisa Heinzelmann Francesco Piraino

Cancer remains a leading cause of mortality worldwide. Patient-derived organoids (PDOs) are three-dimensional (3D) cultures that recapitulate tumor histology, genetics, and cellular heterogeneity, providing physiologically relevant preclinical models. Integrating PDOs with artificial intelligence (AI) and machine learning (ML) enables scalable analysis of high-dimensional datasets, including imaging, transcriptomics, proteomics, and pharmacological readouts. These approaches support prediction of drug sensitivity, biomarker discovery, and patient stratification. Recent advances—such as deep learning (DL), transfer learning, federated learning, and self-supervised learning—enhance phenotypic profiling, cross-institutional model training, and translational prediction. In this review, we summarize the current state of AI-driven PDO research, highlighting methodological approaches, preclinical and clinical applications, challenges, and emerging trends. We also propose strategies for standardization, validation, and multi-modal integration to accelerate patient-specific therapeutic strategies.

Organoids doi: 10.3390/organoids4040029

Authors: Daniel Montoya

As brain organoids and organoid-based computational models grow in complexity, they increasingly exhibit electrophysiological patterns consistent with plasticity and information processing. This article explores a central question at the intersection of neuroscience, synthetic biology, and philosophy of mind: Can intelligent behavior be meaningfully separated from an intelligent being? In other words, can adaptive, goal-directed behavior exist independently of subjective awareness—a question that challenges conventional definitions of cognition and consciousness. Drawing from neuroscience, artificial intelligence, and philosophy, I propose a tiered framework based on neural complexity and environmental responsiveness. This includes a simple level analysis and a context-sensitive benchmark for evaluating intelligence in organoid systems without presupposing sentience. Ethical and ontological implications are also addressed, particularly the risk of anthropomorphizing synthetic cognition and the importance of developing context-aware definitions of intelligence. By distinguishing functional sophistication from subjective experience, the framework aims to guide responsible scientific inquiry while clarifying the boundaries of synthetic cognition.

Organoids doi: 10.3390/organoids4040028

Authors: Kathrin Groeneveld Ralf Mrowka

This review aims to highlight how the study of kidney organoids combined with proteomic analysis can deepen our understanding of renal physiology and disease. Proteomics quantifies proteins in a sample, allowing us to determine which proteins are present, how abundant they are, and how they are modified. These data may reveal the pathways that are active in the kidney organoids and how they change in disease, helping to pinpoint candidate biomarkers. Kidney organoids are three-dimensional structures derived from induced pluripotent stem cells (iPS) that recapitulate many architectural and functional features of the adult organ. Because they can be generated in large numbers under defined conditions, organoids provide a promising platform for testing how genetic mutations, environmental stresses, or drugs affect kidney development and pathology. When proteomic profiles are obtained from mature organoids, researchers can directly link protein-level changes to phenotypic outcomes observed in the model. This integration makes it possible to map disease-related networks at the molecular level and to assess the impact of therapeutic interventions in a system that more closely resembles human kidney tissue than traditional cell lines. A current limitation is that many kidney organoids do not reach the full maturation seen in vivo; they often lack complete segmental differentiation and the functional robustness of adult nephrons. Improving the maturation state of organoids will be essential for accurately modeling chronic kidney diseases and for translating findings into clinically relevant therapies.

Organoids doi: 10.3390/organoids4040027

Authors: Tokiyoshi Matsushita Takahiro Matsuyama Takayuki Kawasaki Fumiaki Uchiumi

Human retinal organoids derived from pluripotent stem cells represent a robust in vitro model for investigating retinal development and disease mechanisms of retinal disorders. However, achieving structural maturation that faithfully recapitulates the intricate architecture of photoreceptors within a feasible and cost-efficient culture timeframe remains a significant challenge. Here, we present an optimized differentiation protocol that enables the generation of retinal organoids exhibiting advanced photoreceptor maturation within 140 days. Photoreceptors in the retinal organoids displayed compartmentalized architecture, including distinct inner and outer segments and connecting cilia. Notably, we observed the emergence of budding calyceal process-like structures—a feature not previously emphasized in photoreceptors derived from pluripotent stem cells. These results suggest that our protocol may promote advanced photoreceptor maturation within a relatively shortened culture period. Thus, this method could serve as a useful model for investigating retinal development and related pathologies, building upon previous protocols.

Organoids doi: 10.3390/organoids4040026

Authors: Arvind Kumar Shukla Sandhya Shukla Raj Kumar Mongre Adarsha Mahendra Upadhyay Govindhan Thiruppathi Chandra Dhar Shukla Shuktika Mishra Sayan Deb Dutta

Breast cancer remains one of the leading causes of cancer morbidity and mortality among women worldwide. Conventional two-dimensional (2D) cell culture models and animal studies often fail to accurately recapitulate the complex tumor microenvironment and heterogeneous nature of breast cancer. Recent advancements in tissue engineering have enabled the development of more physiologically relevant models using three-dimensional (3D) bioprinting and organoid technology. This study focuses on integrating 3D bioprinting with patient-derived organoid models to replicate breast cancer tissue architecture, cellular heterogeneity, and tumor-stroma interactions. Utilizing biomimetic bioinks and customized bioprinting protocols, we successfully fabricated breast cancer tissue constructs embedded with stromal and immune components. These engineered models demonstrated high fidelity in mimicking in vivo tumor pathophysiology, including angiogenesis, epithelial–mesenchymal transition, and extracellular matrix remodeling. Furthermore, the platform allowed for high-throughput drug screening and evaluation of therapeutic responses, revealing differential sensitivities to chemotherapeutics and targeted therapies. Our findings highlight the potential of bioprinted organoid models as powerful tools for personalized medicine, enabling more predictive and reliable cancer research and drug development.

Organoids doi: 10.3390/organoids4040024

Authors: Brady M. Wessel Jenna N. Castro Henry F. Harrison Brian P. Scottoline Margaret C. Wilcox Maureen K. Baldwin Victoria H. J. Roberts

First-trimester placental development comprises many critical yet understudied cellular events that determine pregnancy outcomes. Improper placentation leads to a host of health issues that not only impact the fetal period but also influence later-life offspring health. Thus, an experimental paradigm for studying early placental development is necessary and has spurred the development of new in vitro models. Organoid model systems are three-dimensional structures comprising multiple differentiated cell types that originate from a progenitor population. Trophoblasts are the progenitor cells that serve as the proliferative base for the differentiation and maintenance of the placenta. Due to research constraints, experimental studies on the causal mechanisms underlying pathological pregnancies cannot readily be performed in human subjects. The nonhuman primate (NHP) offers a solution to this problem as it circumvents the limitations of human pregnancy sampling. Importantly, NHPs share many developmental features of human pregnancy, including placenta type and a similar fetal growth trajectory, making longitudinal pregnancy studies feasible and relevant. Since perturbations made in vivo can be validated in vitro, an NHP model of early pregnancy would facilitate mechanistic studies of pregnancy disorders. Herein, we describe the methodology for the establishment of a first-trimester NHP placenta trophoblast organoid model system.

Organoids doi: 10.3390/organoids4040025

Authors: Dilhana S. Badurdeen Zhen Li Jeong-Heon Lee Tao Ma Aditya Vijay Bhagwate Rachel Latanich Arjit Dogiparthi Tamas Ordog Olga Kovbasnjuk Vivek Kumbhari Jennifer Foulke-Abel

Obesity is an epidemic with myriad health effects, but little is understood regarding individual obese phenotypes and how they may respond to therapy. Epigenetic changes associated with obesity have been detected in blood, liver, pancreas, and adipose tissues. Previous work using human organoids found that dietary glucose hyperabsorption is a steadfast trait in cultures derived from some obese subjects, but detailed transcriptional or epigenomic features of the intestinal epithelia associated with this persistent phenotype are unknown. This study evaluated differentially expressed genes and relative chromatin accessibility in intestinal organoids established from donors classified as non-obese, obese, or obese hyperabsorptive by body mass index and glucose transport assays. Transcriptomic analysis indicated that obese hyperabsorptive subject organoids have significantly upregulated dietary nutrient absorption transcripts and downregulated type I interferon targets. Chromatin accessibility and transcription factor footprinting predicted that enhanced HNF4G binding may promote the obese hyperabsorption phenotype. Quantitative RT-PCR assessment in organoids representing a larger subject cohort suggested that intestinal epithelial expression of CUBN, GIP, SLC5A11, and SLC2A5 were highly correlated with hyperabsorption. Thus, the obese hyperabsorption phenotype was characterized by transcriptional changes that support increased nutrient uptake by intestinal epithelia, potentially driven by differentially accessible chromatin. Recognizing unique intestinal phenotypes in obesity provides a new perspective in considering therapeutic targets and options with which to manage the disease.

Organoids doi: 10.3390/organoids4040023

Authors: Ayush Madan Ramandeep Saini Nainci Dhiman Shu-Hui Juan Mantosh Kumar Satapathy

Organoid technology has emerged as a revolutionary tool in cancer research, offering physiologically accurate, three-dimensional models that preserve the histoarchitecture, genetic stability, and phenotypic complexity of primary tumors. These self-organizing structures, derived from adult stem cells, induced pluripotent stem cells, or patient tumor biopsies, recapitulate critical aspects of tumor heterogeneity, clonal evolution, and microenvironmental interactions. Organoids serve as powerful systems for modeling tumor progression, assessing drug sensitivity and resistance, and guiding precision oncology strategies. Recent innovations have extended organoid capabilities beyond static culture systems. Integration with microfluidic organoid-on-chip platforms, high-throughput CRISPR-based functional genomics, and AI-driven phenotypic analytics has enhanced mechanistic insight and translational relevance. Co-culture systems incorporating immune, stromal, and endothelial components now permit dynamic modeling of tumor–host interactions, immunotherapeutic responses, and metastatic behavior. Comparative analyses with conventional platforms, 2D monolayers, spheroids, and patient-derived xenografts emphasize the superior fidelity and clinical potential of organoids. Despite these advances, several challenges remain, such as protocol variability, incomplete recapitulation of systemic physiology, and limitations in scalability, standardization, and regulatory alignment. Addressing these gaps with unified workflows, synthetic matrices, vascularized and innervated co-cultures, and GMP-compliant manufacturing will be crucial for clinical integration. Proactive engagement with regulatory frameworks and ethical guidelines will be pivotal to ensuring safe, responsible, and equitable clinical translation. With the convergence of bioengineering, multi-omics, and computational modeling, organoids are poised to become indispensable tools in next-generation oncology, driving mechanistic discovery, predictive diagnostics, and personalized therapy optimization.

Organoids doi: 10.3390/organoids4040022

Authors: Aleksandar B. Kirov Veerle Lammers Arezo Torang Jan Koster Jan Paul Medema

In many cancers, tumorigenesis is determined in part by the cell type in the tissue that transforms, which has been called the cell of origin. In intestinal cancer, previous observations suggested that transformation can occur from both stem cells and more differentiated cells; in the latter case, this is provided that NF-kB is activated and apoptosis is blocked. However, whether these distinct transformation trajectories yield similar types of cancer remains unresolved. In this study the effect of APC loss within different cellular backgrounds was analyzed. Transformation of either stem-like cells or secretory-like cells, as defined by CD24 or c-KIT expression, by deleting the APC function in organoids in vitro, led to WNT-independent growth of organoids in both cellular populations. Importantly, transformed cultures derived from secretory-like cells had significantly distinct gene expression profiles as compared to the more stem cell-derived (CD44high cells) APC mutant cultures and in fact preserved a level of gene expression that relates back to their original cell lineage. Our data highlights the influence of different cellular backgrounds on the initiation of intestinal cancer and suggests that the cell of origin could be a defining factor in colorectal cancer heterogeneity.

Organoids doi: 10.3390/organoids4030021

Authors: Sarah L. Harbach Bang M. Tran Abderrahman Hachani Samantha Leigh Grimley Damian F. J. Purcell Georgia Deliyannis Joseph Torresi Julie L. McAuley Elizabeth Vincan

COVID-19 has triggered the rapid adoption of human organoid-based tissue culture models to overcome the limitations of the commonly used Vero cell line that did not fully recapitulate SARS-CoV-2 infection of human tissues. As the primary site of SARS-CoV-2 infection, the human nasal epithelium (HNE) cultivated in vitro and differentiated at air–liquid interface (ALI) is an ideal model to study infection processes and for testing anti-viral antibodies and drugs. However, the need for primary basal cells to establish the ALI-HNE limits the scalability of this model system. To try and bypass this bottleneck, we devised an ALI-differentiated form of the human adenocarcinoma cell line Calu-3, reported to model most aspects of authentic SARS-CoV-2 infection, including viral entry. The ALI-Calu-3 were tested for infection by a panel of SARS-CoV-2 variants, including ancestral (VIC01) and early pandemic lineages (VIC2089, Beta, Delta), and Omicron subvariants (BA2.75, BA4, BA5, XBB1.5). All tested lineages infected the ALI-HNE. In stark contrast, infection of the ALI-Calu-3 by Omicron subvariants BA4 and XBB1.5 was reduced. These data support the use of ALI-Calu-3 as a complementary, intermediary model for most but not all SARS-CoV-2 lineages, and places the ALI-HNE as the benchmark culture model for SARS-CoV-2 infection.

Organoids doi: 10.3390/organoids4030020

Authors: Nikki Liddelow Jie Yu Tan Dustin J. Flanagan

The stomach epithelium is a highly dynamic tissue that undergoes continuous self-renewal and responds robustly to injury through tightly regulated repair processes. Organoids have emerged as powerful tools for modelling gastrointestinal biology. This review focuses on the capacity of gastric organoids to model epithelial homeostasis, injury and repair in the stomach. We examine how organoid systems recapitulate key features of in vivo gastric architecture and stem cell dynamics, enabling detailed interrogation of lineage specification, proliferative hierarchies and regional identity. Gastric organoids have proven particularly useful for studying how environmental factors, such as Helicobacter pylori infection or inflammatory cytokines, disrupt epithelial equilibrium and drive metaplastic transformation. Furthermore, we discuss the emerging use of injury-mimicking conditions, co-cultures and bioengineered platforms to model regeneration and inflammatory responses in vitro. While organoids offer unparalleled accessibility and experimental manipulation, they remain limited by the absence of critical niche components such as immune, stromal and neural elements. Nevertheless, advances in multi-cellular and spatially resolved organoid models are closing this gap, making them increasingly relevant for disease modelling and regenerative medicine. Overall, gastric organoids represent a transformative approach to dissecting the cellular and molecular underpinnings of stomach homeostasis and repair.

Organoids doi: 10.3390/organoids4030019

Authors: Marina Alexandrova Mariela Ivanova Martina Metodieva Antonia Terzieva Tanya Dimova

Implantation studies are extremely important to solve reproductive problems since about 60% of abortions occur around this period. The 3D in vitro models emerge as closest to the in vivo structures and processes. Here, we constructed trophoblast Sw71-spheroids as implanting human blastocyst–like surrogates (BLS). The model is well-characterized, standardized, validated tool to study extravillous trophoblast (EVT) invasion/migration during implantation. A limitation is that it is a short-living 3D-culture that must be generated de novo. This study aimed to create and embed Sw71-spheroids in paraffin for permanent histological preparations. The main challenges were the micro-size and the preservation of the intact structure. The standardly generated compact and stable Sw71-spheroids were intact, with blastocyst-like morphology. Histological analysis showed preserved cell morphology, shape, and intact periphery of the embedded Sw71-spheroids. These were usable for immunohistochemistry(IHC) and expressed common EVT markers: EpCAM, HLA-C and and HLA-G. Our protocol for spheroid paraffin embedding is suitable for simultaneous histological analyses of several Sw71-spheroids. It might be further optimized to embed migrating/invading Sw71-BLS as snapshots of trophoblast implantation steps in permanent histological preparations for in depth IHC studies.

Organoids doi: 10.3390/organoids4030018

Authors: Deepak Gulwani Neha Singh Manisha Gupta Ridhima Goel Thoudam Debraj Singh

Organoid and spheroid technologies have rapidly become pivotal in thyroid cancer research, offering models that are more physiologically relevant than traditional two-dimensional culture. In the study of papillary and anaplastic thyroid carcinomas, two subtypes that differ both histologically and clinically, three-dimensional (3D) models offer unparalleled insights into tumor biology, therapeutic vulnerabilities, and resistance mechanisms. These models maintain essential tumor characteristics such as cellular diversity, spatial structure, and interactions with the microenvironment, making them extremely valuable for disease modeling and drug testing. This review emphasizes recent progress in the development and use of thyroid cancer organoids and spheroids, focusing on their role in replicating disease features, evaluating targeted therapies, and investigating epithelial–mesenchymal transition (EMT), cancer stem cell behavior, and treatment resistance. Patient-derived organoids have shown potential in capturing individualized drug responses, supporting precision oncology strategies for both differentiated and aggressive subtypes. Additionally, new platforms, such as thyroid organoid-on-a-chip systems, provide dynamic, high-fidelity models for functional studies and assessments of endocrine disruption. Despite ongoing challenges, such as standardization, limited inclusion of immune and stromal components, and culture reproducibility, advancements in microfluidics, biomaterials, and machine learning have enhanced the clinical and translational potential of these systems. Organoids and spheroids are expected to become essential in the future of thyroid cancer research, particularly in bridging the gap between laboratory discoveries and patient-focused therapies.

Organoids doi: 10.3390/organoids4030017

Authors: Giacomo Masi Camilla Guiducci Francesca Rescigno

Background/Objectives: VitroScreenORA® (by VitroScreen srl) Dermo Papilla spheroids, based on two micro-physiological systems (non-vascularized DP and vascularized VASC-DP), were used to study the molecular mechanisms behind hair cycle regression. Methods: Dermal papilla cells (HFDPC) were cultured to develop both models. Hair cycle regression was induced by exposing DP spheroids to TGF-β1 for 72 h and/or FGF-18 for an additional 24 h. Catagen phase entrance was evaluated by modulating specific genes (FGF7, CCND1, and WNT5B). The VASC-DP model was obtained by sequentially co-culturing HFDPC and primary dermal microvascular endothelial cells (HMDEC), mimicking the surrounding capillary loop. The vascular system’s impact was assessed at 5 and 10 days using IF on CD31 (micro-vessels) and Fibronectin (FN). Nanostring nCounter® technology was applied to investigate the transcriptional signature based on the WNT pathway. Extended culture time up to 11 days simulated natural hair cycle regression, monitored by versican and FN expression (IF). Minoxidil, Doxorubicin, and Retinol-based products were used to modify physiological aging over time. Results: Data shows that the vascular system improves tissue physiology by modulating the associated genes. Extended culture time confirms progressive DP structure degeneration that is partially recoverable with Retinol-based treatments. Conclusions: Both models provide a reliable platform to investigate the hair cycle, paving the way for new testing systems for personalized therapies.

Organoids doi: 10.3390/organoids4030016

Authors: Karnika Yogeswari Makesh Abilash Navaneethan Mrithika Ajay Ganesh Munuswamy-Ramanujam Arulvasu Chinnasamy Dhanavathy Gnanasampanthapandian Kanagaraj Palaniyandi

Organoids are three-dimensional tissue culture models derived from stem cells, and they have become one of the most valuable tools in biomedical research. These self-organizing miniature organs mimic the structure−function properties of their in vivo counterparts and offer an exceptional prospective for disease modeling, drug discovery, and regenerative medicine. By replicating the complexity of human tissue, organoids enable the study of disease pathophysiology, tissue development, and cellular interactions in a highly controlled and manipulable environment. Recent developments in organoid technology have enabled the production of functional organoids of various tissues. These systems have proven to be highly promising tools for personalized medicine. In addition, organoids have also raised hopes for the development of functional transplantable organs, transforming the study of regenerative medicine. This review provides an overview of the current state of organoid technology and its application and prospects and focuses on the transformative impact of organoid technology on biomedical research and its contribution to human health.

Organoids doi: 10.3390/organoids4030015

Authors: Maree C. Faux Chenkai Ma Serena R. Kane Andre Samson Yumiko Hirokawa Ilka Priebe Leah Cosgrove Rajvinder Singh Michael Christie Gregor Brown Kim Y. C. Fung Antony W. Burgess

Current systemic therapies for advanced colorectal cancer (CRC) have limited efficacy, so more effective strategies for the treatment and prevention of CRC are needed. The majority of colorectal cancers are initiated by mutations in Wnt signalling pathway genes, including mutations in the APC gene, which result in a truncated APC protein and lead to excess signalling from β-catenin and the formation of pre-cancerous adenomas. The aim of this study was to determine if targeting the Wnt pathway in combination with pro-apoptotic mimetics altered the proliferative capacity or viability of human colorectal adenoma cells. Patient-derived colorectal adenoma organoid cultures were established from colon adenoma tissue collected by colonoscopy and recapitulated the histopathology of primary colorectal adenoma tissue. The growth of colorectal adenoma organoids is inhibited by the Wnt-signalling antagonist pyrvinium pamoate (PP) and a pro-apoptotic inhibitor of BCL-XL but not BCL-2 (venetoclax) or MCL-1 inhibitors. At low concentrations, the PP and the BCL-XL inhibitor combination demonstrated potent synergy and induced apoptosis in APC-defective patient-derived adenoma organoids, even in the presence of oncogenic KRAS or BRAF mutations, providing a new strategy for colon cancer prevention.

Organoids doi: 10.3390/organoids4020014

Authors: Layla El Bouazzaoui Daniëlle A. E. Raats André Verheem Inne H. M. Borel Rinkes Hugo J. G. Snippert Madelon M. Maurice Onno Kranenburg

The current treatment for refractory BRAF-V600E mutant metastatic colorectal cancer (mCRC) involves combined inhibition of BRAF and the epidermal growth factor receptor (EGFR). However, tumour responses are often short-lived due to a rebound in mitogen-activated protein kinase (MAPK) activity. In this study, we combined short-term cell viability assays with long-term regrowth assays following drug removal over a period of three weeks. This allowed assessment of regrowth after therapy discontinuation. We tested the effect of combined BRAF inhibition (encorafenib) and EGFR inhibition (afatinib) on BRAF-V600E mutant CRC patient-derived organoids (PDOs). Combined EGFR/BRAF inhibition initially caused a major reduction in PDO growth capacity in BRAF-V600E mutant PDOs. This was followed by rapid regrowth after drug removal, mirroring clinical outcomes. EGFR inhibition in BRAF-V600E mutant PDOs led to activation of the insulin receptor (IR) and insulin-like growth factor-1 receptor (IGF1R). The IGF1R/IR inhibitor linsitinib prevented the rebound in MAPK activity following removal of afatinib and encorafenib, prevented regrowth of CRC PDOs, and improved the anti-tumour response in an in vivo model. PDO regrowth assays allow the identification of pathways driving tumour recurrence. IR/IGF1R-inhibition prevents regrowth following golden standard MAPK pathway-targeted therapy and provides a strategy to improve the treatment of BRAF-V600E mutant CRC

Organoids doi: 10.3390/organoids4020013

Authors: Sushmita Mishra Ginia Mondal Murali Kumarasamy

Improved in vitro models are needed to reduce costs and delays in central nervous system (CNS) drug discovery. The FDA Modernization Acts 2.0 and 3.0 require human-centered alternative testing methods to mitigate animal-based experiments and discovery delays, and to ensure human safety. Developing cost-efficient, flexible microfluidic chips is essential to advance organ-on-chip (OoC) technology for drug discovery and disease modeling. While CNC micromilling shows promise for fabricating microfluidic devices, it remains underutilized due to limited accessibility. We present a simple CNC-milled flexible microfluidic chip fabricated from thermoplastic poly (methyl methacrylate) (PMMA). The structure of the microplate included drilled openings for connecting the wells. The chip’s biocompatibility was evaluated with isolated primary neuronal cultures from postnatal Wistar rat pups (p1). Primary cells cultured in the device showed high viability, differentiation, and 3D neurosphere formation, similar to conventional well-plate cultures. Neuronal cultures showed neurite growth and functional markers. Although cleanroom-based methods provide higher accuracy, the chip effectively promotes cell viability, differentiation, and alignment, offering an ideal platform for tissue modeling and OoC applications. It allows cell biologists to quickly create prototypes at lower cost and in less time than required for soft lithography and is a viable alternative to the current manufacturing methods.

Organoids doi: 10.3390/organoids4020012

Authors: Elisabetta Ferrero Jonas Hue Marina Ferrarini Lorenzo Veschini

Tumour-associated angiogenesis plays a key role at all stages of cancer development and progression by providing a nutrient supply, promoting the creation of protective niches for therapy-resistant cancer stem cells, and supporting the metastatic cascade. Therapeutic strategies aimed at vascular targeting, including vessel disruption and/or normalisation, have yielded promising but inconsistent results, pointing to the need to set up reliable models dissecting the steps of the angiogenic process, as well as the ways to interfere with them, to improve patients’ outcomes while limiting side effects. Murine models have successfully contributed to both translational and pre-clinical cancer research, but they are time-consuming, expensive, and cannot recapitulate the genetic heterogeneity of cancer inside its native microenvironment. Non-animal technologies (NATs) are rapidly emerging as invaluable human-centric tools to reproduce the complex and dynamic tumour ecosystem, particularly the tumour-associated vasculature. In the present review, we summarise the currently available NATs able to mimic the vascular structure and functions with progressively increasing complexity, starting from two-dimensional static cultures to the more sophisticated tri-dimensional dynamic ones, patient-derived cultures, the perfused engineered microvasculature, and in silico models. We emphasise the added value of a “one health” approach to cancer research, including studies on spontaneously occurring tumours in companion animals devoid of the ethical concerns associated with traditional animal studies. The limitations of the present tools regarding broader use in pre-clinical oncology, and their translational potential in terms of new target identification, drug development, and personalised therapy, are also discussed.

Organoids doi: 10.3390/organoids4020011

Authors: Klara Beslmüller Rick Rodrigues de Mercado Gijsje H. Koenderink Erik H. J. Danen Thomas Schmidt

The extracellular matrix (ECM) provides structural support to cells, thereby forming a functional tissue. In cancer, the growth of the tumor creates internal mechanical stress, which, together with the remodeling activity of tumor cells and fibroblasts, alters the ECM structure, leading to an increased stiffness of the pathological ECM. The enhanced ECM stiffness, in turn, stimulates tumor growth and activates tumor-promoting fibroblasts and tumor cell migration, leading to metastasis and increased therapy resistance. While the relationship between matrix stiffness and migration has been studied before, their connection to internal tumor stress remains unresolved. Here we used 3D ECM-embedded spheroids and hydrogel particle stress sensors to quantify and correlate internal tumor spheroid pressure, ECM stiffness, ECM remodeling, and tumor cell migration. We note that 4T1 breast cancer spheroids and SV80 fibroblast spheroids showed increased invasion—described by area, complexity, number of branches, and branch area—in a stiffer, cross-linked ECM. On the other hand, changing ECM stiffness only minimally changed the radial alignment of fibers but highly changed the amount of fibers. For both cell types, the pressure measured in spheroids gradually decreased as the distance into the ECM increased. For 4T1 spheroids, increased ECM stiffness resulted in a further reach of mechanical stress into the ECM, which, together with the invasive phenotype, was reduced by inhibition of ROCK-mediated contractility. By contrast, such correlation between ECM stiffness and stress-reach was not observed for SV80 spheroids. Our findings connect ECM stiffness with tumor invasion, ECM remodeling, and the reach of tumor-induced mechanical stress into the ECM. Such mechanical connections between tumor and ECM are expected to drive early steps in cancer metastasis.

Organoids doi: 10.3390/organoids4020010

Authors: Piotr Jung Adam J. Wolpaw

Basic and translational cancer biology research requires model systems that recapitulate the features of human tumors. While two-dimensional (2D) cell cultures have been foundational and allowed critical advances, they lack the organizational complexity, cellular interactions, and extracellular matrix present in vivo. Mouse models have thus remained the gold standard for studying cancer. In addition to high cost and low throughput, mouse models can also suffer from reduced tumor heterogeneity and species-specific differences. Three-dimensional (3D) culture models have emerged as a key intermediary between 2D cell lines and mouse models, with lower cost and greater flexibility than mouse models and a more accurate representation of the tumor microenvironment than 2D cell lines. In neuroblastoma, an aggressive childhood cancer, 3D models have been applied to study drug responses, cell motility, and tumor–matrix interactions. Recent advances include the integration of immune cells for immunotherapy studies, mesenchymal stromal cells for tumor–stroma interactions, and bioprinted systems to manipulate matrix properties. This review examines the use of 3D culture systems in neuroblastoma, highlighting their advantages and limitations while emphasizing their potential to bridge gaps between in vitro, preclinical, and clinical applications. By improving our understanding of neuroblastoma biology, 3D models hold promise for advancing therapeutic strategies and outcomes in this childhood cancer.

Organoids doi: 10.3390/organoids4020009

Authors: Michael W. Nestor Richard L. Wilson

The development of brain organoids from human-induced pluripotent stem cells (iPSCs) has expanded research into neurodevelopment, disease modeling, and drug testing. More recently, the concept of organoid intelligence (OI) has emerged, proposing that these constructs could evolve to support learning, memory, or even sentience. While this perspective has driven enthusiasm in the field of organoid research and suggested new applications in fields such as neuromorphic computing, it also introduces significant scientific and conceptual concerns. Current brain organoids lack the anatomical complexity, network organization, and sensorimotor integration necessary for intelligence or sentience. Despite this, claims surrounding OI often rely on oversimplified interpretations of neural activity, fueled by neurorealist and reification biases that misattribute neurophysiological properties to biologically limited systems. Beyond scientific limitations, the framing of OI risks imposing ethical and regulatory challenges based on speculative concerns rather than empirical evidence. The assumption that organoids might possess sentience, or could develop it over time, could lead to unnecessary restrictions on legitimate research while misrepresenting their actual capabilities. Additionally, comparing biological systems to silicon-based computing overlooks fundamental differences in scalability, efficiency, and predictability, raising questions about whether organoids can meaningfully contribute to computational advancements. The field must recognize the limitations of these models rather than prematurely defining OI as a distinct research domain. A more cautious, evidence-driven approach is necessary to ensure that brain organoids remain valuable tools for neuroscience without overstating their potential. To maintain scientific credibility and public trust, it is essential to separate speculative narratives from grounded research, thus allowing for continued progress in organoid studies without reinforcing misconceptions about intelligence or sentience.

Organoids doi: 10.3390/organoids4020008

Authors: Eyad Awad Matthew Bedding-Tyrrell Alberto Coccarelli Feihu Zhao

Recent research works have shown the effect of nutrient concentration on cell activity, such as proliferation and differentiation. In 3D cell culture, the impact of scaffold geometry, including pore size, strut diameter, and pore shape, on the concentration gradient within scaffolds under two different loading conditions—constant fluid perfusion and non-fluid perfusion—in a perfusion bioreactor is investigated by developing an in silico model of scaffolds. In this study, both triply periodic minimal surface (TPMS) (with gyroid struts) and non-TPMS (with cubic and spherical pores) scaffolds were investigated. Two types of criteria are applied to the scaffolds: static and perfusion culture conditions. In a static environment, the scaffold in a perfusion bioreactor is loaded with a fluid velocity of 0 mm/s, whereas in a dynamic environment, perfusion flow with a velocity of 1 mm/s is applied. The results of in silico simulation indicate that the concentration gradient within the scaffold is significantly influenced by pore size, strut diameter, pore shape, and fluid flow, which in turn affects the diffusion rate during drug delivery.

Organoids doi: 10.3390/organoids4020007

Authors: Ania Bogoslowski Joice Ren Clément Quintard Josef M. Penninger

Lymphoid organs are critical for organizing the development of the immune system, generating immune tolerance, and orchestrating the adaptive immune response to foreign antigens. Defects in their structure and function can lead to immunodeficiency, hypersensitivity, cancer, or autoimmune diseases. To better understand these diseases and assess potential therapies, complex models that recapitulate the anatomy and physiology of these tissues are required. Organoid models possess a number of advantages, including complex 3D microarchitecture, scalability, and personalization, which make them ideal for modelling lymphoid organs and related pathologies. Organoids have been developed for both primary and secondary lymphoid tissues; however, these models possess several limitations, including immature phenotypes and incomplete stromal cell populations. Furthermore, these organoids are often heterogeneous in both structure and function. Several lymphoid organs, such as the spleen, do not yet have robust organoid models, offering opportunities for breakthroughs in the field. Overall, development of lymphoid organoids will pave the way for the rapid development and testing of novel therapies, organ modelling, and personalized medicine. This review summarizes current advances in models for the primary lymphoid organ—bone marrow and thymus—as well as the secondary lymphoid organs of the lymph node and spleen.

Organoids doi: 10.3390/organoids4010006

Authors: Toshio Takahashi Yuta Takase

In cell biology, the stem cell niche is the dynamic microenvironment in which stem cells reside and receive signals that determine their behavior and fate. The stem cell niche has largely been a theoretical construct due to the difficulty in identifying and manipulating individual stem cells and their surroundings. Recent technical advances have made it possible to characterize the niches that maintain and control stem cell activity in several organs, including the small intestine. Although the small intestine has a relatively simple architecture, it has an extraordinary capacity for fast self-renewal. Thus, the organ is a unique model for studying intestinal stem cells (ISCs) and their niche. The intestinal epithelium maintains the intestine, enabling it to perform its absorption, secretion, and barrier functions. ISCs reside at the base of crypts adjacent to Paneth cells. In vivo, ISCs are surrounded by the microenvironment that makes up the niche, which provides a variety of stimuli that determine the fate of the cells. Research on stem cell niches is beginning to deepen our understanding of ISC regulation at the cellular and molecular levels and is expected to provide insights that can be applied to ISC therapy. Intestinal organoids originate from a group of crypt base ISCs. These organoids possess a three-dimensional (3D) cell structure made up of the lumen facing inward. Therefore, 3D intestinal organoids are often digested and seeded in a two-dimensional (2D) manner to form confluent organoid monolayers. Here, we not only review our current understanding of ISC niches with a focus on systems that are well-characterized at the cellular and mechanistic levels, but we also summarize the current applications of intestinal organoids.

Organoids doi: 10.3390/organoids4010005

Authors: Lucia Miranda Luigi Mandrich Simona Massa Teresa Nutile Clotilde Crovella Ilaria De Rosa Raffaella Lucci Filippo De Rosa Pasquale Somma Vincenzo Mercadante Ciro Abate Salvatore Arbucci Luigi Panico Emilia Caputo

In 2023, at the Center for Biological Resources (CRB) at the Institute of Genetics and Biophysics (IGB, Naples, Italy) of the National Research Council (CNR), the Breast Cancer Tissues and Organoids Biobank (BCTO BioBank) was founded. This is a new generation Biobank, dedicated to the collection, characterization, storage, and distribution of tissues and their 3D ‘organoid’ patients-derived. Tumor and healthy tissues from breast cancer patients have been collected from surgeons at Monaldi Hospital (Naples, Italy) and used to generate the corresponding tumor and healthy organoids from the same patient. After their establishment in culture, both organoids were characterized for their receptor status on a microfluidic 2-lane OrganoPlate, by immunofluorescence. The resulting data were compared with the expression profile obtained by immunohistochemistry on respective parental tissues. These data allowed us to phenotypically validate the generated organoids and classify them in a dedicated database, where also the clinical data of the corresponding patients were collected. During the six months of activities, we collected and characterized 27 samples. The continuous BCTO BioBank activity is fundamental to generating a high number of samples, for a broader and efficiently elaborated patient stratification at molecular level, biomarker discovery investigations, and for tailored treatment protocols design.

Organoids doi: 10.3390/organoids4010004

Authors: Huong Nguyen Francesca Di Cara Jun Wang Andrew W. Stadnyk

The healthy gut masks a dynamic balance between pro- and anti-inflammatory activities, largely due to microbial factors in the lumen. IL-10 is vital among the anti-inflammatory mediators, yet confirming constitutive versus stimulated secretion in any cell type is difficult due to the cellular complexity in the gut. Seeking to determine whether intestinal epithelial cells are programmed to constitutively make IL-10, we confirmed that IL-10 mRNA was present in enteroids from C57BL/6 mice and IL-10 protein was co-localized with a Paneth cell marker but not with markers for goblet or tuft cells. Paneth cells positive for IL-10 also possessed apical and basal IL-10RA, while cells negative for IL-10 had only basal IL-10RA, suggesting a possible autocrine role for IL-10. Indeed, Paneth cells in IL-10 gene knockout (IL-10KO) enteroids possessed lower levels of anti-microbial protein mRNAs, which could not be restored by adding IL-10. Enteroids passaged onto Transwell® filters to form monolayers were treated with IL-10 and STAT3 phosphorylation was measured. Apically applied IL-10 resulted in a stronger STAT3 signal than basally applied cytokine. Our results indicate that a subpopulation of Paneth cells constitutively secrete IL-10 apically, which binds apical IL-10RA, impacting the expression of anti-microbial proteins unique to Paneth cells.

Organoids doi: 10.3390/organoids4010003

Authors: Arisa Ishimura Ken Iwatsuki Hiroo Imai

Intestinal organoids are useful for the in vitro investigation of the properties of intestinal epithelial cells and their interaction with the gut microbiome. In this study, we cultured cecal and colonic organoids from common marmosets, which are highlighted as model nonhuman primates but are susceptible to gastrointestinal diseases. The organoids established were capable of passaging and long-term culture. The results of quantitative reverse transcription PCR and immunostaining showed that the organoids differentiated into major cell types (colonocytes, goblet cells, and enteroendocrine cells) in the intestinal epithelium, enabling the in vitro analysis of these cells in marmosets. The organoids could therefore represent a useful model for the investigation of gut physiology in relation to gastrointestinal diseases and host-microbiome interactions, further expanding medical, biological, and veterinary research in the future.

Organoids doi: 10.3390/organoids4010002

Authors: Harleen Kaur Josephine A. Wright Daniel L. Worthley Elizabeth Murphy Susan L. Woods

Peritoneal carcinomatosis from gastrointestinal tumours is considered a poor prognostic factor, with a median overall survival of six to nine months in the absence of intervention. The advent of patient-derived organoid cultures (PDOs) has provided a breakthrough in personalised medicine, allowing researchers and clinicians to model the complexity and heterogeneity of individual tumours in vitro. PDOs hold great promise in this field, as variations in the management of peritoneal carcinomatosis due to differences in the method of delivery of chemotherapeutics, drug selection, exposure duration, and tumour pathology make it impractical to use a single, standardised treatment regimen. We aim to summarise the methodologies and limitations of studies encapsulating organoids derived from peritoneal metastases to encourage design considerations that may improve future clinical relevance, standardise protocols, and address translational challenges in personalising treatment strategies.

Organoids doi: 10.3390/organoids4010001

Authors: Sarah Handcock Kay Richards Timothy J. Karle Pamela Kairath Alita Soch Carolina A. Chavez Steven Petrou Snezana Maljevic

Summary Statement: A tailored image analysis workflow was applied to quantify cortical organoid health, development, morphology and cellular composition over time. The assessment of cellular composition and viability of stem cell-derived organoid models is a complex but essential approach to understanding the mechanisms of human development and disease. Aim: Our study was motivated by the need for an image-analysis workflow, including high-cell content, high-throughput methods, to measure the architectural features of developing organoids. We assessed stem cell-derived cortical organoids at 4 and 6 months post-induction using immunohistochemistry-labelled sections as the analysis testbed. The workflow leveraged fluorescence imaging tailored to classify cells as viable and dying or non-viable and assign neuronal and astrocytic perinuclear markers to count cells. Results/Outcomes: Image acquisition was accelerated by capturing the organoid slice in 3D using widefield-fluorescence microscopy. This method used computational clearing to resolve nuclear and perinuclear markers and retain their spatial information within the organoid’s heterogeneous structure. The customised workflow analysed over 1.5 million cells using DAPI-stained nuclei, filtering and quantifying viable and non-viable cells and the necrotic-core regions. Temporal analyses of neuronal cell number derived from perinuclear labelling were consistent with organoid maturation from 4 to 6 months of in vitro differentiation. Overall: We have provided a comprehensive and enhanced image analysis workflow for organoid structural evaluation, creating the ability to gather cellular-level statistics in control and disease models.

Organoids doi: 10.3390/organoids3040018

Authors: Tamara Dal-Mora Najla Adel Saleh Veridiana Pacheco Goulart Martinazzo Maria Luiza Carneiro Buchele Michele Patrícia Rode Adny Henrique Silva Laura Sartori Assunção Tânia Beatriz Creczynski-Pasa Fabiola Branco Filippin-Monteiro

Adipogenesis is a complex process influenced by various cellular interactions within adipose tissue, which plays a critical role in metabolic homeostasis. This study aimed to develop a novel in vitro three-dimensional (3D) co-culture model using murine 3T3-L1 pre-adipocytes, J774 macrophages, and NIH-3T3 fibroblasts to investigate adipogenic differentiation and inflammatory pathways. We first validated an adipogenic differentiation protocol in a two-dimensional (2D) model, where 3T3-L1 pre-adipocytes were subjected to a hormonal medium containing 3-isobutyl-1-methylxanthine, dexamethasone and insulin. After 7 days, differentiated cells were analyzed using Oil Red O and Nile Red staining, confirming lipid accumulation. Subsequently, spheroids were formed in 3D cultures, with monospheroids and heterospheroids maintained in either control medium or MDI for 11 days. Size measurements indicated significant growth in heterospheroids, particularly in the 3T3-L1:J774 combination, underscoring the importance of cellular interactions. Confocal microscopy and flow cytometry analyses demonstrated that even in the absence of hormonal stimuli, control spheroids exhibited adipogenic differentiation, evidenced by a notable proportion of Nile Red-positive cells (75.7 ± 1.7%). Inflammatory profiling revealed that the heterospheroid 3:J produced the highest levels of nitric oxide (NO), with no significant differences observed between control and MDI conditions. This study highlights the potential of 3D co-culture systems for elucidating the intricate interactions among adipocytes, macrophages, and fibroblasts. The findings may provide valuable insights into novel therapeutic targets for metabolic disorders.

Organoids doi: 10.3390/organoids3040017

Authors: Raphael S. Werner Jae-Hwi Jang Markus Rechsteiner Michaela B. Kirschner Isabelle Opitz

Background: Recent advances in the personalized treatment of non-small cell lung cancer (NSCLC) require representative in vitro model systems that reflect tumor heterogeneity and maintain the characteristic genetic aberrations. We therefore aimed to establish patient-derived NSCLC organoids that offer a reliable platform for further investigations. Methods: NSCLC organoids were cultured between May 2020 and February 2022 from surgically resected NSCLC tissue specimens. After histological and immunohistochemical validation, genetic validation was performed by targeted next-generation sequencing of tissue and organoid specimens using the Oncomine Focus Assay (ThermoFisher Scientific). Results: From 37 resected NSCLC samples, 18 primary organoid cultures were successfully established and expanded during early passages. Upon histomorphological validation, organoids showed complementary characteristics when compared to the resected parental tumor, including adenocarcinoma, squamous cell carcinoma, mucoepidermoid carcinoma, and lung carcinoid differentiation. Among nine parental tumors, traceable genetic alterations were detected, and three corresponding organoids lines retained this mutational profile, including a KRAS p.Gly12Val mutation, KRAS p.Gly12Cys mutation, and RET-fusion. Conclusions: The establishment of primary NSCLC organoids from surgically resected tissue is feasible. Histological, immunohistochemical, and genetic validation is essential to identify representative NSCLC organoids that maintain the characteristics of the parental tumor. Overall, low establishment rates remain a challenge for broad clinical applications.

Organoids doi: 10.3390/organoids3040016

Authors: Tharindie N. Silva Josephine A. Wright Daniel L. Worthley Susan L. Woods

Gastric cancer (GC) presents a significant health challenge and ranks as the fifth most common cancer in the world. Unfortunately, most patients with GC exhaust standard care treatment options due to late diagnosis and tumour heterogeneity that leads to drug resistance, resulting in poor survival outcomes. Potentially, this situation can be improved by personalising treatment choice. Organoids are an emerging cell model system that recapitulates tumour heterogeneity and drug responses. Coupled with genomic analysis, organoid culture can be used to guide personalised medicine. The GC organoid field, however, lacks standardised methodologies for assessing organoid drug sensitivities. Comparing results across different GC organoid studies and correlating organoid drug responses with patient outcomes is challenging. Hence, we aim to summarise the methodologies used in GC organoid drug testing and correlation with clinical outcomes and discuss design considerations and limitations to enhance the robustness of such studies in the future.

Organoids doi: 10.3390/organoids3040015

Authors: Fuad Gandhi Torizal Syarifah Tiara Noorintan Zakiya Gania

Tooth and periodontal organoids from stem and progenitor cells represent a significant advancement in regenerative dentistry, offering solutions for tooth loss and periodontal diseases. These organoids, which mimic the architecture and function of real organs, provide a cutting-edge platform for studying dental biology and developing therapies. Recent methodologies have been developed to optimize conditions for organoid production, advancing dental regenerative medicine, disease modeling, and developmental studies. The integration of bioengineering strategies with culture techniques enhances both our understanding and the therapeutic potential of these organoids. Additionally, factors such as the extracellular matrix, growth factors, and culture systems profoundly influence organoid formation and maturation. This review explores various bioengineering approaches for generating organoids, emphasizing the pivotal role of stem and progenitor cells.

Organoids doi: 10.3390/organoids3030014

Authors: Gowtham Reddy Cheruku Chloe Veronica Wilson Suriya Raviendran Qingzhong Xiao

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.

Organoids doi: 10.3390/organoids3030013

Authors: Valérie M. Wouters Ciro Longobardi Jan Paul Medema

Colorectal cancer (CRC) is the third most common cancer worldwide and it is the second leading cause of cancer death. In CRC, as in most cancers, the formation of metastasis through the migration and invasion of cancer cells to distant organs is associated with a dismal prognosis. The study of the mechanisms associated with cancer, and, in particular, CRC, changed in the last decade due to the introduction of organoids. These represent a step forward in terms of complexity from cell lines and allowed the use of mouse models in cancer research to be limited. Although organoids faithfully model the cellular complexity of CRC, current protocols do not allow for the use of organoids in some crucial processes of metastasis, such as migration and invasion. In this study, a method to study migration and invasion using mouse intestinal organoids in vitro is presented. This protocol provides researchers with the opportunity to investigate the migratory behavior of organoid lines and study the impact of distinct mutations on the migratory and invasive capacity of cancer cells.

Organoids doi: 10.3390/organoids3030012

Authors: Brady M. Wessel Jenna N. Castro Victoria H. J. Roberts

First trimester placental development comprises some of the most critical yet understudied events that impact fetal development. Improper placentation leads to a host of health issues that not only impact the fetal period but also influence offspring throughout their lives. Thus, a paradigm to study early placental development is necessary, and this has spurred on the pursuit of new in vitro model systems that recapitulate specific aspects of placentation. One of the most complex and translationally valid models to arise are organoids, three-dimensional structures comprising multiple differentiated cell types that originate from a common progenitor population. Trophoblasts are the progenitor cells of the placenta, serving as the proliferative base for placental development. Recent advances have enabled the derivation of organoids from primary tissue, yet access to first trimester human samples is ethically constrained; derivation from established trophoblast stem cell lines is an alternative source. Organoids have already proven useful in generating insights into molecular events that underlie trophoblast differentiation, with the identification of new cell subtypes that are primed to differentiate down different paths. In this review, (1) we recap early pregnancy development events, (2) provide an overview of the cellular complexity of the placenta, (3) discuss the generation of organoids from tissue versus cellular sources, (4) highlight the value of translational animal models, and (5) focus on the complexities of the molecular regulation of trophoblast organoid development, differentiation, and function.

Organoids doi: 10.3390/organoids3030011

Authors: Payal Ganguly

Globally, a number of diseases impact us and while treatment options exist, it is often found that similar treatments have variable effects on different patients with the same disease. Particularly in the case of conditions that are closely associated with genetics (like cancer), the intensity and results of a treatment vary between patients. Even for diseases like arthritis it is not uncommon for only a fraction of patients to achieve remission with the same therapeutic approach. With millions suffering from diseases like cancer and arthritis, precision medicine (PM) has been at the forefront of biomedical and pharmaceutical research since 2015. PM focusses on understanding the genetic and environmental factors affecting the patients and has several platforms. One of the platforms is the use of three-dimensional (3D) in vitro models, especially those derived from the patient themselves. These models, like organ-on-chip (OOC), organoid and spheroid models, 3D biomaterial scaffolds and others, have several advantages over traditional two-dimensional (2D) cell culture approaches. In this opinion paper, the author briefly discusses the different platforms used for PM. Then, the advantages that 3D in vitro models have over traditional 2D models and in vivo models are considered and an overview of their applications is provided. Finally, the author outlines the challenges and future directions and shares their opinion about using 3D in vitro models as a tool for PM towards enhanced patient outcomes.

Organoids doi: 10.3390/organoids3030010

Authors: Michelle N. H. Tang Mariya Moosajee Najam A. Sharif G. Astrid Limb Karen Eastlake

In zebrafish and various mammalian species, HB-EGF has been shown to promote Müller glia proliferation and activation of repair mechanisms that have not been fully investigated in human retina. In the current study, 70- to 90-day-old human retinal organoids were treated with 20 μM 4-hydroxytamoxifen (4-OHT), and CRX, REC, NRL, PAX6, VIM, GFAP, and VSX2 gene and protein expression were assessed at various times points after treatment. Organoids with or without 4-OHT-induced damage were then cultured with HB-EGF for 7 days. We showed that 20 μM 4-OHT caused a reduction in the number of recoverin-positive cells; an increase in the number of TUNEL-positive cells; and downregulation of the photoreceptor gene markers CRX, NRL, and REC. Culture of organoids with HB-EGF for 7 days after 4-OHT-induced damage caused a marked reduction in the number of TUNEL-positive cells and small increases in the number of Ki67-positive cells and PAX6 and NOTCH1 gene expression. The current results suggest that treatment of human ESC-derived retinal organoids with 4-OHT may be used as a model of retinal degeneration in vitro. Furthermore, HB-EGF treatment of human retinal organoids increases proliferating Müller cells, but only after 4-OHT induced damage, and may be an indication of Muller reactivity in response to photoreceptor damage. Further studies will aim to identify factors that may induce Müller cell-mediated regeneration of the human retina, aiding in the development of therapies for retinal degeneration.

Organoids doi: 10.3390/organoids3020009

Authors: Thomas P. Rudibaugh Ryan W. Tam R. Chris Estridge Samantha R. Stuppy Albert J. Keung

The mesolimbic pathway connects ventral tegmental area dopaminergic neurons and striatal medium spiny neurons, playing a critical role in reward and stress behaviors. Exposure to substances of abuse during development and adulthood has been linked to adverse outcomes and molecular changes. The rise of human cell repositories and whole-genome sequences enables human functional genomics ‘in a dish’, offering insights into human-specific responses to substances of abuse. Continued development of new models is needed, and the characterization of in vitro models is also necessary to ensure appropriate experimental designs and the accurate interpretation of results. This study introduces new culture conditions for generating medium spiny neurons and dopaminergic neurons with an early common media, allowing for coculture and assembloid generation. It then provides a comprehensive characterization of these and prior models and their responses to substances of abuse. Single-cell analysis reveals cell-type-specific transcriptomic responses to dopamine, cocaine, and morphine, including compound and cell-type-specific transcriptomic signatures related to neuroinflammation and alterations in signaling pathways. These findings offer a resource for future genomics studies leveraging human stem cell-derived models.

Organoids doi: 10.3390/organoids3020008

Authors: Héloïse Castiglione Lucie Madrange Thomas Lemonnier Jean-Philippe Deslys Frank Yates Pierre-Antoine Vigneron

In recent years, 3D cell culture systems have emerged as sophisticated in vitro models, providing valuable insights into human physiology and diseases. The transition from traditional 2D to advanced 3D cultures has introduced novel obstacles, complicating the characterization and analysis of these models. While the lactate dehydrogenase (LDH) activity assay has long been a standard readout for viability and cytotoxicity assessments in 2D cultures, its applicability in long-term 3D cultures is hindered by inappropriate normalization and low LDH stability over time. In response to these challenges, we propose an optimization of LDH assays, including a crucial normalization step based on total protein quantification and a storage method using an LDH preservation buffer. We applied it to compare unexposed cerebral organoids with organoids exposed to a toxic dose of valproic acid, and showed efficient normalization of cellular viability as well as enhanced LDH stability within the buffer. Importantly, normalized LDH activity results obtained were independent of organoid dimension and cell density. This refined LDH assay, tailored to address 3D culture constraints, allows for the transposition of this routine test from 2D to 3D cultures.

Organoids doi: 10.3390/organoids3020007

Authors: Xavier Palmer Cyril Akafia Eleasa Woodson Amanda Woodson Lucas Potter

Organoids present immense promise for studying organ systems and their functionality. Recently, they have become the subject of exploration outside of purely biomedical uses in multiple directions. We will explore the rapidly evolving landscape of organoid research over the 21st century, discussing significant advancements in organoid research and highlighting breakthroughs, methodologies, and their transformative impact on our understanding of physiology and modeling. In addition, we will explore their potential use for biocomputing and harnessing organoid intelligence, investigate how these miniaturized organ-like structures promise to create novel computational models and processing platforms allowing for innovative approaches in drug discovery, personalized medicine, and disease prediction. Lastly, we will address the ethical dilemmas surrounding organoid research by dissecting the intricate ethical considerations related to the creation, use, and potential implications of these in vitro models. Through this work, the goal of this paper is to provide introductory perspectives and bridges that will connect organoids to cybersecurity applications and the imperative ethical discourse accompanying its advancements with commentary on future uses.

Organoids doi: 10.3390/organoids3020006

Authors: Flavia C. M. Oliveira Annemarie W. Y. Voorbij Elisa C. Pereira Leonor M. M. Alves e Almeida Geanne R. Moraes Joana T. De Oliveira Boyd H. T. Gouw Sabrina A. M. Legatti Hans S. Kooistra Bart Spee Andre M. C. Meneses Louis C. Penning

For over 150 years, researchers have studied the (patho)physiology of the endocrine pancreas and devised treatment options for diabetes mellitus (DM). However, no cure has been developed so far. In dogs, diabetes mellitus type 1 (T1DM) is the most common presentation. Treatment consists of twice daily insulin injections, monitored by spatial blood glucose measurements. Even though dogs were instrumental in the discovery of insulin and islet transplantations, the treatment in diabetic dogs has remained unchanged for decades. Providing twice daily insulin injections is demanding for both owners and dogs and may result in hypoglycaemic events, creating the need for new treatment strategies. Novel regenerative medicine-based tools, such as improved β-cell culture protocols and artificial devices, have sparked hope for a cure. In human medicine, emerging technologies such as the transplantation of insulin-producing β-cells, generated by stem cell differentiation, with or without an encapsulation device, are currently tested in phase I/II clinical trials. As the pathogenesis of T1DM is remarkably similar between humans and dogs, novel treatment methods could be implemented in canine medicine. This review briefly summarises the physiology of the canine endocrine pancreas and the pathophysiology of canine DM before exploring current and possible future treatment options for canine DM.

Organoids doi: 10.3390/organoids3010005

Authors: Bas van Rijn Diane Van Opstal Nicole van Koetsveld Maarten Knapen Joost Gribnau Olivier Schäffers

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.

Organoids doi: 10.3390/organoids3010004

Authors: Anna Rebecca Dorn Sara Neff Sophia Hupp Melissa Engelhardt Eric Pion Ulrich Lenze Carolin Knebel Anna Duprée Simone Schewe Markus Weber Christian Wulbrand Axel Hillmann Florian Weber Phillip Clarke Philipp Kainz Thiha Aung Silke Haerteis

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.

Organoids doi: 10.3390/organoids3010003

Authors: Süleyman Ergün Organoids Editorial Office Organoids Editorial Office

In this issue, we are pleased and honored to have an interview with Professor Hans Clevers, who is the Advisory Board Member of Organoids [...]

Organoids doi: 10.3390/organoids3010002

Authors: Julio Carrera Montoya Simon Collett Daniel Fernandez Ruiz Linda Earnest Melissa A. Edeling Ashley Huey Yiing Yap Chinn Yi Wong James P. Cooney Kathryn C. Davidson Jason Roberts Steven Rockman Bang M. Tran Julie L. McAuley Georgia Deliyannis Samantha L. Grimley Damian F. J. Purcell Shafagh A. Waters Dale I. Godfrey Dhiraj Hans Marc Pellegrini Jason M. Mackenzie Elizabeth Vincan William R. Heath Joseph Torresi

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.

Organoids doi: 10.3390/organoids3010001

Authors: Andreas Ettner-Sitter Agata Montagner Jonas Kuenzel Kathrin Brackmann Maximilian Schäfer Robert Schober Florian Weber Thiha Aung Christina Hackl Silke Haerteis

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.

Organoids doi: 10.3390/organoids2040019

Authors: Xinhui Wang Brent Bijonowski Nicholas Kurniawan

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.

Organoids doi: 10.3390/organoids2040018

Authors: Hans-Werner Denker

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.

Organoids doi: 10.3390/organoids2040017

Authors: Clara Elena López Vásquez Clint Gray Claire Henry Matthew J. Munro

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.

Organoids doi: 10.3390/organoids2040016

Authors: Yi Xiao Qiaoling Huang Jesse W. Collins Julie Brouchon Jeffery A. Nelson Zachary Niziolek Alison O’Neil Fangfu Ye David A. Weitz John A. Heyman

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.

Organoids doi: 10.3390/organoids2040015

Authors: Na Qu Abdelkader Daoud Braxton Jeffcoat Jorge O. Múnera

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.

Organoids doi: 10.3390/organoids2040014

Authors: Remi Yokoi Nami Nagafuku Yuto Ishibashi Naoki Matsuda Ikuro Suzuki

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.

Organoids doi: 10.3390/organoids2040013

Authors: R. Chris Estridge Jennifer E. O’Neill Albert J. Keung

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.

Organoids doi: 10.3390/organoids2030012

Authors: Georg Csukovich Janina Huainig Selina Troester Barbara Pratscher Iwan Anton Burgener

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.

Organoids doi: 10.3390/organoids2030011

Authors: Ania Bogoslowski Meilin An Josef M. Penninger

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.

Organoids doi: 10.3390/organoids2030010

Authors: Magdalena Rausch Neelam Iqbal Shelly Pathak Heather E. Owston Payal Ganguly

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.

Organoids doi: 10.3390/organoids2020009

Authors: Elizabeth Vincan

“Organoids Are Us” is an annual one-day symposium organised to highlight the advances in science and medicine that are the direct result of organoid technology [...]

Organoids doi: 10.3390/organoids2020008

Authors: Kathryn Futrega Md. Shafiullah Shajib Pamela G. Robey Michael R. Doran

(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.

Organoids doi: 10.3390/organoids2020007

Authors: Pin Shie Quah Bang M. Tran Vincent D.A. Corbin Jessie J.-Y. Chang Chinn Yi Wong Andrés Diaz-Méndez Carol A. Hartley Weiguang Zeng Eric Hanssen Zlatan Trifunovic Patrick C. Reading David C. Jackson Elizabeth Vincan Lachlan J.M. Coin Georgia Deliyannis

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.

Organoids doi: 10.3390/organoids2020006

Authors: Philipp Wörsdörfer Süleyman Ergün

Organoids are taking the scientific world by storm, revolutionizing the ways in which we study complex biological systems [...]

Organoids doi: 10.3390/organoids2010005

Authors: Sparshita Nag Ashleigh S. Boyd

Chronic Kidney Disease (CKD) is a major cause of morbidity and mortality characterized by progressive renal fibrosis, and in extreme cases, renal failure. Human CKD models that replicate the biological complexity of the kidney and CKD are lacking and will be invaluable in identifying drugs to revert and/or prevent fibrosis. To address this unmet need, we developed 3D renal organoids where human induced pluripotent stem cells (hiPSCs) were differentiated to renal progenitors within a renal extracellular matrix (rECM) gel, based on the premise that an rECM could recreate the renal niche to facilitate hiPSC-derived renal progenitor generation. We used mouse kidneys as a source of rECM and identified that superior detergent-mediated decellularization of mouse kidneys was achieved with a combination of 0.5% w/v Sodium Dodecyl Sulphate and 1% v/v Triton-X and mechanical agitation for 60 h. HiPSCs that underwent specification to become metanephric mesenchyme (MM) were subsequently cultured within the rECM gel and, notably, mesenchymal to epithelial transition (MET) was observed, as judged by expression of nephron markers K-cadherin, Nephrin and WT1. These data demonstrate a role for rECM gel in developing human renal organoids from hiPSCs, which will aid the further development of a human disease model for renal fibrosis.

Organoids doi: 10.3390/organoids2010004

Authors: Eliza Goddard Eva Tomaskovic-Crook Jeremy Micah Crook Susan Dodds

Human brain organoids provide a remarkable opportunity to model prenatal human brain biology in vitro by recapitulating features of in utero molecular, cellular and systems biology. An ethical concern peculiar to human brain organoids is whether they are or could become capable of supporting sentience through the experience of pain or pleasure and/or consciousness, including higher cognitive abilities such as self-awareness. Identifying the presence of these traits is complicated by several factors, beginning with consciousness—which is a highly contested concept among neuroscientists, cognitive scientists, and philosophers and so there is no agreed definition. Secondly, given human brain organoids are disembodied, there is no practical way to identify evidence of consciousness as we might in humans or animals. What would count as evidence of organoid consciousness is an emerging area of research. To address concerns about consciousness and human brain organoids, in this paper we clarify the morally relevant aspects of human consciousness, phenomenal experience and embodied development and explore the empirical basis of consciousness to develop a defensible framework for informed decision-making on the moral significance and utility of brain organoids, which can also guide regulation and future research of these novel biological systems.

Organoids doi: 10.3390/organoids2010003

Authors: Matthew J. Munro Swee T. Tan Clint Gray

Organoids are 3D organ-like structures grown from stem cells in vitro that mimic the organ or disease from which they are derived. Due to their stem cell origin, organoids contain a heterogeneous population of cells reflecting the diversity of cell types seen in vivo. Similarly, tumour organoids reflect intratumoural heterogeneity in a way that traditional 2D cell culture and cell lines do not, and, therefore, they show greater promise as a more relevant model for effective disease modelling and drug testing. Tumour organoids arise from cancer stem cells, which contribute to many of the greatest challenges to cancer treatment, including therapy resistance, tumour recurrence, and metastasis. In this review, we outline methods for generating colon organoids from patient-derived normal and tumour tissues. Furthermore, we discuss organoid biobanking, applications of organoids in disease modelling, and a range of platforms applicable to high-throughput drug testing, including apical-out/reverse-polarity colon organoids.

Organoids doi: 10.3390/organoids2010002

Authors: Eva Tomaskovic-Crook Sarah Liza Higginbottom Binbin Zhang Justin Bourke Gordon George Wallace Jeremy Micah Crook

Human brain organoids present a new paradigm for modeling human brain organogenesis, providing unprecedented insight to the molecular and cellular processes of brain development and maturation. Other potential applications include in vitro models of disease and tissue trauma, as well as three-dimensional (3D) clinically relevant tissues for pharmaceuticals development and cell or tissue replacement. A key requirement for this emerging technology in both research and medicine is the simple, scalable, and reproducible generation of organoids using reliable, economical, and high-throughput culture platforms. Here we describe such a platform using a defined, clinically compliant, and readily available hydrogel generated from gelatin methacrylate (GelMA). We demonstrate the efficient production of organoids on GelMA from human induced pluripotent stem cells (iPSCs), with scalable production attained using 3D printed GelMA-based multiwell arrays. The differentiation of iPSCs was systematic, rapid, and direct to enable iPSCs to form organoids in their original position following seeding on GelMA, thereby avoiding further cell and organoid disruption. Early neural precursors formed by day 5, neural rosettes and early-stage neurons by day 14, and organoids with cellular and regional heterogeneity, including mature and electrophysiologically active neurons, by day 28. The optimised method provides a simplified and well-defined platform for both research and translation of iPSCs and derivative brain organoids, enabling reliable 3D in vitro modelling and experimentation, as well as the provision of clinically relevant cells and tissues for future therapeutics.

Organoids doi: 10.3390/organoids2010001

Authors: Susanne Ramm Robert Vary Twishi Gulati Jennii Luu Karla J. Cowley Michael S. Janes Nicholas Radio Kaylene J. Simpson

Technical advances in microscopy and automation have enabled image-based phenotypic screening of spheroids and organoids to become increasingly high throughput and high content at the same time. In particular, matrix-embedded 3D structures can recapitulate many aspects of parent (e.g., patient) tissues. Live-cell imaging of growing structures allows tremendous insight into population heterogeneity during drug treatment. However, screening for targeted markers and more detailed morphological analyses typically require fixation of 3D structures, and standard formaldehyde (FA) incubation conditions can dissolve collagen-based extracellular matrices such as Matrigel. The dislocation and clumping of the spheroids make image-based segmentation very difficult and the tracking of structures from the live cell stage to their fixed cell location virtually impossible. In this method, we present a fixation and staining protocol that is gentle enough to maintain 3D structures exactly in their live-cell location and does not alter their morphology. This opens up analytical strategies that connect the spheroid’s growth kinetics and heterogeneity of treatment responses with the more targeted fixed cell stains. Furthermore, we optimized the automated seeding and imaging of spheroids so that screening and phenotypic characterization can be performed in high-throughput at either low or high magnification and yield the same result, independent of the microscope used.

Organoids doi: 10.3390/organoids1020013

Authors: Sara Noorani Shannon R. Nelson Neil T. Conlon Justine Meiller Ekaterina Shcheglova Alice Usai Jojanneke Stoof Letizia Palanga Fiona O’Neill Sandra Roche Maura B. Cotter Niall Swan Naomi Walsh

Pancreatic cancer is a highly lethal disease. Therapeutic resistance to chemotherapy is a major cause of treatment failure and recurrence in pancreatic cancer. Organoids derived from cancer stem cells (CSC) are promising models for the advancement of personalised therapeutic responses to inform clinical decisions. However, scaling-up of 3D organoids for high-throughput screening is time-consuming and costly. Here, we successfully developed organoid-derived cell lines (2.5D) from 3D organoids; the cells were then expanded and recapitulated back into organoids known as cell line organoids (CLOs). The 2.5D lines were cultured long term into 2D established cell lines for downstream comparison analysis. Experimental characterisation of the models revealed that the proliferation of CLOs was slightly faster than that of parental organoids. The therapeutic response to chemotherapeutic agents in 3D CLOs and organoids showed a similar responsive profile. Compared to 3D CLOs and organoids, 2D cell lines tended to be less responsive to all the drugs tested. Stem cell marker expression was higher in either 3D CLOs or organoids compared to 2D cell lines. An in vivo tumorigenicity study found CLOs form tumours at a similar rate to organoids and retain enhanced CSC marker expression, indicating the plasticity of CSCs within the in vivo microenvironment.

Organoids doi: 10.3390/organoids1020012

Authors: Yichen Zhu Elliot Kang Matthew Wilson Taylor Basso Evelynn Chen Yanqi Yu Yan-Ruide Li

The intricate microenvironment in which malignant cells reside is essential for the progression of tumor growth. Both the physical and biochemical features of the tumor microenvironment (TME) play a critical role in promoting the differentiation, proliferation, invasion, and metastasis of cancer cells. It is therefore essential to understand how malignant cells interact and communicate with an assortment of supportive tumor-associated cells including macrophages, fibroblasts, endothelial cells, and other immune cells. To study the complex mechanisms behind cancer progression, 3D spheroid and organoid models are widely in favor because they replicate the stromal environment and multicellular structure present within an in vivo tumor. It provides more precise data about the cell–cell interactions, tumor characteristics, drug discovery, and metabolic profile of cancer cells compared to oversimplified 2D systems and unrepresentative animal models. This review provides a description of the key elements of the tumor microenvironment as well as early research using cell-line derived, 3D spheroid tumor models that paved the way for the adoption of patient-derived spheroid and organoid models. In particular, 3D spheroid and organoid models provide a method for drug screening with a particular emphasis on influence of the TME in cancer immunotherapy.

Organoids doi: 10.3390/organoids1020011

Authors: Zhong Alan Li Jiangyinzi Shang Shiqi Xiang Eileen N. Li Haruyo Yagi Kanyakorn Riewruja Hang Lin Rocky S. Tuan

Organoids offer a promising strategy for articular tissue regeneration, joint disease modeling, and development of precision medicine. In this study, two types of human stem cells—primary mesenchymal stem cells (MSCs) and induced pluripotent stem cells (iPSCs)—were employed to engineer organoids that mimicked bone, cartilage and adipose tissue, three key tissue components in articular joints. Prior to organoidogenesis, the iPSCs were first induced into mesenchymal progenitor cells (iMPCs). After characterizing the MSCs and iMPCs, they were used to generate cell-embedded extracellular matrix (ECM) constructs, which then underwent self-aggregation and lineage-specific differentiation in different induction media. Hydroxyapatite nanorods, an osteoinductive bioceramic, were leveraged to generate bone and osteochondral organoids, which effectively enhanced mineralization. The phenotypes of the generated organoids were confirmed on the basis of gene expression profiling and histology. Our findings demonstrate the feasibility and potential of generating articular tissue-recapitulating organoids from MSCs and iPSCs.

Organoids doi: 10.3390/organoids1020010

Authors: Hélène Vignes Guillaume Conzatti Guoqiang Hua Nadia Benkirane-Jessel

Walking, running, jumping, or even just standing up are habits that we all have to perform in our everyday lives. However, defects in tissues composing the knee joint can drastically alter our ability to complete those simple actions. The knee joint is made up of the interaction between bones (femur, tibia, and patella), tendons, ligaments, and the two menisci (lateral and medial) in order to ensure smooth body movements. The meniscus corresponds to a crescent-shaped fibrocartilaginous tissue, which is found in the knee joint between the femoral condyles and the tibial plateau. It plays a key role in the stability of the knee joint. However, it is quite vulnerable and therefore tears can occur within this tissue and compromise the proper function of the knee. Recently, numerous efforts have been made in order to find solutions to repair and regenerate the meniscus, supported by both bioengineering researchers and orthopedic surgeons. However, due to its poor healing capacity and its complex structure, the reconstruction of the meniscus remains particularly challenging. In this review, the current treatment options will be explained and the possibility of using organoids as building blocks for implant formation or as an in vitro three-dimensional model will be highlighted.

Organoids doi: 10.3390/organoids1020009

Authors: Olivier J. M. Schäffers Catherine Dupont Eric M. Bindels Diane Van Opstal Dick H. W. Dekkers Jeroen A. A. Demmers Joost Gribnau Bas B. van Rijn

Trophoblast organoids (TOs) hold great promise for elucidating human placental development and function. By deriving TOs in ongoing pregnancies using chorionic villus sampling (CVS), we established a platform to study trophoblast differentiation and function in early pregnancy, including pregnancies with different fetal genetic abnormalities. We addressed cellular heterogeneity of CVS-derived TOs by providing a single-cell transcriptomic atlas and showed that CVS-TOs recapitulate key aspects of the human placenta, including syncytial fusion and hormone synthesis. This study demonstrates the utility of trophoblast organoids for investigating genetic defects in the placenta and describes an experimental platform for future personalized placental medicine approaches, including genotype–phenotype mapping.

Organoids doi: 10.3390/organoids1010008

Authors: Elena Richiardone Valentin Van den Bossche Cyril Corbet

Organoid technologies represent a major breakthrough in biomedical research since they offer increasingly sophisticated models for studying biological mechanisms supporting human development and disease. Organoids are three-dimensional (3D) physiological in vitro systems that recapitulate the genetic, histological and functional features of the in vivo tissues of origin more accurately than classical cell culture methods. In the last decade, organoids have been derived from various healthy and diseased tissues and used for a wide range of applications in basic and translational research, including (cancer) tissue biology, development, regeneration, disease modeling, precision medicine, gene editing, biobanking and drug screening. Here, we report the current applications of organoid models to study (stem) cell metabolism in several pathophysiological contexts such as cancer and metabolic diseases. More precisely, we discuss the relevance and limitations of these 3D cultures to model and study metabolic (dys)functions associated with hepatic, renal or pancreatic disorders, as well as tumor development and progression. We also describe the use of organoids to understand the dynamic interaction between diet, microbiota and the intestinal epithelium. Finally, this review explores recent methodological improvements in organoid culture that may help to better integrate the influence of microenvironmental conditions in the study of tumor cell metabolic phenotypes.

Organoids doi: 10.3390/organoids1010007

Authors: Patrycja Sokolowska Agnieszka Zuchowska Zbigniew Brzozka

Preclinical studies are the first stage of introducing a new potential drug to the pharmaceutical market. Many of the compounds with promising results approved in the preclinical stage show poor prognosis during the first stage of clinical studies, which is connected with inadequate in vitro and in vivo models used in this stage. Both basic in vitro models, and in vivo animal models do not represent the human conditions. Therefore, scientists work on creating an appropriate model that will highly reproduce the characteristics of the human body. The solution could be an organoids model: a laboratory-produced human miniature organ, grown in a specially designed Organ-on-Chip microfluidic tools. This review focuses on characterizing the 3D cell culture types, focusing mainly on organoids, the Organ-on-Chip approach, and presenting the latest reports about the application of their combination in biological research, including toxicological studies.

Organoids doi: 10.3390/organoids1010006

Authors: Tamar Evron-Levy Michal Caspi Amnon Wittenstein Yamit Shorer-Arbel Olga Shomron Koret Hirschberg Revital Kariv Rina Rosin-Arbesfeld

Human colonic organoids derived from adult tissue biopsies are based on the ability of isolated somatic epithelial stem cells to reconstitute the structure and function of the colon, offering new opportunities for studying the biology of the large intestine in both health and disease. These colonoids may also function as efficient platforms for drug screening and discovery. Here, we describe the establishment of human colonic organoids derived from healthy, and adenomatous polyp tissues. We then demonstrate that organoids grown from adenomas of familial adenomatous polyposis (FAP) patients harboring nonsense mutations in the tumor suppressor gene adenomatous polyposis coli (APC), can be used to establish a personalized therapeutic strategy which relies on nonsense mutation readthrough therapy.

Organoids doi: 10.3390/organoids1010005

Authors: Sven Schmidt Yvonne Alt Nikita Deoghare Sarah Krüger Anna Kern Anna Frederike Rockel Nicole Wagner Süleyman Ergün Philipp Wörsdörfer

Blood vessel organoids are an important in vitro model to understand the underlying mechanisms of human blood vessel development and for toxicity testing or high throughput drug screening. Here we present a novel, cost-effective, and easy to manufacture vascular organoid model. To engineer the organoids, a defined number of human induced pluripotent stem cells are seeded in non-adhesive agarose coated wells of a 96-well plate and directed towards a lateral plate mesoderm fate by activation of Wnt and BMP4 signaling. We observe the formation of a circular layer of angioblasts around days 5–6. Induced by VEGF application, CD31+ vascular endothelial cells appear within this vasculogenic zone at approximately day 7 of organoid culture. These cells arrange to form a primitive vascular plexus from which angiogenic sprouting is observed after 10 days of culture. The differentiation outcome is highly reproducible, and the size of organoids is scalable depending on the number of starting cells. We observe that the initial vascular ring forms at the interface between two cell populations. The inner cellular compartment can be distinguished from the outer by the expression of GATA6, a marker of lateral plate mesoderm. Finally, 14-days-old organoids were transplanted on the chorioallantois membrane of chicken embryos resulting in a functional connection of the human vascular network to the chicken circulation. Perfusion of the vessels leads to vessel wall maturation and remodeling as indicated by the formation of a continuous layer of smooth muscle actin expressing cells enwrapping the endothelium. In summary, our organoid model recapitulates human vasculogenesis, angiogenesis as well as vessel wall maturation and therefore represents an easy and cost-effective tool to study all steps of blood vessel development and maturation directly in the human setting without animal experimentation.

Organoids doi: 10.3390/organoids1010004

Authors: Süleyman Ergün Philipp Wörsdörfer

The organs and tissues of our bodies consist of a specific set of cell types [...]

Organoids doi: 10.3390/organoids1010003

Authors: Antonie Fuhr Andreas Kurtz Christian Hiepen Sabine Müller

Organoids derived from human stem cell lines represent genetically mostly identical models of their donors. Their use as personalized in vitro miniature twins of living individuals creates challenges of reproducibility, comparability and standardization. To fully exploit personalization, it is essential to assess individual variabilities in organoid function, morphology or maturity. There is a need to establish platforms to compare individual organoids and to link them to data elements related to the individual donor. Moreover, principal ethical issues arise because of their infinite repetition for an unlimited period of time and global dissemination. This infinite temporal and spatial space applies to the biological material but also to the data associated with it. It increases the possibility of uses that are unpredictable at the time of donation, and thus, beyond the donor’s consented choices. We propose an open data platform to address the issue of authenticity and persistent comparability of the biological organoid models, and of preserving the ethical provenance information. The platform would collect standardized donors, organoids and ethical information to create a system suitable for quality control of individual organoids. We discuss whether the human pluripotent stem cell registry (hPSCreg), a well-established resource for stem cell data, provides a suitable model platform.

Organoids doi: 10.3390/organoids1010002

Authors: Bang M. Tran Georgia Deliyannis Abderrahman Hachani Linda Earnest Joseph Torresi Elizabeth Vincan

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which was classified as a pandemic in March 2020. As of 22 January 2022, globally more than 347 million cases of COVID-19 have been diagnosed, with 5.6 million deaths, making it the deadliest pandemic since the influenza pandemic in 1918. The clinical presentation of COVID-19-related illness spans from asymptomatic to mild respiratory symptoms akin to influenza infection to acute symptoms, including pneumonia necessitating hospitalisation and admission to intensive care units. COVID-19 starts in the upper respiratory tract and lungs but in severe cases can also involve the heart, blood vessels, brain, liver, kidneys and intestine. The increasing global health and economic burden of COVID-19 necessitates an urgent and global response. Understanding the functional characteristics and cellular tropism of SARS-CoV-2, and the pathogenesis that leads to multi-organ failure and death, has prompted an unprecedented adoption of organoid models. Successful drug discovery and vaccine development rely on pre-clinical models that faithfully recapitulate the viral life cycle and the host cell response to infection. Human stem cell-derived organoids fulfill these criteria. Here we highlight the role of organoids in the study of SARS-CoV-2 infection and modelling of COVID-19 pathogenesis.

Organoids doi: 10.3390/organoids1010001

Authors: Constanze Schelhorn

Organoids are stem cell-derived 3D multicellular tissue constructs that mimic some of the structures and functions of a corresponding organ [...]