Organoids, Volume 5, Issue 1 (March 2026) – 9 articles

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