Accessible luminal interface of bovine rectal organoids generated from cryopreserved biopsy tissues.

Developing precise species-specific in vitro models that closely resemble in vivo intestinal tissues is essential for advancing our understanding of gastrointestinal physiology and associated diseases. This is especially crucial in examining host-pathogen interactions, particularly in bovines, a known reservoir for microbes and pathogens posing substantial public health threats. This research investigated the viability of producing bovine rectal organoids from cryopreserved tissues. We compared two cryopreservation methods with a traditional technique using fresh tissues, evaluating their effectiveness through growth rates, long-term viability, and comprehensive structural, cellular, and genetic analyses. These assessments utilized phase-contrast imaging, immunofluorescence imaging, and RT-qPCR assays. Additionally, the study developed a sophisticated method for forming a functional epithelial barrier from organoid-derived bovine rectal monolayers, incorporating a wide range of epithelial cells. This methodology employed transepithelial electrical resistance (TEER), parallel artificial membrane permeability assay (Papp), confocal microscopy, and advanced imaging techniques like scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Our findings decisively show that bovine rectal organoids can be effectively generated from cryopreserved biopsy tissues. Moreover, we formulated a robust and optimized protocol for creating functional rectal monolayers from these organoids. This significant progress is particularly relevant given the susceptibility of the bovine rectum to various enteric pathogens of public health concern, marking a vital step forward in veterinary and biomedical research. The creation of accurate species specific in vitro models that faithfully mimic in vivo intestinal tissues is critical for enhancing our understanding of gut physiology and related pathologies. This is particularly relevant in studying the interactions between hosts and microbes or pathogens with significant public health risks where bovine can be the major reservoir.

iPSCs-Derived Neurons and Brain Organoids from Patients.

Induced pluripotent stem cells (iPSCs) can be differentiated into specific neurons and brain organoids by adding induction factors and small molecules in vitro, which carry human genetic information and recapitulate the development process of human brain as well as physiological, pathological, and pharmacological characteristics. Hence, iPSC-derived neurons and organoids hold great promise for studying human brain development and related nervous system diseases in vitro, and provide a platform for drug screening. In this chapter, we summarize the development of the differentiation techniques for neurons and brain organoids from iPSCs, and their applications in studying brain disease, drug screening, and transplantation.

Thyroid organoids: Advances and applications.

Organoids are derived from stem cells under three-dimensional culture conditions through self-assembly, and they can recapitulate the structural and functional characteristics of organs in vivo during culture. Organoids can be generated from both normal and malignant tissues. Those derived from normal tissues are widely used in the field of regenerative medicine. Meanwhile, tumour-derived organoids retain the phenotypic heterogeneity and atypia of the primary tumour, thereby providing a reliable in vitro model for the study of tumour pathogenesis and treatment. The thyroid gland is one of the most important endocrine organs regulating the body's energy metabolism and growth; however, it is also associated with a high incidence of malignancy. Organoid is an effective tool for thyroid research. Thyroid tumour-derived organoids can inherit the histopathological properties of primary tumours, and thyroid tissue-derived organoids can form follicular structures and secrete thyroid hormones. The above characteristics of organoids provide a reliable way to study the mechanism of thyroid genesis and tumour development in vitro. In this review, we focus on current knowledge and strategies for the establishment of thyroid organoids in thyroid regeneration and tumour research aiming to increase our understanding of the pathogenesis of thyroid tumours and the regenerative treatment of patients with hypothyroidism.

Light and electron microscopy continuum-resolution imaging of 3D cell cultures.

3D cell cultures, in particular organoids, are emerging models in the investigation of healthy or diseased tissues. Understanding the complex cellular sociology in organoids requires integration of imaging modalities across spatial and temporal scales. We present a multi-scale imaging approach that traverses millimeter-scale live-cell light microscopy to nanometer-scale volume electron microscopy by performing 3D cell cultures in a single carrier that is amenable to all imaging steps. This allows for following organoids' growth, probing their morphology with fluorescent markers, identifying areas of interest, and analyzing their 3D ultrastructure. We demonstrate this workflow on mouse and human 3D cultures and use automated image segmentation to annotate and quantitatively analyze subcellular structures in patient-derived colorectal cancer organoids. Our analyses identify local organization of diffraction-limited cell junctions in compact and polarized epithelia. The continuum-resolution imaging pipeline is thus suited to fostering basic and translational organoid research by simultaneously exploiting the advantages of light and electron microscopy.

Liver organoids: established tools for disease modeling and drug development.

In the past decade, liver organoids have evolved rapidly as valuable research tools, providing novel insights into almost all types of liver diseases, including monogenic liver diseases, alcohol-associated liver disease, metabolic-associated fatty liver disease, various types of (viral) hepatitis, and liver cancers. Liver organoids in part mimic the microphysiology of the human liver and fill a gap in high-fidelity liver disease models to a certain extent. They hold great promise to elucidate the pathogenic mechanism of a diversity of liver diseases and play a crucial role in drug development. Moreover, it is challenging but opportunistic to apply liver organoids for tailored therapies of various liver diseases. The establishment, applications, and challenges of different types of liver organoids, for example, derived from embryonic, adult, or induced pluripotent stem cells, to model different liver diseases, are presented in this review.

Give Them Vasculature and Immune Cells: How to Fill the Gap of Organoids.

Valid and relevant models are critical for research to have biological relevance or to proceed in the right path. As well-established two-dimensional cell cultures lack niches and cues and rodent models differ in species, three-dimensional organoids emerged as a powerful platform for research. Cultured in vitro from stem cells, organoids are heterogeneous in cells and closely resemble the in vivo settings. Organoids also recapitulate the unique human features if cultured from a human source and are subjected to genetic modification. However, one type of organoid possesses only a limited selection of cells. In particular, the absence of vasculature and immune cells restricts the organoids from nutrition, cues, or critical interactions, undermining the validity of organoids as physiological or pathological models. To fill the current gap, there is an urgent need to provide organoids with vasculature and immune cells. In this paper, we review the methods to generate physiological and pathological organoid models and summarize ways to vascularize or immunize them. Our discussion continues with some advantages and disadvantages of each method and some emerging solutions to current problems.

Advanced Technologies for Studying Microbiome-Female Reproductive Tract Interactions: Organoids, Organoids-on-a-Chip, and Beyond.

The female reproductive tract (FRT) is home to diverse microbial communities that play a pivotal role in reproductive health and disorders such as infertility, endometriosis, and cervical cancer. To understand the complex host-microbiota interactions within the FRT, models that authentically replicate the FRT's environment, including the interplay between the microbiota, mucus layer, immune system, and hormonal cycle, are key. Recent strides in organoid and microfluidic technologies are propelling research in this domain, offering insights into FRT-microbiota interactions and potential therapeutic avenues. This review delves into the current state of FRT organoid models and microbe integration techniques, evaluating their merits and challenges for specific research objectives. Emphasis is placed on innovative approaches and applications, including integrating organoids with microfluidics, and using patient-derived biobanks, as this offers potential for deeper mechanistic insights and personalized therapeutic strategies. Modeling various FRT properties in organoids is explored, from encompassing age-related epithelial features, oxygen levels, and hormonal effects to mucus layers, immune responses, and microbial interactions, highlighting their potential to transform reproductive health research and predict possible outcomes.

Lung Organoids in Smoking Research: Current Advances and Future Promises.

Tobacco smoking has been established to contribute to the pathogenesis of various respiratory diseases including chronic obstructive pulmonary disease (COPD), lung cancer, and asthma. However, major hurdles in mechanistic studies on the role of smoking in human lungs remain in part due to the lack of ex vivo experimental models and ambiguous data from animal models that can best recapitulate the architecture and pathophysiology of the human lung. Recent development of the lung organoid culture system has opened new avenues for respiratory disease research as organoids are proving to be a sophisticated ex vivo model that functionally and structurally mimics the human lungs better than other traditionally used models. This review will discuss how recent advances in lung organoid systems may help us better determine the injurious and immunological effect of smoking on human lungs and will provide some suggestions for future research directions.

Advances in liver organoids: model systems for liver disease.

Reliable in vitro models with human-derived cells that recapitulate in vivo-like physiologies are required for drug discovery and development to reduce the gap between the results of cell-based drug testing, animal testing, and human clinical trials. Liver organoid models have emerged as novel tools for hepatotoxicity evaluation, liver disease modeling, and drug screening. Liver organoids can be generated from biopsies of liver tissues or pluripotent stem cells and can be applied to various liver diseases, including metabolic associated fatty liver disease, infectious liver disease, genetic liver disease, and liver cancer. This review focuses on recent studies on organoids to model human liver diseases and discusses the advantages and limitations of current liver organoids for translational applications.

Protocol to generate mouse oviduct epithelial organoids for viral transduction and whole-mount 3D imaging.

Epithelial cells lining the oviduct/fallopian tube are essential in reproduction and have been identified as the cell-of-origin in high-grade serous ovarian carcinoma (HGSOC). This protocol describes the generation of organoids from mouse oviduct epithelial cells, providing a powerful in vitro tool to study epithelial homeostasis and malignant transformation. We also outline a protocol for whole-mount immunofluorescence and 3D confocal imaging. In addition, we describe approaches of viral transduction to investigate gene function in organoid development and epithelial cell behavior. For complete details on the use and execution of this profile, please refer to Ford et al. (2021).

An in vitro chronic damage model impairs inflammatory and regenerative responses in human colonoid monolayers.

Acute damage to the intestinal epithelium can be repaired via de-differentiation of mature intestinal epithelial cells (IECs) to a stem-like state, but there is a lack of knowledge on how intestinal stem cells function after chronic injury, such as in inflammatory bowel disease (IBD). We developed a chronic-injury model in human colonoid monolayers by repeated rounds of air-liquid interface and submerged culture. We use this model to understand how chronic intestinal damage affects the ability of IECs to (1) respond to microbial stimulation, using the Toll-like receptor 5 (TLR5) agonist FliC and (2) regenerate and protect the epithelium from further damage. Repeated rounds of damage impair the ability of IECs to regrow and respond to TLR stimulation. We also identify mRNA expression and DNA methylation changes in genes associated with IBD and colon cancer. This methodology results in a human model of recurrent IEC injury like that which occurs in IBD.

Kidney organoids recapitulate human basement membrane assembly in health and disease.

Basement membranes (BMs) are complex macromolecular networks underlying all continuous layers of cells. Essential components include collagen IV and laminins, which are affected by human genetic variants leading to a range of debilitating conditions including kidney, muscle, and cerebrovascular phenotypes. We investigated the dynamics of BM assembly in human pluripotent stem cell-derived kidney organoids. We resolved their global BM composition and discovered a conserved temporal sequence in BM assembly that paralleled mammalian fetal kidneys. We identified the emergence of key BM isoforms, which were altered by a pathogenic variant in COL4A5. Integrating organoid, fetal, and adult kidney proteomes, we found dynamic regulation of BM composition through development to adulthood, and with single-cell transcriptomic analysis we mapped the cellular origins of BM components. Overall, we define the complex and dynamic nature of kidney organoid BM assembly and provide a platform for understanding its wider relevance in human development and disease.

The Origin and Contribution of Cancer-Associated Fibroblasts in Colorectal Carcinogenesis.

BACKGROUND & AIMS: Cancer-associated fibroblasts (CAFs) play an important role in colorectal cancer (CRC) progression and predict poor prognosis in CRC patients. However, the cellular origins of CAFs remain unknown, making it challenging to therapeutically target these cells. Here, we aimed to identify the origins and contribution of colorectal CAFs associated with poor prognosis. METHODS: To elucidate CAF origins, we used a colitis-associated CRC mouse model in 5 different fate-mapping mouse lines with 5-bromodeoxyuridine dosing. RNA sequencing of fluorescence-activated cell sorting-purified CRC CAFs was performed to identify a potential therapeutic target in CAFs. To examine the prognostic significance of the stromal target, CRC patient RNA sequencing data and tissue microarray were used. CRC organoids were injected into the colons of knockout mice to assess the mechanism by which the stromal gene contributes to colorectal tumorigenesis. RESULTS: Our lineage-tracing studies revealed that in CRC, many ACTA2+ CAFs emerge through proliferation from intestinal pericryptal leptin receptor (Lepr)+ cells. These Lepr-lineage CAFs, in turn, express melanoma cell adhesion molecule (MCAM), a CRC stroma-specific marker that we identified with the use of RNA sequencing. High MCAM expression induced by transforming growth factor ß was inversely associated with patient survival in human CRC. In mice, stromal Mcam knockout attenuated orthotopically injected colorectal tumoroid growth and improved survival through decreased tumor-associated macrophage recruitment. Mechanistically, fibroblast MCAM interacted with interleukin-1 receptor 1 to augment nuclear factor κB-IL34/CCL8 signaling that promotes macrophage chemotaxis. CONCLUSIONS: In colorectal carcinogenesis, pericryptal Lepr-lineage cells proliferate to generate MCAM+ CAFs that shape the tumor-promoting immune microenvironment. Preventing the expansion/differentiation of Lepr-lineage CAFs or inhibiting MCAM activity could be effective therapeutic approaches for CRC.

IFITM1 expression determines extracellular vesicle uptake in colorectal cancer.

The majority of colorectal cancer (CRC) patients carry mutations in the APC gene, which lead to the unregulated activation of the Wnt pathway. Extracellular vesicles (EV) are considered potential therapeutic tools. Although CRC is a genetically heterogeneous disease, the significance of the intra-tumor heterogeneity in EV uptake of CRC cells is not yet known. By using mouse and patient-derived organoids, the currently available best model of capturing cellular heterogeneity, we found that Apc mutation induced the expression of interferon-induced transmembrane protein 1 (Ifitm1), a membrane protein that plays a major role in cellular antiviral responses. Importantly, organoids derived from IFITM1high CRC cells contained more proliferating cells and they had a markedly reduced uptake of fibroblast EVs as compared to IFITM1low/- cells. In contrast, there was no difference in the intensity of EV release between CRC subpopulations with high and low IFITM1 levels. Importantly, the difference in cell proliferation between these two subpopulations disappeared in the presence of fibroblast-derived EVs, proving the functional relevance of the enhanced EV uptake by IFITM1low CRC cells. Furthermore, inactivating IFITM1 resulted in an enhanced EV uptake, highlighting the importance of this molecule in establishing the cellular difference for EV effects. Collectively, we identified CRC cells with functional difference in their EV uptake ability that must be taken into consideration when using EVs as therapeutic tools for targeting cancer cells.

Live cell molecular analysis of primary prostate cancer organoids identifies persistent androgen receptor signaling.

Prostate Cancer (PC) is a disease with remarkable tumor heterogeneity that often manifests in significant intra-patient variability with regards to clinical outcomes and treatment response. Commonly available PC cell lines do not accurately reflect the complexity of this disease and there is critical need for development of new models to recapitulate the intricate hierarchy of tumor pathogenesis. In current study, we established ex vivo primary patient-derived cancer organoid (PDCO) cultures from prostatectomy specimens of patients with locally advanced PC. We then performed a comprehensive multi-parameter characterization of the cellular composition utilizing a novel approach for live-cell staining and direct imaging in the integrated microfluidic Stacks device. Using orthogonal flow cytometry analysis, we demonstrate that primary PDCOs maintain distinct subsets of epithelial cells throughout culture and that these cells conserve expression of androgen receptor (AR)-related elements. Furthermore, to confirm the tumor-origin of the PDCOs we have analyzed the expression of PC-associated epigenetic biomarkers including promoter methylation of the GSTP1, RASSF1 and APC and RARb genes by employing a novel microfluidic rare-event screening protocol. These results demonstrate that this ex vivo PDCO model recapitulates the complexity of the epithelial tumor microenvironment of multifocal PC using orthogonal analyses. Furthermore, we propose to leverage the Stacks microfluidic device as a high-throughput, translational platform to interrogate phenotypic and molecular endpoints with the capacity to incorporate a complex tumor microenvironment.

3D in vitro culture system to study collective migration in mammary organoid epithelium.

We recently established an in vitro culture system in which mammary gland organoid undergoes directional migration in response to an FGF10 concentration gradient. Here, we describe a step-by-step protocol for preparing organoids, the setup of the 3D culture system, and the image acquisition approach. The technical difficulties in conducting the 3D migration assay are choosing epithelial organoids of appropriate sizes and manually paring organoids and beads pre-soaked in FGF10 within a desirable distance (∼100 µm). For complete details on the use and execution of this protocol, please refer to Lu et al. (2020).

Low-viscosity matrix suspension culture enables scalable analysis of patient-derived organoids and tumoroids from the large intestine.

Cell embedment into a solid support matrix is considered essential for the culture of intestinal epithelial organoids and tumoroids, but this technique presents challenges that impede scalable culture expansion, experimental manipulation, high-throughput screening and diagnostic applications. We have developed a low-viscosity matrix (LVM) suspension culture method that enables efficient establishment and propagation of organoids and tumoroids from the human large intestine. Organoids and tumoroids cultured in LVM suspension recapitulate the morphological development observed in solid matrices, with tumoroids reflecting the histological features and genetic heterogeneity of primary colorectal cancers. We demonstrate the utility of LVM suspension culture for organoid and tumoroid bioreactor applications and biobanking, as well as tumoroid high-throughput drug sensitivity testing. These methods provide opportunities for the study and use of patient-derived organoids and tumoroids from the large intestine.

Engineering mechanobiology through organoids-on-chip: A strategy to boost therapeutics.

The mechanical environment of living cells is as critical as chemical signaling. Mechanical stimuli play a pivotal role in organogenesis and tissue homeostasis. Unbalances in mechanotransduction pathways often lead to diseases, such as cancer, cystic fibrosis, and neurodevelopmental disorders. Despite its inherent relevance, there is a lack of proper mechanoresponsive in vitro study systems. In this context, there is an urge to engineer innovative, robust, dynamic, and reliable organotypic technologies to better connect cellular processes to organ-level function and multi-tissue cross-talk. Mechanically active organoid-on-chip has the potential to surpass this challenge. These systems converge microfabrication, microfluidics, biophysics, and tissue engineering fields to emulate key features of living organisms, hence, reducing costs, time, and animal testing. In this review, we intended to present cutting-edge organ-on-chip platforms that integrate biomechanical stimuli as well as novel multicellular culture, such as organoids. We focused on its application in two main fields: precision medicine and drug development. Moreover, we also discussed the state of the art for the development of an engineered model to assess patient-derived tumor organoid metastatic potential. Finally, we highlighted the current drawbacks and emerging opportunities to match the industry needs. We envision the use of mechanoresponsive organotypic-on-chip microdevices as an indispensable tool for precision medicine, drug development, disease modeling, tissue engineering, and developmental biology.

Intestinal organoid-based 2D monolayers mimic physiological and pathophysiological properties of the pig intestine.

Gastrointestinal infectious diseases remain an important issue for human and animal health. Investigations on gastrointestinal infectious diseases are classically performed in laboratory animals leading to the problem that species-specific models are scarcely available, especially when it comes to farm animals. The 3R principles of Russel and Burch were achieved using intestinal organoids of porcine jejunum. These organoids seem to be a promising tool to generate species-specific in vitro models of intestinal epithelium. 3D Organoids were grown in an extracellular matrix and characterized by qPCR. Organoids were also seeded on permeable filter supports in order to generate 2D epithelial monolayers. The organoid-based 2D monolayers were characterized morphologically and were investigated regarding their potential to study physiological transport properties and pathophysiological processes. They showed a monolayer structure containing different cell types. Moreover, their functional activity was demonstrated by their increasing transepithelial electrical resistance over 18 days and by an active glucose transport and chloride secretion. Furthermore, the organoid-based 2D monolayers were also confronted with cholera toxin derived from Vibrio cholerae as a proof of concept. Incubation with cholera toxin led to an increase of short-circuit current indicating an enhanced epithelial chloride secretion, which is a typical characteristic of cholera infections. Taken this together, our model allows the investigation of physiological and pathophysiological mechanisms focusing on the small intestine of pigs. This is in line with the 3R principle and allows the reduction of classical animal experiments.

Recombinant soluble thrombomodulin accelerates epithelial stem cell proliferation in mouse intestinal organoids and promotes the mucosal healing in colitis.

BACKGROUND AND AIM: Epithelial regeneration, a critical step for the mucosal healing in inflammatory bowel disease, is tightly regulated by stem cells. Therefore, identification of the specific factors that induce stem cell proliferation could contribute to the development of effective strategies for treating inflammatory bowel disease. Recombinant soluble thrombomodulin (rsTM) has previously been shown to promote cell proliferation in skin and corneal wound healing in murine models, but its effects on intestinal epithelial cell proliferation remains unclear. METHODS: Mouse intestinal organoids and dextran sulfate sodium (DSS)-induced colitis mouse model were used to assess the effects of rsTM on proliferation of intestinal epithelial cells. The size and budding morphologies of organoids were studied by confocal microscopy. The gene expression levels were analyzed by quantitative real-time polymerase chain reaction and immunofluorescence analysis. The effects of rsTM on DSS-induced colitis were investigated by evaluating body weight changes, colon length, histological score, and survival rate. RESULTS: The rsTM markedly stimulated the growth of intestinal organoids, thereby increasing the surface areas and budding phenotypes of the organoids. rsTM also significantly upregulated the gene expression of intestinal stem cell-specific and epithelial cell-specific markers in a dose-dependent manner. Furthermore, the treatment with high concentrations of rsTM significantly improved the recovery of body weight, histological outcomes, colon length shortening, and prolonged the survival of mice with colitis. CONCLUSIONS: The rsTM promotes intestinal stem cell proliferation in intestinal organoids and enhances the mucosal healing during recovery phase in DSS-induced colitis.