Organoid Technology Research Articles

Cryopreservation of organoids: Strategies, innovation, and future prospects.

Organoid technology has demonstrated unique advantages in multidisciplinary fields such as disease research, tumor drug sensitivity, clinical immunity, drug toxicology, and regenerative medicine. It will become the most promising research tool in translational research. However, the long preparation time of organoids and the lack of high-quality cryopreservation methods limit the further application of organoids. Although the high-quality cryopreservation of small-volume biological samples such as cells and embryos has been successfully achieved, the existing cryopreservation methods for organoids still face many bottlenecks. In recent years, with the development of materials science, cryobiology, and interdisciplinary research, many new materials and methods have been applied to cryopreservation. Several new cryopreservation methods have emerged, such as cryoprotectants (CPAs) of natural origin, ice-controlled biomaterials, and rapid rewarming methods. The introduction of these technologies has expanded the research scope of cryopreservation of organoids, provided new approaches and methods for cryopreservation of organoids, and is expected to break through the current technical bottleneck of cryopreservation of organoids. This paper reviews the progress of cryopreservation of organoids in recent years from three aspects: damage factors of cryopreservation of organoids, new protective agents and loading methods, and new technologies of cryopreservation and rewarming.

Neuroimmune mechanisms in autism etiology - untangling a complex problem using human cellular models.

Autism spectrum disorder (ASD) affects 1 in 36 people and is more often diagnosed in males than in females. Core features of ASD are impaired social interactions, repetitive behaviors and deficits in verbal communication. ASD is a highly heterogeneous and heritable disorder, yet its underlying genetic causes account only for up to 80% of the cases. Hence, a subset of ASD cases could be influenced by environmental risk factors. Maternal immune activation (MIA) is a response to inflammation during pregnancy, which can lead to increased inflammatory signals to the fetus. Inflammatory signals can cross the placenta and blood brain barriers affecting fetal brain development. Epidemiological and animal studies suggest that MIA could contribute to ASD etiology. However, human mechanistic studies have been hindered by a lack of experimental systems that could replicate the impact of MIA during fetal development. Therefore, mechanisms altered by inflammation during human pre-natal brain development, and that could underlie ASD pathogenesis have been largely understudied. The advent of human cellular models with induced pluripotent stem cell (iPSC) and organoid technology is closing this gap in knowledge by providing both access to molecular manipulations and culturing capability of tissue that would be otherwise inaccessible. We present an overview of multiple levels of evidence from clinical, epidemiological, and cellular studies that provide a potential link between higher ASD risk and inflammation. More importantly, we discuss how stem cell-derived models may constitute an ideal experimental system to mechanistically interrogate the effect of inflammation during the early stages of brain development.

Modelling neurodegeneration and inflammation in early diabetic retinopathy using 3D human retinal organoids

PurposeDiabetic retinopathy (DR) is a complication of diabetes and a primary cause of visual impairment amongst working-age individuals. DR is a degenerative condition in which hyperglycaemia results in morphological and functional changes in certain retinal cells. Existing treatments mainly address the advanced stages of the disease, which involve vascular defects or neovascularization. However, it is now known that retinal neurodegeneration and inflammation precede these vascular changes as early events of DR. Therefore, there is a pressing need to develop a reliable human in vitro model that mimics the early stage of DR to identify new therapeutic approaches to prevent and delay its progression.MethodsHere, we used human-induced pluripotent stem cells (hiPSCs) differentiated into three-dimensional (3D) retinal organoids, which resemble the complexity of the retinal tissue. Retinal organoids were subjected to high-glucose conditions to generate a model of early DR.ResultsOur model showed well-established molecular and cellular features of early DR, such as (i) loss of retinal ganglion and amacrine cells; (ii) glial reactivity and inflammation, with increased expression of the vascular endothelial-derived growth factor (VEGF) and interleukin-1β (IL-1β), and monocyte chemoattractant protein-1 (MCP-1) secretion; and (iii) increased levels of reactive oxygen species accompanied by activation of key enzymes involved in antioxidative stress response.ConclusionThe data provided highlight the utility of retinal organoid technology in modelling early-stage DR. This offers new avenues for the development of targeted therapeutic interventions on neurodegeneration and inflammation in the initial phase of DR, potentially slowing the disease’s progression.

Establishment of human cerebral organoid systems to model early neural development and assess the central neurotoxicity of environmental toxins.

JOURNAL/nrgr/04.03/01300535-202501000-00032/figure1/v/2024-05-14T021156Z/r/image-tiff Human brain development is a complex process, and animal models often have significant limitations. To address this, researchers have developed pluripotent stem cell-derived three-dimensional structures, known as brain-like organoids, to more accurately model early human brain development and disease. To enable more consistent and intuitive reproduction of early brain development, in this study, we incorporated forebrain organoid culture technology into the traditional unguided method of brain organoid culture. This involved embedding organoids in matrigel for only 7 days during the rapid expansion phase of the neural epithelium and then removing them from the matrigel for further cultivation, resulting in a new type of human brain organoid system. This cerebral organoid system replicated the temporospatial characteristics of early human brain development, including neuroepithelium derivation, neural progenitor cell production and maintenance, neuron differentiation and migration, and cortical layer patterning and formation, providing more consistent and reproducible organoids for developmental modeling and toxicology testing. As a proof of concept, we applied the heavy metal cadmium to this newly improved organoid system to test whether it could be used to evaluate the neurotoxicity of environmental toxins. Brain organoids exposed to cadmium for 7 or 14 days manifested severe damage and abnormalities in their neurodevelopmental patterns, including bursts of cortical cell death and premature differentiation. Cadmium exposure caused progressive depletion of neural progenitor cells and loss of organoid integrity, accompanied by compensatory cell proliferation at ectopic locations. The convenience, flexibility, and controllability of this newly developed organoid platform make it a powerful and affordable alternative to animal models for use in neurodevelopmental, neurological, and neurotoxicological studies.

DEVELOPMENT OF A PERSONALIZED HUMAN PANETH CELL MODEL USING INTESTINAL ORGANOIDS

Abstract INTRODUCTION Paneth cells are a specialized intestinal epithelial cell subtype whose major function is to produce antimicrobial peptides (AMPs). Alterations in Paneth cell function are associated with Crohn’s disease (CD) but the confounding influences of environmental factors and/or genetic variations makes it difficult to identify the cause of this. Human intestinal organoid (HIO) technology now allows for the generation of intestinal epithelium from any individual but there are relatively few protocols that allow for the enrichment of Paneth cells within organoids. Our goal was to specifically enrich the Paneth cell population in HIOs derived from induced pluripotent stem cells (iPSCs). METHODS iPSCs from a control individual and 2 CD patients were directed to HIOs and then dissociated to generate purified populations of epithelial only-HIOs (eHIOs). eHIOs were passaged weekly in proliferation medium (EGF, Noggin and CHIR99021) and the γ-secretase inhibitor, DAPT, was added to direct towards a Paneth cell fate. Flow cytometry, qPCR and immunocytochemistry were used to determine Paneth cell numbers, and gene and protein expression of various AMPs respectively. RESULTS iPSC-derived eHIOs from the control individual, which were used to develop this enrichment protocol, could be maintained for at least 4 months in proliferation medium. Flow cytometry analysis of the Paneth marker lysozyme revealed that the population of Paneth cells in eHIOs significantly increased from ~1% in proliferation media to ~28% upon treatment with DAPT. qPCR analysis demonstrated that DAPT treatment significantly increased the expression of the Paneth cell associated genes DEFA5, DEFA6, ITLN2, REG3A and PLA2G2A. Immunocytochemistry revealed that DAPT treated eHIOs were enriched for cells possessing granulated lysozyme, and also for additional AMPs such as DEFA5, ITLN2 and REG3A. The presence of the bacterial sensors TLR2, TLR5 and NOD2 were also detected in our Paneth cells. Finally, to confirm the applicability to CD patients, we applied our protocol and found the population of Paneth cells in CD eHIOs significantly increased from ~1% in proliferation media to ~10% upon treatment with DAPT, had significantly increased expression of the Paneth cell related genes and were also were enriched for cells with granulated AMPs. CONCLUSION We have successfully developed a methodology to enrich Paneth cells in iPSC-derived HIOs from both healthy and CD patients. Given that iPSCs can be generated from donor cells stored in well characterized biorepositories or obtained from a small blood draw from any CD patient, this modeling system now opens up a new avenue of research by allowing an examination of how environmental factors (microbes/cytokines) and/or genetic variations influence human Paneth cell function in a personalized manner. Fluorescent image showing the presence of granulated DEFA5 (green) in E-cadherin+ (red) human iPSC-derived intestinal organoids treated with DAPT (X60). qPCR analysis of Paneth cell related genes in proliferation media compared to DAPT treatment. *P < .05, **P < .01, ***P < .001, and ****P < .0001 as compared to gene expression in proliferation media.

Organoids as a tool to study the impact of heterogeneity in gastrointestinal epithelium on host-pathogen interactions.

The epithelium of the gastrointestinal (GI) tract has been extensively characterized using advanced histological and RNA sequencing techniques, which has revealed great cellular diversity. Pathogens, such as viruses and bacteria, are highly adapted to their host and often exhibit not only species-specificity but also a preference or tropism for specific GI segments or even cell types-some of these preferences are so specific, that these pathogens still cannot be cultured invitro. Organoid technology now provides a tool to generate human cell types, which enables the study of host cell tropism. Focussing on the GI tract, we provide an overview about cellular differentiation in vivo and in organoids and how differentiation in organoids and their derived models is used to advance our understanding of viral, bacterial, and parasitic infection. We emphasize that it is central to understand the composition of the model, as the alteration of culture conditions yields different cell types which affects infection. We examine future directions for wider application of cellular heterogeneity and potential advanced model systems for GI tract infection studies.

NAD+ dependent UPRmt activation underlies intestinal aging caused by mitochondrial DNA mutations

Aging in mammals is accompanied by an imbalance of intestinal homeostasis and accumulation of mitochondrial DNA (mtDNA) mutations. However, little is known about how accumulated mtDNA mutations modulate intestinal homeostasis. We observe the accumulation of mtDNA mutations in the small intestine of aged male mice, suggesting an association with physiological intestinal aging. Using polymerase gamma (POLG) mutator mice and wild-type mice, we generate male mice with progressive mtDNA mutation burdens. Investigation utilizing organoid technology and in vivo intestinal stem cell labeling reveals decreased colony formation efficiency of intestinal crypts and LGR5-expressing intestinal stem cells in response to a threshold mtDNA mutation burden. Mechanistically, increased mtDNA mutation burden exacerbates the aging phenotype of the small intestine through ATF5 dependent mitochondrial unfolded protein response (UPRmt) activation. This aging phenotype is reversed by supplementation with the NAD+ precursor, NMN. Thus, we uncover a NAD+ dependent UPRmt triggered by mtDNA mutations that regulates the intestinal aging.

Organoids: An Emerging Precision Medicine Model for Prostate Cancer Research.

Prostate cancer (PCa) has been known as the most prevalent cancer disease and the second leading cause of cancer mortality in men almost all over the globe. There is an urgent need for establishment of PCa models that can recapitulate the progress of genomic landscapes and molecular alterations during development and progression of this disease. Notably, several organoid models have been developed for assessing the complex interaction between PCa and its surrounding microenvironment. In recent years, PCa organoids have been emerged as powerful in vitro 3D model systems that recapitulate the molecular features (such as genomic/epigenomic changes and tumor microenvironment) of PCa metastatic tumors. In addition, application of organoid technology in mechanistic studies (i.e., for understanding cellular/subcellular and molecular alterations) and translational medicine has been recognized as a promising approach for facilitating the development of potential biomarkers and novel therapeutic strategies. In this review, we summarize the application of PCa organoids in the high-throughput screening and establishment of relevant xenografts for developing novel therapeutics for metastatic, castration resistant, and neuroendocrine PCa. These organoid-based studies are expected to expand our knowledge from basic research to clinical applications for PCa diseases. Furthermore, we also highlight the optimization of PCa cultures and establishment of promising 3D organoid models for in vitro and in vivo investigations, ultimately facilitating mechanistic studies and development of novel clinical diagnosis/prognosis and therapies for PCa.

Expanding the evo-devo toolkit: generation of 3D mammary tissue from diverse mammals.

The varying pathways of mammary gland development across species and evolutionary history are underexplored, largely due to a lack of model systems. Recent progress in organoid technology holds the promise of enabling in-depth studies of the developmental adaptations that have occurred throughout the evolution of different species, fostering beneficial phenotypes. The practical application of this technology for mammary glands has been mostly confined to rodents and humans. In the current study, we have successfully created next-generation 3D mammary gland organoids from eight eutherian mammals and the first branched organoid of a marsupial mammary gland. Using mammary organoids, we identified a role for ROCK protein in regulating branching morphogenesis, a role that manifests differently in organoids from different mammals. This finding demonstrates the utility of the 3D organoid model for understanding the evolution and adaptations of signaling pathways. These achievements highlight the potential for organoid models to expand our understanding of mammary gland biology and evolution, and their potential utility in studies of lactation or breast cancer.

Progress and perspective of organoid technology in breast cancer research.

Breast cancer, a malignant tumor with a high incidence in women, lacks in vitro research models that can represent the biological functions of breast tumors in vivo. As a new biological tool, the organoid model has unique advantages over traditional methods, such as cell culture and patient-derived xenografts. Combining organoids with other emerging technologies, such as gene engineering and microfluidic chip technology, provides an effective method to compensate for the deficiencies in organoid models of breast cancer in vivo. The emergence of breast cancer organoids has provided new tools and research directions in precision medicine, personality therapy, and drug research. In this review, we summarized the merits and demerits of organoids compared to traditional biological models, explored the latest developments in the combination of new technologies and organoid models, and discussed the construction methods and application prospects of different breast organoid models.

Advances and Applications of Lung Organoids in the Research on Acute Respiratory Distress Syndrome (ARDS).

Acute Respiratory Distress Syndrome (ARDS) is a sudden onset of lung injury characterized by bilateral pulmonary edema, diffuse inflammation, hypoxemia, and a low P/F ratio. Epithelial injury and endothelial injury are notable in the development of ARDS, which is more severe under mechanical stress. This review explains the role of alveolar epithelial cells and endothelial cells under physiological and pathological conditions during the progression of ARDS. Mechanical injury not only causes ARDS but is also a side effect of ventilator-supporting treatment, which is difficult to model both in vitro and in vivo. The development of lung organoids has seen rapid progress in recent years, with numerous promising achievements made. Multiple types of cells and construction strategies are emerging in the lung organoid culture system. Additionally, the lung-on-a-chip system presents a new idea for simulating lung diseases. This review summarizes the basic features and critical problems in the research on ARDS, as well as the progress in lung organoids, particularly in the rapidly developing microfluidic system-based organoids. Overall, this review provides valuable insights into the three major factors that promote the progression of ARDS and how advances in lung organoid technology can be used to further understand ARDS.

Adult Animal Stem Cell-Derived Organoids in Biomedical Research and the One Health Paradigm.

Preclinical biomedical research is limited by the predictiveness of in vivo and in vitro models. While in vivo models offer the most complex system for experimentation, they are also limited by ethical, financial, and experimental constraints. In vitro models are simplified models that do not offer the same complexity as living animals but do offer financial affordability and more experimental freedom; therefore, they are commonly used. Traditional 2D cell lines cannot fully simulate the complexity of the epithelium of healthy organs and limit scientific progress. The One Health Initiative was established to consolidate human, animal, and environmental health while also tackling complex and multifactorial medical problems. Reverse translational research allows for the sharing of knowledge between clinical research in veterinary and human medicine. Recently, organoid technology has been developed to mimic the original organ's epithelial microstructure and function more reliably. While human and murine organoids are available, numerous other organoids have been derived from traditional veterinary animals and exotic species in the last decade. With these additional organoid models, species previously excluded from in vitro research are becoming accessible, therefore unlocking potential translational and reverse translational applications of animals with unique adaptations that overcome common problems in veterinary and human medicine.

Brain organoids and organoid intelligence from ethical, legal, and social points of view.

Human brain organoids, aka cerebral organoids or earlier "mini-brains", are 3D cellular models that recapitulate aspects of the developing human brain. They show tremendous promise for advancing our understanding of neurodevelopment and neurological disorders. However, the unprecedented ability to model human brain development and function in vitro also raises complex ethical, legal, and social challenges. Organoid Intelligence (OI) describes the ongoing movement to combine such organoids with Artificial Intelligence to establish basic forms of memory and learning. This article discusses key issues regarding the scientific status and prospects of brain organoids and OI, conceptualizations of consciousness and the mind-brain relationship, ethical and legal dimensions, including moral status, human-animal chimeras, informed consent, and governance matters, such as oversight and regulation. A balanced framework is needed to allow vital research while addressing public perceptions and ethical concerns. Interdisciplinary perspectives and proactive engagement among scientists, ethicists, policymakers, and the public can enable responsible translational pathways for organoid technology. A thoughtful, proactive governance framework might be needed to ensure ethically responsible progress in this promising field.

Organoids in concert: engineering in vitro models toward enhanced fidelity

AbstractOrganoids have emerged as a powerful platform for studying complex biological processes and diseases in vitro. However, most studies have focused on individual organoids, overlooking the inter‐organ interactions in vivo and limiting the physiological relevance of the models. To address this limitation, the development of a multi‐organoid system has gained considerable attention. This system aims to recapitulate inter‐organ communication and enable the study of complex physiological processes. This review provides a comprehensive overview of the recent advancements in organoid engineering and the emerging strategies for constructing a multi‐organoid system. First, we highlight the critical mechanical, structural, and biochemical factors involved in designing suitable materials for the growth of different organoids. Additionally, we discuss the incorporation of dynamic culture environments to enhance organoid culture and enable inter‐organoid communication. Furthermore, we explore techniques for manipulating organoid morphogenesis and spatial positioning of organoids to establish effective inter‐organoid communication networks. We summarize the achievements in utilizing organoids to recapitulate inter‐organ communication in vitro, including assembloids and microfluidic multi‐organoid platforms. Lastly, we discuss the existing challenges and opportunities in developing a multi‐organoid system from its technical bottlenecks in scalability to its applications toward complex human diseases.

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.

3D Cell Culture: Techniques For and Beyond Organoid Applications.

In the rapidly evolving landscape of cell biology and biomedical research, three-dimensional (3D) cell culture has contributed not only to the diversification of experimental tools available but also to their improvement toward greater physiological relevance. 3D cell culture has emerged as a revolutionary technique that bridges the long-standing gap between traditional two-dimensional (2D) cell culture and the complex microenvironments found in living organisms. By providing conditions for establishing critical features of in vivo environment, such as cell-cell and cell-extracellular matrix interactions, 3D cell culture enables proper tissue-like architecture and differentiated function of cells. Since the early days of 3D cell culture in the 1970s, the field has witnessed remarkable progress, with groundbreaking discoveries, novel methodologies, and transformative applications. One particular 3D cell culture technique has caught the attention of many scientists and has experienced an unprecedented boom and enthusiastic application in both basic and translational research over the past decade - the organoid technology. This book chapter provides an introduction to the fundamental concepts of 3D cell culture including organoids, an overview of 3D cell culture techniques, and an overview of methodological- and protocol-oriented chapters in the book 3D Cell Culture.

IPSC-derived and Patient-Derived Organoids: Applications and challenges in scalability and reproducibility as pre-clinical models

Recent advancements in stem cell technology have led to the development of organoids – three-dimensional (3D) cell cultures that closely mimic the structural and functional characteristics of human organs. These organoids represent a significant improvement over traditional two-dimensional (2D) cell cultures by preserving native tissue architecture and cellular interactions critical for physiological relevance.This review provides a comprehensive comparison between two main types of organoids: induced Pluripotent Stem Cell (iPSC)-derived and Adult Stem Cell (ASC)-derived (also known as Patient-Derived Organoids, PDOs). iPSC-derived organoids, derived from reprogrammed cells, exhibit remarkable plasticity, and can model a wide range of tissues and developmental stages. They are particularly valuable for studying early human development, genetic disorders, and complex diseases. However, challenges such as prolonged differentiation protocols and variability in maturation levels remain significant hurdles. In contrast, ASC-derived organoids, generated directly from patient tissues, faithfully recapitulate tissue-specific characteristics and disease phenotypes. This fidelity makes them indispensable for personalized medicine applications, including drug screening, disease modeling, and understanding individualized treatment responses.The review highlights the unique advantages and limitations of each organoid type, emphasizing their roles in advancing biomedical research and drug discovery. It addresses key challenges in organoid technology, such as scalability, reproducibility, and the need for standardized culture protocols. Furthermore, it explores recent innovations in scaffold-guided organoid engineering and the integration of organoids with advanced technologies like artificial intelligence and high-throughput screening.The integration of organoids with cutting-edge technologies holds promise for enhancing their utility in modeling complex human diseases and accelerating drug discovery and development. By providing more physiologically relevant models of human organs, organoid technology is poised to revolutionize biomedical research, offering new insights into disease mechanisms and personalized therapeutic strategies.

CRISPR Knock-Ins in Organoids to Track Tumor Cell Subpopulations.

The integration of CRISPR/Cas9 genome editing techniques with organoid technology has revolutionized the field of tumor modeling, enabling the creation of diverse tumor models with distinct mutational profiles. This protocol details the application of CRISPR knock-ins to engineer tumor organoids with reporter cassettes, which are regulated by endogenous promoters of specific genes of interest. This approach facilitates the precise fluorescent labeling, isolation, and subsequent manipulation of targeted tumor cell subpopulations. The utilization of these knock-in reporter cassettes not only allows the visualization and purification of specific tumor cell subsets but also enables conditional cell ablation and lineage tracing studies. In this chapter, we provide a comprehensive guide for the design, construction, delivery, and validation of CRISPR/Cas9 tools tailored for knock-in reporter cassette integration into specific marker genes of interest. By following this protocol, researchers can harness the potential of engineered tumor organoids to decipher intricate tumor heterogeneity, track metastatic trajectories, and unveil novel therapeutic vulnerabilities linked to specific tumor cell subpopulations.

Renal Organoids from Whole Kidney Cells.

Organoid technology, as a three-dimensional (3D) culture method, provides a feasible tool to self-organize multiple types of organ-specific cells, construct inherent anatomic structures, and display functional biological activities. For the purpose of renal regeneration, renal organoids are considered as predictive options to form functional kidney substitutions in vitro. Here, we describe an accessible and convenient way to generate renal organoids without differentiation procedures using whole kidney cells.

Leveraging Patient-Derived Organoids for Personalized Liver Cancer Treatment.

Primary liver cancer (PLC) is a primary cause of cancer-related death worldwide, and novel treatments are needed due to the limited options available for treatment and tumor heterogeneity. 66 surgically removed PLC samples were cultured using the self-developed 2:2 method, and the final success rate for organoid culture was 40.9%. Organoid performance has been evaluated using comprehensive molecular measurements, such as whole-exome and RNA sequencing, as well as anticancer drug testing. Multiple organoids and their corresponding tumor tissues contained several of the same mutations, with all pairs sharing conventional TP53 mutations. Regarding copy number variations and gene expression, significant correlations were observed between the organoids and their corresponding parental tumor tissues. Comparisons at the molecular level provided us with an assessment of organoid-to-tumor concordance, which, in combination with drug sensitivity testing provided direct guidance for treatment selection. Finally, we were able to determine an appropriate pharmacological regimen for a patient with ICC, demonstrating the clinical practicality in tailoring patient-specific drug regimens. Our study provides an organoid culture technology that can cultivate models that retain most of the molecular characteristics of tumors and can be used for drug sensitivity testing, demonstrating the broad potential application of organoid technology in precision medicine for liver cancer treatment.