Organoids

The myocardium possesses one of the highest vascular densities in the body. The outermost wall layer of large and medium-sized vessels, the adventitia, forms a critical interface between the vasculature and the myocardium and serves as a reservoir for stem and progenitor cells capable of differentiating into all vascular wall lineages as well as innate immune cells, including macrophages. Current cardiac organoid models intrinsically develop networks of endothelial cords and small capillary-like structures that resemble cardiac microvessels. However, these microvessels mostly lack an adventitial compartment in vivo. Here, we present a potential alternative assembloid strategy that combines vascular segments from mouse and human origin with either cardiomyocytes or cardiac spheroids derived from human induced pluripotent stem cells, thereby incorporating large diameter vessels and the vascular adventitia into a cardiac tissue model. Within the assembloids, the myocardial component remained contractile and connected to the vascular adventitia, which displayed cellular sprouting toward the hiPSC-derived cardiac tissue. Immunostaining for vascular and immune markers revealed that the adventitia gave rise to endothelial sprouts and macrophage-like cells which integrated into the myocardial tissue. In summary, we present proof of concept for complex assembloids composed of vessel segments and human iPSC-derived cardiomyocytes which contain and maintain an in vivo-like adventitial compartment. We suggest this model may serve as a platform for investigating myocardial–stromal interactions, cardiac tissue repair, and functional remodeling under both physiological and pathological conditions. Furthermore, the incorporation of large-lumen vessel segments may enable future experimental perfusion, rendering the model particularly suitable for drug testing via intravascular delivery. Full article

The intracellular accumulation of amyloid beta 42 (iAβ42) has been proposed as an early pathological indicator of familial Alzheimer’s disease (FAD). DJ-1 is a multifunctional protein sensitive to oxidative stress (OS) that has been associated with neurodegeneration; however, its role in iAβ42 pathology is unclear. In this study, we examined whether oxidized (sulfonic) DJ-1 (Cys¹⁰⁶-SO₃H) drives iAβ42 accumulation using postmortem brain samples and in vitro 3D iPSC-derived cerebral organoids (COs) or 2D induced pluripotent stem cells (iPSC)-derived ChLNs (cholinergic-like neurons) models from a PSEN1 E280A patient and a healthy volunteer (as a control sample). Post-mortem analyses of the temporal and frontal cortices and hippocampus from FAD PSEN1 E280A patients revealed strong intracellular co-localization of sulfonic DJ-1 and iAβ42, which was absent in control samples. To validate these findings, we generated COs from an iPSC PSEN1 E280A FAD patient and a healthy donor. In these organoids, we observed the co-localization of oxidized DJ-1 and Aβ42 in the absence of extracellular fibrils or plaques, as confirmed by BTA-1 staining. To further support these observations, 2D iPSC PSEN1 E280A-derived ChLNs cultures showed that intracellular Aβ42 accumulates progressively in direct correlation with increasing DJ-1 oxidation, as demonstrated by immunofluorescence microscopy and Western blotting analysis. These results indicate that DJ-1 oxidation accompanies the earliest intracellular stages of Aβ42 pathology. Furthermore, complementary in silico molecular docking analysis revealed a higher affinity between Aβ42 and oxidized sulfonic DJ-1 (DJ-1 Cys¹⁰⁶-SO₃H) compared to sulfenic (DJ-1 Cys¹⁰⁶-SOH) or sulfinic acid (DJ-1 Cys¹⁰⁶-SO₂H) forms. Likewise, ELISA tests and seeding assays confirmed that oxidized DJ-1 binds to and decelerates Aβ42 aggregation kinetics. Together, our results identify DJ-1 oxidation as a critical molecular event in the accumulation of iAβ42 in FAD. These findings suggest that oxidized DJ-1 represents not only a potential early biomarker of intracellular pathology but also a pharmacological target. Preventing the oxidation of DJ-1 or its pathological aggregation could provide new biomarkers and therapeutic strategies for reducing the intracellular accumulation of Aβ42 and neurodegeneration in FAD. Full article

Tumor heterogeneity and microenvironmental complexity remain fundamental barriers to genomics-centered precision oncology, frequently causing discordance between molecular alterations and real-world therapeutic responses. Here, we reviewed patient-derived organoid (PDO) technologies as functional platforms that complement molecular profiling by directly investigating patient-specific sensitivity, resistance, and microenvironment dependent vulnerability. We first summarize why conventional preclinical systems, two-dimensional cell lines and patient-derived xenografts, are limited by reduced biological fidelity, impractical turnaround time, and scalability for clinical decision support. We then synthesized organoid-based evidence across three representative disease malignancies with distinct precision-medicine bottlenecks. Across these settings, we highlight advances that extend the PDO capability beyond the tumor epithelium alone, including air–liquid interface cultures, immune and stromal co-cultures, and microfluidic organoid-on-chip systems, as well as integration with multi-omics and artificial intelligence for scalable analytics. Finally, we discuss the key translational requirements, standardization of culture matrices and assay readouts, quality control, automation to reduce turnaround time, and regulatory/ethical frameworks, required to transition organoid-guided testing from proof-of-concept to routine implementation. Collectively, this review reframes organoids as functional stratification platforms supporting the integration of functional response profiling alongside genomics-guided precision oncology approaches. Full article

Patient-derived organoids (PDOs) have rapidly transitioned from research tools into promising platforms for clinical translation. In this review, we analyze 139 PDO-related clinical trials registered between 2023 and 2025 and contrast them with recent advances in disease modelling. Our analysis revealed a predominance of oncology-focused studies, with translational maturity spanning from foundational research to studies in which PDOs directly informed clinical decision-making. In contrast, non-oncology areas show extensive preclinical progress but remain trial-poor. We found that trial registration is geographically concentrated in a small number of countries, reflecting uneven global adoption. We then explored advances in disease modeling, mainly confined to preclinical studies, including immune-competent PDOs, complex organ-on-a-chip systems, synthetic matrices, AI-enabled platforms, and therapeutic transplantation. Based on these findings, we propose a conceptual framework outlining the trajectory of PDO adoption in clinical trials. This trajectory can be understood as three overlapping waves of translation: the first wave, focusing on oncology, has already demonstrated impacts on patient care; the second, targeting non-oncology diseases, is scientifically advanced but has not achieved widespread clinical application; and the third, involving frontier technologies, remains in the preclinical stage. Understanding these trajectories underscores the promise and challenges of PDOs that must be addressed for broader clinical adoption. Full article

Treatment-refractory rectal cancer liver metastasis represents a major therapeutic challenge, particularly in the absence of actionable genomic biomarkers. Functional precision oncology approaches integrating genomic profiling with patient-derived organoid (PDO) drug testing may provide biologically informed therapeutic prioritization. A 50-year-old female patient with KRAS/TP53-mutant, microsatellite-stable (MSS) rectal adenocarcinoma refractory to FOLFIRINOX was enrolled. A liver metastasis from a treatment-refractory rectal cancer patient was processed to establish three-dimensional patient-derived organoids. Histopathological concordance was assessed using H&E and p53 immunohistochemistry. Comprehensive genomic profiling was performed using a 637-gene targeted next-generation sequencing panel, enabling detection of single-nucleotide variants, indels, copy number variations, microsatellite instability, and tumor mutational burden. Functional drug sensitivity profiling was conducted in parallel 2D and 3D platforms using a customized 17-agent panel, followed by exploratory combinatorial validation. The organoids demonstrated high phenotypic and genomic concordance with the parental tumor, preserving key driver alterations (KRAS^A146T, TP53^R175H, APC frameshifts, CCNE1 amplification), microsatellite stability, and low tumor mutational burden (TMB: 6.37 mut/Mb). Functional screening identified selective sensitivity to bevacizumab (IC₅₀: 0.130 μM), doxorubicin (IC₅₀: 0.570 μM), carboplatin (IC₅₀: 0.950 μM), and topotecan (IC₅₀: 1.600 μM) in the 3D organoid model, with consistent cross-platform validation. An exploratory combination assay further supported enhanced viability suppression under bevacizumab-based regimens. Critically, at the time of manuscript preparation, the patient demonstrated radiological disease stabilization under bevacizumab plus trastuzumab deruxtecan, consistent with the organoid-derived response profile. These findings highlight the capacity of integrated genomic and organoid-based profiling to uncover therapeutic vulnerabilities beyond standard biomarker assessment. This proof-of-concept case report study demonstrates the feasibility and translational relevance of an established organoid-based functional precision oncology platform for therapeutic prioritization in metastatic rectal cancer. Full article

Inherited retinal dystrophies (IRDs) comprise a diverse group of genetic disorders that frequently result in irreversible vision loss due to photoreceptor dysfunction or degeneration. Among them, retinitis pigmentosa (RP) and achromatopsia (ACHM) are, in some cases, associated with pathogenic variants in PDE6A and PDE6C, respectively, which are key components of the phototransduction cascade. As most of IRDs still lack effective therapies, retinal organoids (ROs) provide a valuable in vitro model for the investigation of disease-associated mechanisms. Here, we generated induced pluripotent stem cell (iPSC)-derived ROs from an RP patient carrying compound heterozygous PDE6A mutations and from a patient with ACHM harboring a homozygous PDE6C mutation, along with their corresponding CRISPR/Cas9-corrected isogenic controls, which, to our knowledge, represent the first patient-derived RO models reported for the PDE6A and PDE6C genes. The mutant PDE6A line exhibited impaired neuroretinal vesicle formation and RO differentiation; however, a subset of RP-derived ROs matured appropriately and retained photoreceptor features. Moreover, the specific isoform expression pattern detected in retinal tissues reflected differences across developmental maturation stages that could influence disease severity. In contrast, the PDE6C_mutant ROs displayed normal structure and maturation, although cGMP hydrolysis within photoreceptors was likely compromised. In both models, CRISPR/Cas9-mediated correction restored the disease-associated phenotype resembling wild-type ROs. Collectively, these findings provide new insights into PDE6-associated pathogenesis, underscore the utility of patient-specific and gene-corrected ROs for elucidating IRD mechanisms, and support gene editing as a promising therapeutic strategy. Full article

Advanced Glycation End Products (AGEs) are reactive compounds formed through the non-enzymatic glycation of proteins, lipids, or nucleic acids due to exposure to reducing sugars. They accumulate through endogenous metabolic dysregulation and exogenous dietary intake, particularly high-fat and high-sugar foods prepared at high temperatures. The interaction between AGEs and their receptor, RAGE (receptor for Advanced Glycation End Products), has been implicated in a range of pathological conditions, including diabetes and metabolic syndrome. However, the impact of AGEs accumulation on neurodevelopment remains poorly understood. In this study, we investigated the effects of AGEs on human-induced pluripotent stem cell (iPSC)-derived cerebral organoids comprising neurons, astrocytes, and microglia. Our findings reveal that AGEs induce RAGE expression, leading to microglial activation, increased deposition of amyloid-beta (Aβ) aggregates, and impaired neurodevelopment. Additionally, elevated levels of AGE-modified proteins, along with altered microglial polarization, were observed in cerebral organoids modeling Western Pacific Amyotrophic Lateral Sclerosis and Parkinsonism–Dementia Complex (ALS-PDC). These findings demonstrate AGEs as active drivers of neurodevelopmental disruption and establish a mechanistic link between metabolic stress and increased susceptibility to neurodegenerative disease. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article

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. Full article