Abstract Organoids have revolutionized the field of biomedical research by allowing the recapitulation of organ architecture in three dimensions. Though biochemical factors have long played an established role in organoid culture, it is presently recognized that mechanical forces are equally indispensable in the self-organization of tissues and the determination of stem cell and disease phenotypes. Here, this review integrates the biophysics of cellular mechanotransduction with the biology of organoid systems, examining how forces encoded in extracellular matrix stiffness, viscoelasticity, fluid shear, cyclic stretch, and geometric confinement are sensed and transduced by a specialized molecular machinery—including integrin–focal adhesion kinase (FAK) complexes, mechanosensitive Piezo1/2 ion channels, and the linker of nucleoskeleton and cytoskeleton (LINC) complex—to ultimately regulate Yes-associated protein/transcriptional coactivator with PDZ-binding motif (YAP/TAZ)-mediated transcriptional programs. How engineered hydrogels with tunable mechanical properties, microfluidic organ-on-chip platforms, and bioreactor systems have enabled in-depth exploration of these forces in gut, brain, cardiac, and kidney organoids will be well discussed. Emerging work highlights Piezo channels as critical gatekeepers of intestinal stem cell (ISC) fate, extracellular matrix (ECM) stiffness as a driver of aberrant gyrification in lissencephaly brain organoids, and viscoelastic stress relaxation as a key determinant of neural progenitor maturation. Organoid mechanobiology is set to revolutionize human disease modeling and regenerative medicine with its convergence with patient-derived models, computational mechanics, and Good Manufacturing Practice (GMP)-compatible bioengineering platforms.
Abstract The modernization of traditional Chinese medicine (TCM) confronts a persistent bottleneck: interpreting its dynamic, holistic “formula–human body” system in the language of modern science. The multi-component, multi-target pharmacology of TCM compound formulas resists reductionist analysis, and conventional experimental models fall short of capturing the systemic regulatory effects that underlie clinical efficacy. Recent advances in artificial intelligence (AI) and organoid technology now provide complementary tools to address this long-standing challenge. AI supplies the computational power to model complex, high-dimensional biological networks; organoids furnish biologically faithful platforms to validate predictions in human-relevant contexts. Together, these technologies enable a closed-loop research paradigm—computational prediction, experimental verification, model iteration—that is high-throughput, quantifiable, and predictive. This Comment argues that the convergence of AI and organoids can drive TCM research from an experience-driven tradition toward a data-driven, mechanism-verified framework, bridging millennia of accumulated clinical wisdom with contemporary biomedical science.
Patient-derived lung cancer organoid provides a valuable platform for various applications. Histopathological examination of organoids is crucial to verify their consistency with the original tissue. Traditional methods require collecting multiple Matrigel domes and dissociating organoids from the matrix before histopathology. In this study, we report a fast and convenient method for making a paraffin block using only one Matrigel droplet. Organoids cultured in a 24-well plate were fixed with formalin in situ for 72 hours, pre-embedded in 3% agarose to block the Matrigel dome, and processed through routine dehydration, embedding, sectioning, and staining. We compared this method with others, including traditional small specimen paper wrapping and collecting dissociated organoids in EP tubes before embedding. Using only a single droplet, this approach yields a large number of organoids per section. Our method eliminates the need for Matrigel dissociation, effectively reduces organoids loss during fixation and paraffin embedding processes. In lung cancer organoids, tumor markers were consistent with the corresponding original tumor tissues. This novel protocol allows in-situ processing of patient-derived lung cancer organoids from a single Matrigel dome. It significantly reduces culture costs and time and is particularly useful for the histological examination of early-stage organoids.
Colorectal cancer (CRC), a malignancy originating in the colon or rectum, has seen significant progress in treatment outcomes due to advances in therapeutic strategies. Organoids, as a cutting-edge research tool, have emerged as a transformative platform for understanding CRC mechanisms and optimizing personalized treatment approaches. We systematically review the traditional and new methodologies for establishing colorectal cancer organoids and the co-culture systems. Furthermore, we explore the utility of colorectal cancer organoids in drug screening, therapeutic development, and fundamental cancer research. While the integration of organoid technology into clinical practice holds great promise for personalized therapy, several technical and translational challenges remain to be addressed. The continued refinement and application of this technology are expected to deepen our understanding of CRC biology and accelerate the development of targeted therapeutic strategies, ultimately reshaping the landscape of cancer treatment.
The Transforming Growth Factor-beta (TGF-u03B2) signaling pathway is a master regulator of cellular processes, including proliferation, differentiation and apoptosis, with profound implications for tissue homeostasis and disease pathogenesis. This review delves into the mechanisms and applications of TGF-u03B2 signaling within organoids, three-dimensional (3D) cultures that mimic human tissue architecture and function. We systematically explore the multifaceted role of TGF-u03B2 in organoid initiation, growth, and differentiation, highlighting its influence on stem cell self-renewal and lineage specification. The interplay between TGF-u03B2 and other signaling pathways, such as Wnt/u03B2-catenin and Notch, is critical for maintaining tissue homeostasis and regulating stem cell niches. Furthermore, we discuss the significance of TGF-u03B2 signaling in modeling diseases like cancer and fibrosis, where its dysregulation is often implicated. Modulating TGF-u03B2 signaling in organoids holds vast therapeutic potential, offering insights into disease pathogenesis, predicting drug responses, and developing personalized treatment strategies. As our understanding of TGF-u03B2 signaling and organoid technology advances, these systems hold promise for groundbreaking biomedical research, potentially revolutionizing regenerative medicine and personalized therapeutics.
Human adult hepatocytes are essential for metabolic homeostasis but remain challenging to expand in vitro without losing functional maturity or acquiring ductal traits. In a recent landmark study published in Nature, Igarashi et al. describe the generation of human adult hepatocyte organoids (HHOs) that maintain long-term proliferative capacity while recapitulating key metabolic functions and spatial zonation. By harnessing Wnt and STAT3 signaling, the authors overcame the trade-off between cell replication and hepatic identity, creating organoids capable of sustained growth, functional maturation, and genetic manipulation. These HHOs represent a transformative platform for modeling metabolic liver diseases, drug toxicity, and regenerative therapies, bridging the gap between in vitro scalability and in vivo physiological relevance.
In recent years, the study of complex cellular interactions has been hampered by the limitations of traditional two-dimensional (2D) and single-cell type culture systems, which fail to accurately mimic the intricate dynamics of human tissues. To bridge this gap, assembloid technology has emerged as a transformative approach. Assembloids are self-organizing three-dimensional (3D) systems formed by integrating multiple organoids or cell types, providing a more accurate model for studying inter-tissue and inter-organ communication. Here, we categorize current assembloids into four types based on assembly strategiesu2014multi-region, multi-lineage, multi-gradient, and multi-layeru2014each designed to replicate specific biological phenomena with high fidelity. We also explore the diverse applications of assembloids across various human systems, demonstrating the broad application scope of assembloids. Finally, we highlight the challenges faced by assembloid technology and outline its future prospects. Overall, assembloids represent a powerful platform for advancing research in developmental biology, disease modeling, and drug discovery.
Patient-derived organoids (PDOs) are revolutionizing cancer research, serving as invaluable models for tumor biology and therapeutic screening. The fidelity and applicability of these organoids are fundamentally shaped by the tissue dissociation techniques employed, namely mechanical dissociation and enzymatic digestion. This comprehensive review delves into the nuances of these two methods, scrutinizing their effects on solid tumor organoid properties, including stemness, heterogeneity, long-term culturing. We discuss the advantages and limitations of each technique, with a focus on their impact on tumor microenvironment preservation, their application in drug screening and cancer modeling. Moreover, we examine how recent technological breakthroughs have bolstered the efficiency and scalability of organoid production through these methods. Our analysis is designed to assist researchers in choosing the optimal tissue dissociation strategy for their research objectives and to fuel the evolution of organoid-based cancer models.
Intrahepatic cholangiocarcinoma (ICC) is a highly lethal malignancy associated with significant morbidity, necessitating the urgent development of an effective chemotherapeutic assay for ICC patients. In this study, we have successfully established an advanced culture method for ICC organoids that can be utilized with both single-cell assembly and tissue fragmentation initiation techniques. These ICC organoids maintain the morphological characteristics, including mutation profiles and frequency (46.9% in organoid and 48.5% in tumor tissue) of IDH1 genes, and 1733 high-frequent overlapped mutated genes (94.2%). Additionally, ICC biomarkers such as CK7 and CK19 also maintain a similar pattern compared with the original tissue. Furthermore, RNA-seq analysis reveals upregulation of immune-related genes in single-cell assembly organoids. The significantly changed genes including IL9R (4.4-fold), IL2RB (3.2-fold), CCR4 (3.5-fold), TESPA1 (4.4-fold), ZAP70 (4.3-fold) and CD6 (4.3-fold) in log scale. These evidence both indicating the presence of viable and active immune cells. Overall, our findings present an advanced and user-friendly culture approach for generating ICC organoids adaptable to diverse experimental objectives.
Glioblastoma (GBM) is a highly aggressive brain tumor characterized by rapid growth and high heterogeneity, posing challenges for fundamental research and personalized drug screening due to the lack of suitable models. GBM organoids serve as an innovative research tool, providing a valuable model for studying the biological characteristics of GBM. In this study, we successfully generated 4 GBM organoids and employed enzymatic digestion and mechanical fragmentation techniques for subsequent cultivation. Through continuous observation, pathological assessment, and RNA sequencing (RNA-seq), we observed that all the organoids generated through both methods demonstrated good growth characteristics. The organoids derived from mechanical fragmentation not only achieved a two-dimensional (2D) area of ~ 1.5 mm2 but also exhibited distinct vascular structures. The organoids derived from enzymatic digestion achieved a 2D area of approximately 0.8 mm2. Furthermore, RNA-seq analysis has revealed that organoids cultured using two distinct methods exhibit a heterogeneous cellular composition, comprising a total of 20 cell types (endothelial, immune cells ...). Our studies show that both methods successfully maintained the essential characteristics of GBM, encompassing its distinctive tissue structure and gene expression patterns. Each method exhibits its own attributes, contributing to the understanding of GBM organoids.
Ovarian cancer, a common gynecologic tumor, is associated with a high mortality, due to challenges in early detection within the reproductive system. According to our previous research, cultivating patient-specific organoids from mechanically sheared tissues can be utilized for drug response evaluation but has limitations for high-throughput screening efficiency due to their inconsistent size. In this research, we focused on organoids developed from single-cell suspensions to address the critical requirement for uniformity in organoid size. By the day 3 of culture, single-cell suspensions rapidly and spontaneously aggregated into spherical structures with a more consistent size. Notably, the organoids of sample OVA-37 were ten times larger after 8 days of culture. Transcriptomic analysis was used to compare the two organoid culture techniques, demonstrating that the variations between different organoid culture methods were minimal, with higher variability observed among patients. Gene set enrichment analysis (GSEA) revealed only minor discrepancies in specific pathways, such as TGF-u03B2 and tight junctions. Furthermore, treatment with carboplatin in a 96-well plate setup resulted in reproducible drug responses, as evidenced by coefficients of variation lower than 40%. This finding suggests that single-cell suspension-cultured organoids can be employed for reproducible high-throughput drug screening. This approach holds potential for personalized drug screening in ovarian cancer and may contribute to the development of novel therapeutic strategies.
