Organoid Research Research Articles

AI-organoid integrated systems for biomedical studies and applications.

In this review, we explore the growing role of artificial intelligence (AI) in advancing the biomedical applications of human pluripotent stem cell (hPSC)-derived organoids. Stem cell-derived organoids, these miniature organ replicas, have become essential tools for disease modeling, drug discovery, and regenerative medicine. However, analyzing the vast and intricate datasets generated from these organoids can be inefficient and error-prone. AI techniques offer a promising solution to efficiently extract insights and make predictions from diverse data types generated from microscopy images, transcriptomics, metabolomics, and proteomics. This review offers a brief overview of organoid characterization and fundamental concepts in AI while focusing on a comprehensive exploration of AI applications in organoid-based disease modeling and drug evaluation. It provides insights into the future possibilities of AI in enhancing the quality control of organoid fabrication, label-free organoid recognition, and three-dimensional image reconstruction of complex organoid structures. This review presents the challenges and potential solutions in AI-organoid integration, focusing on the establishment of reliable AI model decision-making processes and the standardization of organoid research.

3D Cell Culture Method in Channel-Free Water-in-Oil Droplets.

A new channel-free water-in-oil (WO) droplet 3D cell culture method is proposed to address the challenges while maintaining the advantages of the conventional 3D cell culture methods. The proposed WO method can fundamentally solve the constraint of spheroids size, a common challenge in conventional 3D culture, by using droplet size controllability. The 3D cell culture performance of the WO method is verified by comparing it with the conventional 3D cell culture methods. A systematic investigation of the culture conditions of the WO method confirms the working range of cell concentration and droplet size, as well as the scalability of spheroid size. Adjusting droplet size and cell concentration enables rapid spheroid formation with large and high cell concentration droplets or fast spheroid growth with small and low cell concentration droplets, providing control over the spheroid size and growth rate according to the purpose. Furthermore, long-term culture is demonstrated for 1 month with the proposed method, showing the largest spheroid culture and demonstrating the possibility that this method can be used not only for spheroid formation but also for organoid studies. Finally, if a WO-based automated 3D cell culture system is developed, it will be a useful tool for organoid research.

Gelatin-Based Hybrid Hydrogels as Matrices for Organoid Culture.

The application of liver organoids is very promising in the field of liver tissue engineering; however, it is still facing some limitations. One of the current major limitations is the matrix in which they are cultured. The mainly undefined and murine-originated tumor matrices derived from Engelbreth-Holm-Swarm (EHS) sarcoma, such as Matrigel, are still the standard culturing matrices for expansion and differentiation of organoids toward hepatocyte-like cells, which will obstruct its future clinical application potential. In this study, we exploited the use of newly developed highly defined hydrogels as potential matrices for the culture of liver organoids and compared them to Matrigel and two hydrogels that were already researched in the field of organoid research [i.e., polyisocyanopeptides, enriched with laminin-entactin complex (PIC-LEC) and gelatin methacryloyl (GelMA)]. The newly developed hydrogels are materials that have a physicochemical resemblance with native liver tissue. Norbornene-modified dextran cross-linked with thiolated gelatin (DexNB-GelSH) has a swelling ratio and macro- and microscale properties that highly mimic liver tissue. Norbornene-modified chondroitin sulfate cross-linked with thiolated gelatin (CSNB-GelSH) contains chondroitin sulfate, which is a glycosaminoglycan (GAG) that is present in the liver ECM. Furthermore, CSNB-GelSH hydrogels with different mechanical properties were evaluated. Bipotent intrahepatic cholangiocyte organoids (ICOs) were applied in this work and encapsulated in these materials. This research revealed that the newly developed materials outperformed Matrigel, PIC-LEC, and GelMA in the differentiation of ICOs toward hepatocyte-like cells. Furthermore, some trends indicate that an interplay of both the chemical composition and the mechanical properties has an influence on the relative expression of certain hepatocyte markers. Both DexNB-GelSH and CSNB-GelSH showed promising results for the expansion and differentiation of intrahepatic cholangiocyte organoids. The stiffest CSNB-GelSH hydrogel even significantly outperformed Matrigel based on ALB, BSEP, and CYP3A4 gene expression, being three important hepatocyte markers.

Advancing Organoid Engineering for Tissue Regeneration and Biofunctional Reconstruction.

Tissue damage and functional abnormalities in organs have become a considerable clinical challenge. Organoids are often applied as disease models and in drug discovery and screening. Indeed, several studies have shown that organoids are an important strategy for achieving tissue repair and biofunction reconstruction. In contrast to established stem cell therapies, organoids have high clinical relevance. However, conventional approaches have limited the application of organoids in clinical regenerative medicine. Engineered organoids might have the capacity to overcome these challenges. Bioengineering-a multidisciplinary field that applies engineering principles to biomedicine-has bridged the gap between engineering and medicine to promote human health. More specifically, bioengineering principles have been applied to organoids to accelerate their clinical translation. In this review, beginning with the basic concepts of organoids, we describe strategies for cultivating engineered organoids and discuss the multiple engineering modes to create conditions for breakthroughs in organoid research. Subsequently, studies on the application of engineered organoids in biofunction reconstruction and tissue repair are presented. Finally, we highlight the limitations and challenges hindering the utilization of engineered organoids in clinical applications. Future research will focus on cultivating engineered organoids using advanced bioengineering tools for personalized tissue repair and biofunction reconstruction.

Interpretation of the past, present, and future of organoid technology: an updated bibliometric analysis from 2009 to 2024.

Organoid technology has been developed rapidly in the past decade, which involves the exploration of the mechanism of development, regeneration and various diseases, and intersects among multiple disciplines. Thousands of literature on 3D-culture or organoids have been published in the research areas of cell biology tissue engineering, nanoscience, oncology and so on, resulting in it being challenging for researchers to timely summarize these studies. Bibliometric statistics is a helpful way to help researchers clarify the above issues efficiently and manage the whole landscape systematically. In our study, all original articles on organoids were included in the Web of Science database from January 2009 to May 2024, and related information was collected and analyzed using Excel software, "bibliometrix" packages of the R software, VOSviewer and CiteSpace. As results, a total of 6222 papers were included to classify the status quo of the organoids and predict future research areas. Our findings highlight a growing trend in publications related to organoids, with the United States and Netherlands leading in this field. The University of California System, Harvard University, Utrecht University and Utrecht University Medical Center have emerged as pivotal contributors and the key authors in the field include Clevers, H, Beekman, JM and Spence JR. Our results also revealed that the research hotspots and trends of organoids mainly focused on clinical treatment, drug screening, and the application of materials and technologies such as "hydrogel" and "microfluidic technology" in organoids. Next, we had an in-depth interpretation of the development process of organoid research area, including the emergence of technology, the translation from bench to bedsides, the profiles of the most widely studied types of organoids, the application of materials and technologies, and the emerging organoid-immune co-cultures trends. Furthermore, we also discussed the pitfalls, challenges and prospects of organoid technology. In conclusion, this study provides readers straightforward and convenient access to the organoid research field.

Rapid and efficient generation of mature retinal organoids derived from human pluripotent stem cells via optimized pharmacological modulation of Sonic hedgehog, activin A, and retinoic acid signal transduction.

Human retinal organoids have become indispensable tools for retinal disease modeling and drug screening. Despite its versatile applications, the long timeframe for their differentiation and maturation limits the throughput of such research. Here, we successfully shortened this timeframe by accelerating human retinal organoid development using unique pharmacological approaches. Our method comprised three key steps: 1) a modified self-formed ectodermal autonomous multizone (SEAM) method, including dual SMAD inhibition and bone morphogenetic protein 4 treatment, for initial neural retinal induction; 2) the concurrent use of a Sonic hedgehog agonist SAG, activin A, and all-trans retinoic acid for rapid retinal cell specification; and 3) switching to SAG treatment alone for robust retinal maturation and lamination. The generated retinal organoids preserved typical morphological features of mature retinal organoids, including hair-like surface structures and well-organized outer layers. These features were substantiated by the spatial immunostaining patterns of several retinal cell markers, including rhodopsin and L/M opsin expression in the outermost layer, which was accompanied by reduced ectopic cone photoreceptor generation. Importantly, our method required only 90 days for retinal organoid maturation, which is approximately two-thirds the time necessary for other conventional methods. These results indicate that thoroughly optimized pharmacological interventions play a pivotal role in rapid and precise photoreceptor development during human retinal organoid differentiation and maturation. Thus, our present method may expedite human retinal organoid research, eventually contributing to the development of better treatment options for various degenerative retinal diseases.

Development of Organoids to Study Infectious Host Interactions.

Emerging organoid research is paving way for studies in infectious diseases. Described here is a technique for the generation of stem-cell derived organoids for human small intestine and lung together with methods to infect such organoids with a mock pathogen (Cryptosporidium parvum). Such systems are amenable to imaging and processing for molecular biological analyses. It is the intent of this chapter to provide a simple, routine organoid procedure so that in vitro studies with Borrelia such as cell invasion and dissemination can be conducted.

Deciphering the Tumor Microenvironment of Colorectal Cancer and Guiding Clinical Treatment With Patient-Derived Organoid Technology: Progress and Challenges.

Colorectal cancer (CRC) is one of the most prevalent malignant tumors of the digestive tract worldwide. Despite notable advancements in CRC treatment, there is an urgent requirement for preclinical model systems capable of accurately predicting drug efficacy in CRC patients, to identify more effective therapeutic options. In recent years, substantial strides have been made in the field of organoid technology, patient-derived organoid models can phenotypically replicate the original intra-tumor and inter-tumor heterogeneity of CRC, reflecting cellular interactions of the tumor microenvironment. Patient-derived organoid models have become an indispensable tool for investigating the pathogenesis of CRC and facilitating translational research. This review focuses on the application of organoid technology in CRC modeling, tumor microenvironment, and guiding clinical treatment, particularly in drug screening and personalized medicine. It also examines the existing challenges encountered in clinical organoid research and provides a prospective outlook on the future development directions of clinical organoid research, encompassing the standardization of organoid culture technology and the application of tissue engineering technology.

Standardization and quality assessment for human intestinal organoids.

To enhance the practical application of intestinal organoids, it is imperative to establish standardized guidelines. This proposed standardization outlines a comprehensive framework to ensure consistency and reliability in the development, characterization, and application of intestinal organoids. The recommended guidelines encompass crucial parameters, including culture conditions, critical quality attributes, quality control measures, and functional assessments, aimed at fostering a standardized approach across diverse research initiatives. The implementation of these guidelines is anticipated to significantly contribute to the reproducibility and comparability of results in the burgeoning field of intestinal organoid research.

Advances in Microfluidic Technologies in Organoid Research.

Organoids have emerged as major technological breakthroughs and novel organ models that have revolutionized biomedical research by recapitulating the key structural and functional complexities of their in vivo counterparts. The combination of organoid systems and microfluidic technologies has opened new frontiers in organoid engineering and offers great opportunities to address the current challenges of existing organoid systems and broaden their biomedical applications. In this review, the key features of the existing organoids, including their origins, development, design principles, and limitations, are described. Then the recent progress in integrating organoids into microfluidic systems is highlighted, involving microarrays for high-throughput organoid manipulation, microreactors for organoid hydrogel scaffold fabrication, and microfluidic chips for functional organoid culture. The opportunities in the nascent combination of organoids and microfluidics that lie ahead to accelerate research in organ development, disease studies, drug screening, and regenerative medicine are also discussed. Finally, the challenges and future perspectives in the development of advanced microfluidic platforms and modified technologies for building organoids with higher fidelity and standardization are envisioned.

Industry News (Jul 2023 to Dec 2023)

This article provides an insightful overview of recent advancements and significant events in organoid research and technology. It covers the transition of HUB Organoids to a private company, the inaugural meeting of the International Society of Organoid Research in Singapore, and developments in organoid group standards at Tsinghua Changgeng Hospital. Additionally, it highlights breakthroughs in automated culture technology, major acquisitions such as Roche's purchase of Herophilus, and substantial funding rounds like Seraxis's $50 million for diabetes organoid therapy, demonstrating the dynamic growth and potential of this field.

Human brain organoid: trends, evolution, and remaining challenges.

Advanced brain organoids provide promising platforms for deciphering the cellular and molecular processes of human neural development and diseases. Although various studies and reviews have described developments and advancements in brain organoids, few studies have comprehensively summarized and analyzed the global trends in this area of neuroscience. To identify and further facilitate the development of cerebral organoids, we utilized bibliometrics and visualization methods to analyze the global trends and evolution of brain organoids in the last 10 years. First, annual publications, countries/regions, organizations, journals, authors, co-citations, and keywords relating to brain organoids were identified. The hotspots in this field were also systematically identified. Subsequently, current applications for brain organoids in neuroscience, including human neural development, neural disorders, infectious diseases, regenerative medicine, drug discovery, and toxicity assessment studies, are comprehensively discussed. Towards that end, several considerations regarding the current challenges in brain organoid research and future strategies to advance neuroscience will be presented to further promote their application in neurological research.

Lipidomic Phenotyping Of Human Small Intestinal Organoids Using Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging.

In the past decade, interest in organoids for biomedical research has surged, resulting in a higher demand for advanced imaging techniques. Traditional specimen embedding methods pose challenges, such as analyte delocalization and histological assessment. Here, we present an optimized sample preparation approach utilizing an Epredia M-1 cellulose-based embedding matrix, which preserves the structural integrity of fragile small intestinal organoids (SIOs). Additionally, background interference (delocalization of analytes, nonspecific (histological) staining, matrix ion clusters) was minimized, and we demonstrate the compatibility with matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI). With our approach, we can conduct label-free lipid imaging at the single-cell level, thereby yielding insights into the spatial distribution of lipids in both positive and negative ion modes. Moreover, M-1 embedding allows for an improved coregistration with histological and immunohistochemical (IHC) stainings, including MALDI-IHC, facilitating combined untargeted and targeted spatial information. Applying this approach, we successfully phenotyped crypt-like (CL) and villus-like (VL) SIOs, revealing that PE 36:2 [M - H]- (m/z 742.5) and PI 38:4 [M - H]- (m/z 885.5) display higher abundance in CL organoids, whereas PI 36:1 [M - H]- (m/z 863.6) was more prevalent in VL organoids. Our findings demonstrate the utility of M-1 embedding for advancing organoid research and unraveling intricate biological processes within these in vitro models.

OctoShaker: A versatile robotic biomechanical agitator for cellular and organoid research.

Mechanical forces have increasingly beenrecognized as a key regulator in the fate of cellular development and functionality. Different mechanical transduction methods, such as substrate stiffness and magnetic bead vibration, have been experimented with to understand the interaction between the biophysical cues and cellular outcome. In the exploration and utilization of the intrinsic cellular mechanism, bio-shakers, traditionally invented for stirring liquid, have garnered more interest as a tool to provide precise mechanical stimuli to aid in this study. Nonetheless, despite the usefulness of current bio-shaking technology, each type of shaker often offers a single mode of motion, insufficient for generating complex force dynamics needed to resemble the actual physical condition that occurs inside living organisms. In this study, we present OctoShaker, a robotic instrument capable of creating a multitude of motions that could be sequenced or programmed to mimic sophisticated hemodynamics in vivo. We demonstrated the programmed motion of circular convection and investigated its influence on micro-particle distribution in 96-well culture microplates. Biological samples, including HeLa cells and organoids, were tested, and unique resultant patterns were observed. We anticipate the open-source dissemination of OctoShaker in diverse biological applications, encompassing biomechanical studies for cellular and organoid research, as well as other disciplines that demand dynamic mechanical force generation.

Adult Bovine-Derived Small and Large Intestinal Organoids: In Vitro Development and Maintenance.

Recent progress in bovine intestinal organoid research has expanded opportunities for creating improved in vitro models to study intestinal physiology and pathology. However, the establishment of a culture condition capable of generating organoids from all segments of the cattle intestine has remained elusive. Although previous research has described the development of bovine jejunal, ileal, and colonic organoids, this study marks the first report of successful bovine duodenal and rectal organoid development. Maintenance of these organoids through serial passages and cryopreservation was achieved, with higher success rates observed in large intestinal organoids compared to their small intestinal counterparts. A novel approach involving the use of biopsy forceps during initial tissue sampling streamlined the subsequent tissue processing, simplifying the procedure compared to previously established protocols in cattle. Additionally, our study introduced a more cost-effective culture medium based on Advanced DMEM/F12, diverging from frequently used commercially available organoid culture media. This enhancement improves accessibility to organoid technology by reducing culture costs. Crucially, the derived organoids from jejunum, ileum, colon and rectum faithfully preserved the structural, cellular, and genetic characteristics of in vivo intestinal tissue. This research underscores the significant potential of adult bovine intestinal organoids as a physiologically and morphologically relevant in vitro model. Such organoids provide a renewable and sustainable resource for a broad spectrum of studies, encompassing investigations into normal intestinal physiology in cattle and the intricate host-pathogen interactions of clinically and economically significant enteric pathogens.

Harnessing mechanobiology for kidney organoid research.

Recently, organoids have emerged as revolutionizing tools with the unprecedented potential to recreate organ-specific microanatomy in vitro. Upon their derivation from human pluripotent stem cells (hPSCs), organoids reveal the blueprints of human organogenesis, further allowing the faithful recapitulation of their physiology. Nevertheless, along with the evolution of this field, advanced research exposed the organoids' shortcomings, particularly regarding poor reproducibility rates and overall immatureness. To resolve these challenges, many studies have started to underscore the relevance of mechanical cues as a relevant source to induce and externally control hPSCs differentiation. Indeed, established organoid generation protocols from hPSCs have mainly relyed on the biochemical induction of fundamental signalling pathways present during kidney formation in mammals, whereas mechanical cues have largely been unexplored. This review aims to discuss the pertinence of (bio) physical cues within hPSCs-derived organoid cultures, while deciphering their effect on morphogenesis. Moreover, we will explore state-of-the-art mechanobiology techniques as revolutionizing means for understanding the underlying role of mechanical forces in biological processes in organoid model systems.

Are human brain organoids cloned human individuals? An ethical analysis

In recent years, there has been much ethical debate about human brain organoid research. However, the possibility that human brain organoids might be human individuals has not been examined. This is an important issue because if human brain organoids are human individuals, then the creation of human brain organoids could be a form of human reproductive cloning. We examine the implications of two mainstream bioethical views on the nature of human individuals for human brain organoids: (1) Functional Integration Theory, the view that the capacity to integrate vital functions is the criterion for human individuality, and (2) Mental State Theory, the view that some mental capacity is the criterion for human individuality. We find that under either theory of human individuality, human brain organoids could be human individuals in the future. According to a version of Functional Integration Theory that locates the integrative function in the human brainstem (Brainstem Theory), a well-developed human brainstem organoid can be a human individual. Further, on Mental State Theory, a human brain organoid with certain mental capacities can be a human individual. Our results, combined with the ban on human reproductive cloning, may provide some reasons to be more cautious in human brain organoid research. Alternatively, in light of the progress in human brain organoid research, it may be necessary to reconsider the ethics and laws surrounding human reproductive cloning. This research paves the way for the first time to examine the question of great ethical importance whether human brain organoids can be human individuals, and encourages further consideration.

A Strainer-Based Platform for the Collection and Immunolabeling of Porcine Epidemic Diarrhea Virus-Infected Porcine Intestinal Organoid.

The development of organoid research has raised new requirements for this methodology. In a previous study, we demonstrated that an emerging protocol achieved the collection, loading, and programmed immunolabeling of mouse intestinal organoids based on a strainer platform. To uncover the applied potential of this novel methodology on organoids from other species, the strainer platform was utilized to characterize the porcine epidemic diarrhea virus (PEDV)-infected porcine intestinal organoid model. Based on a previous study, some steps were changed to improve the efficiency of the assay by simplifying the reagent addition procedure. In addition, we redefined the range of strainer sizes on porcine intestinal organoids, showing that strainers with pore sizes of 40 and 70 μm matched the above protocol well. Notably, the strainer platform was successfully used to label viral proteins, laying the foundation for its application in the visualization of viral infection models. In summary, the potential of the strainer platform for organoid technology was explored further. More extensive exploration of this platform will contribute to the development of organoid technology.

Leveraging mechanobiology and biophysical cues in lung organoids for studying lung development and disease

Lung organoids have emerged as powerful tools for studying lung distal diseases by recapitulating the cellular diversity and microenvironment of the lung tissue. This review article highlights the advancements in leveraging mechanobiology and biophysical cues in lung organoid engineering to improve their physiological relevance and disease modelling capabilities. We discuss the role of mechanobiology in lung development and homeostasis, as well as the integration of biophysical cues in the design and culture of lung organoids. Furthermore, we explore how these advancements have contributed to the understanding of lung distal diseases pathogenesis. We also discuss the challenges and future directions in harnessing mechanobiology and biophysical cues in lung organoid research. This review showcases the potential of lung organoids as a platform to investigate the underappreciated impacts of biophysical and biomechanical properties in enhancing lung organoids complexity and functionality, and ultimately provide new insight into embryonic lung development and pulmonary distal diseases pathogenesis.

When is a brain organoid a sentience candidate?

It would be unwise to dismiss the possibility of human brain organoids developing sentience. However, scepticism about this idea is appropriate when considering current organoids. It is a point of consensus that a brainstem-dead human is not sentient, and current organoids lack a functioning brainstem. There are nonetheless troubling early warning signs, suggesting organoid research may create forms of sentience in the near future. To err on the side of caution, researchers with very different views about the neural basis of sentience should unite behind the “brainstem rule”: if a neural organoid develops or innervates a functioning brainstem that registers and prioritizes its needs, regulates arousal, and leads to sleep-wake cycles, then it is a sentience candidate. If organoid research leads to the creation of sentience candidates, a moratorium or indefinite ban on the creation of the relevant type of organoid may be appropriate. A different way forward, more consistent with existing approaches to animal research, would be to require ethical review and harm-benefit analysis for all research on sentience candidates.