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Primary liver cancer is a leading cause of death worldwide. To create advanced treatments for primary liver cancer, studies have utilized models such as 2D cell culture and in vivo animal models. Recent developments in cancer organoids have created the possibility for 3D in vitro cultures that recapitulates the cancer cell structure and operation as well as the tumor microenvironment (TME). However, before organoids can be directly translated to clinical use, tissue processing and culture medium must be standardized with unified protocols to decrease variability in results. Herein, we present the wide variety of published methodologies used to derive liver cancer organoids from patient tumor tissues. Additionally, we summarize validation methodologies for organoids in terms of marker expression levels with immunohistochemistry as well as the presence of mutations and variants through RNA-sequencing. Primary liver cancer organoids have exciting applications allowing for faster drug testing at a larger scale. Primary liver cancer organoids also assisit in uncovering new mechanisms. Through the coculture of different immune cells and cancer organoids, organoids are now better able to recapitulate the liver cancer TME. In addition, it further aids in the investigation of drug development and drug resistance. Lastly, we posit that the usage of liver cancer organoids in animal models provides researchers a methodology to overcome the current limitations of culture systems.

Because hair loss is a common concern for many individuals, potential regenerative therapies of hair follicles have been extensively researched. Induced pluripotent stem cells (iPSCs) are a promising avenue for hair follicle regeneration. This review explores current iPSC-based approaches and highlights their potential applications and challenges in hair restoration. The principles of iPSC technology, iPSC differentiation into hair follicle precursor cells, and potential clinical implications for hair follicle regeneration are also discussed. This overview of iPSCs and their applications aims to contribute to our understanding of their role in hair restoration and potential future therapeutic applications.

Bioprinting is a groundbreaking technology that enables precise distribution of cell-containing bioinks to construct organoid models that accurately reflect the characteristics of tumors in vivo. By incorporating different types of tumor cells into the bioink, the heterogeneity of tumors can be replicated, enabling studies to simulate real-life situations closely. Precise reproduction of the arrangement and interactions of tumor cells using bioprinting methods provides a more realistic representation of the tumor microenvironment. By mimicking the complexity of the tumor microenvironment, the growth patterns and diffusion of tumors can be demonstrated. This approach can also be used to evaluate the response of tumors to drugs, including drug permeability and cytotoxicity, and other characteristics. Therefore, organoid models can provide a more accurate oncology research and treatment simulation platform. This review summarizes the latest advancements in bioprinting to construct tumor organoid models. First, we describe the bioink used for tumor organoid model construction, followed by an introduction to various bioprinting methods for tumor model formation. Subsequently, we provide an overview of existing bioprinted tumor organoid models.

ABSTRACT: The continuous expansion of intestinal stem cells (ISCs) is crucial for maintaining the renewal of the intestinal epithelium, particularly in inflammatory conditions. It remains largely unknown how the internal microbiota repair damage to the internal mucosal barrier. Hence, investigating potential anti-inflammatory probiotics from the intestinal symbolic microbes of broilers and analyzing their mechanism of action to support the intestinal mucosal barrier function can offer novel regulatory tools to alleviate broiler enteritis. In this research, we utilized in vivo broilers plus ex vivo organoids model to thoroughly examine the effectiveness of Lactobacillus reuteri (LR) in protecting the integrity of the intestinal mucosa during lipopolysaccharide-induced (LPS-induced) enteritis in broilers. The findings indicated that LR feeding maintained intestinal morphological and structural integrity, enhanced proliferation of intestinal epithelial cells, and inhibited cell apoptosis and inflammatory response against the deleterious effects triggered by LPS. Simultaneously, LR enhanced ISCs activity and stimulated intestinal epithelial regeneration to protect the intestinal barrier during LPS-induced injury conditions. The coculture system of LR and ileum organoids revealed that LR increased the growth of organoids and attenuated LPS-stimulated damage to organoids. Furthermore, the LPS-induced decrease in ISC activity was rescued by reactivation of Wnt/β-catenin signaling by LR ex vivo and in vivo. This research revealed that LR promoted the expansion of ISCs and intestinal epithelial cell renewal by regulating the Wnt/β-catenin signaling pathway, thereby maintaining the integrity of the intestinal mucosal barrier. This finding provided theoretical support for lactobacillus as a probiotic additive in livestock feed to improve intestinal inflammation and treat intestinal diseases.

Patient-derived organoids (PDOs) have been proposed as a novel in vitro tumor model that can be applied to tumor research and drug screening. However, current tumor organoid models lack components of the tumor microenvironment, particularly tumor-associated macrophages(TAMs).We collected peripheral blood and tumor samples from 6 patients with extrahepatic cholangiocarcinoma(eCCA). Monocytes were induced into TAMs by cytokine and conditioned medium, and then co-cultured with tumor organoids. Our comprehensive analysis and comparison of histopathology and genomics results confirmed that this co-culture model can better capture intra- and inter-tumor heterogeneity retain the specific mutations of the original tumor. Drug sensitivity data in vitro revealed that gemcitabine and cisplatin are effective drugs for cholangiocarcinoma, but TAMs in the tumor microenvironment promote organoids growth and chemotherapy resistance. In conclusion, our organoid model of cholangiocarcinoma co-cultured with TAMs can not only shorten the model construction cycle, but also preserve the heterogeneity of original tumors to improve the accuracy of drug screening, and can also be applied to the researches of TAMs and tumors.

Summary: Three-dimensional brain organoids from human pluripotent stem cells are a powerful tool for studying human neural networks. Here, we present a protocol for generating cortical brain organoid slices (cBOS) derived from regionalized cortical organoids and grown at the air-liquid interphase. We provide steps for slicing organoids and maintaining them in long-term culture. We then detail approaches for quality control including the evaluation of cell death and cellular identity. Finally, we describe procedures for the expression of a genetically encoded nanosensor for ATP.For complete details on the use and execution of this protocol, please refer to Petersilie et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Midbrain organoids provide an innovative cellular source for transplantation therapies of neurodegenerative diseases. Here, we present a protocol for midbrain organoid-derived cell transplantation into a Parkinson’s disease mouse model. We describe steps for midbrain organoid generation, single-cell suspension preparation, and cell transplantation. This approach is valuable for studying the efficacy of midbrain organoids as a potential cellular source for restoring motor function.For complete details on the use and execution of this protocol, please refer to Fu et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

The DNA damage response (DDR) is a fundamental readout for evaluating efficacy of cancer therapeutics, many of which target DNA associated processes. Current techniques to evaluate DDR rely on immunostaining for phosphorylated histone H2AX (γH2AX), which is an indicator of DNA double-strand breaks. While γH2AX immunostaining can provide a snapshot of DDR in fixed cell and tissue samples, this method is technically cumbersome due to temporal monitoring of DDR requiring timepoint replicates, extensive assay development efforts for 3D cell culture samples such as organoids, and time-consuming protocols for γH2AX immunostaining and its evaluation. The goal of this current study is to reduce overall burden on assay duration and development in non-small cell lung cancer (NSCLC) organoids by leveraging label-free multiphoton imaging. In this study, simultaneous label-free autofluorescence multiharmonic (SLAM) microscopy was used to provide rich intracellular information based on endogenous contrasts. SLAM microscopy enables imaging of live samples eliminating the need to generate sacrificial sample replicates and has improved image acquisition in 3D space over conventional confocal microscopy. Predictive modeling between label-free SLAM microscopy and γH2AX immunostained images confirmed strong correlation between SLAM image features and γH2AX signal. Across multiple DNA targeting chemotherapeutics and multiple patient-derived NSCLC organoid lines, the optical redox ratio and third harmonic generation channels were used to robustly predict DDR. Imaging via SLAM microscopy can be used to more rapidly predict DDR in live 3D NSCLC organoids with minimal sample handling and without labeling.

Liver cancer, a common and intractable liver-related disease, is a malignant tumor with a high morbidity, which needs a high treatment cost but still lacks perfect clinical treatment methods. Looking for an effective platform for liver cancer study and drug screening is urgent and important. Traditional analytical methods for liver disease studies mainly rely on the 2D cell culture and animal experiments, which both cannot fully recapitulate physiological and pathological processes of human liver. For example, cell culture can only show basic functions of cells in vitro, while animal models always hold the problem of species divergence. The organoids, a 3D invitro culture system emerged in recent years, is a cell-bound body with different cell types and has partial tissue functions. The organoid technology can reveal the growth state, structure, function and characteristics of the tissue or organ, and plays an important role in reconstructing invitro experimental models that can truly simulate the human liver. In this paper, we will give a brief introduction of liver organoids and review their applications in liver cancer research, especially in liver cancer pathogenesis, drug screening, precision medicine, regenerative medicine, and other fields. We have also discussed advantages and disadvantages of organoids, as well as future directions and perspectives towards liver organoids.

Over the past decades, there has been ongoing effort to develop complex biomimetic tissue engineering strategies for in vitro cultivation and maintenance of organoids. The defined hydrogels can create organoid models for various organs by changing their properties and various active molecules. An increasing number of researches has been done on the application of hydrogels in organoids, and a large number of articles have been published on the topic. Although there have been existing reviews describing the application of hydrogels in the field of organoids, there is still a lack of comprehensive studies summarizing and analyzing the overall research trends in this field. The citation can be used as an indicator of the scientific influence of an article in its field. This study aims to evaluate the application of hydrogels in organoids through bibliometric analysis, and to predict the hotspots and developing trends in this field.

Summary: Herein, we present a protocol for culturing patient-derived organoids (PDOs) of cervical cancer that includes workflows for tumor biopsy/resection tissue and cytobrush-sampled cells. We describe steps for PDO culture initiation, including rinsing, gentle dissociation, Lymphoprep separation, and cell assessment, as well as seeding cells from surgical and cytobrush tissue digestion. We then provide guidance on PDO maintenance and passage and techniques for producing conditioned medium. Overall, this protocol serves as a valuable guide for establishing and maintaining cervical cancer PDOs.For complete details on the use and execution of this protocol, please refer to Colbert et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Evaluating the synergy of drug combinations is crucial in advancing treatment regimens. Here, we present a protocol to establish primary cells and organoids from murine tumors and calculate drug synergy. We describe all necessary cell culture procedures, including establishing primary cultures, setting up treatment groups, and detecting cell viability. We then outline how to calculate the synergy score based on a bioinformatical pipeline. This approach applies to any disease model in which a combination of drugs needs to be evaluated.For complete details on the use and execution of this protocol, please refer to Ku et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Multiple sampling strategies to cover different or dynamic stages of malignant continuum with organoid cultures provide a valuable platform for epithelium homeostasis, transformation, and cancer progression. Here, we present a protocol to initiate, culture, passage, and characterize organoids from normal and cancer-prone human esophageal tissues. We describe steps for multiple sampling of malignant continuum and the initiation and maintenance of multi-stage organoids. We then detail procedures for the histological characterization of organoids and co-culture systems based on organoids and stromal cells.For complete details on the use and execution of this protocol, please refer to Chen et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

The use of animal models for biological experiments is no longer sufficient for research related to human life and disease. The development of organ tissues has replaced animal models by mimicking the structure, function, development and homeostasis of natural organs. This provides more opportunities to study human diseases such as cancer, infectious diseases and genetic disorders. In this study, bibliometric methods were used to analyze organoid-related articles published over the last 20+ years to identify emerging trends and frontiers in organoid research. A total of 13,143 articles from 4125 institutions in 86 countries or regions were included in the analysis. The number of papers increased steadily over the 20-year period. The United States was the leading country in terms of number of papers and citations. Harvard Medical School had the highest number of papers published. Keyword analysis revealed research trends and focus areas such as organ tissues, stem cells, 3D culture and tissue engineering. In conclusion, this study used bibliometric and visualization methods to explore the field of organoid research and found that organ tissues are receiving increasing attention in areas such as cancer, drug discovery, personalized medicine, genetic disease modelling and gene repair, making them a current research hotspot and a future research trend.

Summary: The mandatory usage of extracellular matrix (ECM) gels in 3D cultures limits antibody penetration and increases background, while the removal of ECM gel causes disruption of morphology and sample loss. These factors pose challenges to effective immune labeling-based staining. Here, we present a protocol for whole-mount immunofluorescence staining of gel-embedded pancreatic organoids. We describe steps for sample fixation, blocking, and antibody incubation. We detail procedures for washing antibodies and mounting. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: T cell acute lymphoblastic leukemia (T-ALL) is a rare but aggressive hematological cancer that occurs primarily in children and adolescents. Here, we present a protocol for in vitro co-culture assay that enables robust expansion of primary T-ALL cells. We describe steps for seeding T-ALL and stromal cells in 3D organoids and subsequent flow analysis to capture the T-ALL cell growth for long-term culture. This protocol provides a valuable platform for in vitro functional studies and drug screenings using patient-derived cells.For complete details on the use and execution of this protocol, please refer to Rivera et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Adult humans cannot regenerate the enamel-forming cell type, ameloblasts. Hence, human induced pluripotent stem cell (hiPSC)-derived ameloblasts are valuable for investigating tooth development and regeneration. Here, we present a protocol for generating three-dimensional induced early ameloblasts (ieAMs) utilizing serum-free media and growth factors. We describe steps for directing hiPSCs toward oral epithelium and then toward ameloblast fate. These cells can form suspended early ameloblast organoids. This approach is critical for understanding, treating, and promoting regeneration in diseases like amelogenesis imperfecta.For complete details on the use and execution of this protocol, please refer to Alghadeer et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: A major bottleneck in the progress of Cryptosporidium research is the lack of accessible cryopreservation of Cryptosporidium oocysts. Here, we present a protocol for the cryopreservation of Cryptosporidium isolates using enteroids. We describe the steps for the establishment of enteroid cultures and cryopreservation of C. parvum-infected HCT-8 cultures. We then detail procedures for the recovery and propagation of frozen parasites using enteroids.For complete details on the use and execution of this protocol, please refer to Deng et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Drug sensitivity testing of patient-derived tumor organoids (PDTOs) is a promising tool for personalizing cancer treatment. Here, we present a protocol for generation of and high-throughput drug testing with PDTOs. We describe detailed steps for PDTO establishment from colorectal cancer tissues, preparation of PDTOs for high-throughput drug testing, and quantification of drug testing results using image analysis. This protocol provides a standardized workflow for PDTO testing of standard-of-care therapies, along with exploring the activity of new agents, for translational research.For complete details on the use and execution of this protocol, please refer to Tan et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Enteroids are in vitro models to study gastrointestinal pathologies and test personalized therapeutics; however, the inherent complexity of enteroids often renders standard gene editing approaches ineffective. Here, we introduce a refined lentiviral transfection protocol, ensuring sufficient lentiviral engagement with enteroids while considering spatiotemporal growth variability throughout the extracellular matrix. Additionally, we highlight a selection process for transduced cells, introduce a protocol to accurately measure transduction efficiency, and explore methodologies to gauge effects of gene knockdown on biological processes. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Three-dimensional (3D) models play an increasingly important role in preclinical drug testing as they faithfully mimic interactions between cancer cells and the tumor microenvironment (TME). Here, we present a protocol for generating scaffold-free 3D multicomponent human melanoma spheroids. We describe steps for characterizing models using live-cell imaging and histology, followed by drug testing and assessment of cell death through various techniques such as imaging, luminescence-based assays, and flow cytometry. Finally, we demonstrate the models’ adaptability for co-cultures with immune cells. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Organ-on-chip technology is a powerful tool for in vitro modeling. Combining it with organoids overcomes lumen inaccessibility while preserving cellular diversity and function of the intestinal epithelium. Here, we present a protocol for generating and analyzing organ-on-chips using human and mouse intestinal organoids. This protocol covers organoid line establishment, single-cell dissociation, chip preparation, and seeding. It outlines procedures for permeability assays, RNA isolation, staining, and imaging. Additionally, we describe independent stimulation and sampling of the apical and basal side. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Generating cell types with properties of embryo cells with full developmental potential is of great biological importance. Here, we present a protocol for generating mouse morula-like cells (MLCs) resembling 8- to 16-cell stage embryo cells. We describe steps for induction, via increasing Stat3 activation, and the isolation of MLCs. We then detail procedures for segregating MLCs into blastocyst cell fates and how to create embryo-like structures from them. This system provides a stem-cell-based embryo model to study early embryo development.For complete details on the use and execution of this protocol, please refer to Li et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: In vitro organoids, including cerebral organoids, are usually developed without mechanical compression, which may contribute to a delay in maturation. Here, we present a protocol for encapsulating cerebral organoids with a thin shell of low-concentration alginate hydrogel. We describe steps for organoid generation, microfluidic chip culture, Matrigel coating, expansion culture, and alginate encapsulation. We then detail procedures for maturation culture and organoid characterization. The moderate compressive stimulation that the shell provides promotes cell proliferation and neuronal maturation.For complete details on the use and execution of this protocol, please refer to Tang et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Objectives: FGFR4-variant and wild-type colorectal cancer (CRC) organoids were developed to investigate the effects of FGFR4-targeted drugs, including FGFR4-IN and erdafitinib, on CRC and their possible molecular mechanism. Methods: Clinical CRC tissues were collected, seven CRC organoids were developed, and whole exome sequencing (WES) was performed. CRC organoids were cultured and organoid drug sensitivity studies were conducted. Finally, an FGFR4-variant (no wild-type) CRC patient-derived orthotopic xenograft mouse model was developed. Western blot measured ERK/AKT/STAT3 pathway-related protein levels. Results: WES results revealed the presence of FGFR4-variants in 5 of the 7 CRC organoids. The structural organization and integrity of organoids were significantly altered under the influence of targeted drugs (FGFR4-IN-1 and erdafitinib). The effects of FGFR4 targeted drugs were not selective for FGFR4 genotypes. FGFR4-IN-1 and erdafitinib significantly reduced the growth, diameter, and Adenosine Triphosphate (ATP) activity of organoids. Furthermore, chemotherapeutic drugs, including 5-fluorouracil and cisplatin, inhibited FGFR4-variant and wild-type CRC organoid activity. Moreover, the tumor volume of mice was significantly reduced at week 6, and p-ERK1/2, p-AKT, and p-STAT3 levels were down-regulated following FGFR4-IN-1 and erdafitinib treatment. Conclusions: FGFR4-targeted and chemotherapeutic drugs inhibited the activity of FGFR4-variant and wild-type CRC organoids, and targeted drugs were more effective than chemotherapeutic drugs at the same concentration. Additionally, FGFR4 inhibitors hindered tumorigenesis in FGFR4-variant CRC organoids through ERK1/2, AKT, and STAT3 pathways. However, no wild-type control was tested in this experiment, which need further confirmation in the next study.

Organoids are three-dimensional structures derived from primary tissue or tumors that closely mimic the architecture, histology, and function of the parental tissue. In recent years, patient-derived organoids (PDOs) have emerged as powerful tools for modeling tumor heterogeneity, drug screening, and personalized medicine. Although most cancer organoids are derived from primary tumors, the ability of organoids from metastatic cancer to serve as a model for studying tumor biology and predicting the therapeutic response is an area of active investigation. Recent studies have shown that organoids derived from metastatic sites can provide valuable insights into tumor biology and may be used to validate predictive models of the drug response. In this comprehensive review, we discuss the feasibility of culturing organoids from multiple metastatic cancers and evaluate their potential for advancing basic cancer research, drug development, and personalized therapy. We also explore the limitations and challenges associated with using metastasis organoids for cancer research. Overall, this review provides a comprehensive overview of the current state and future prospects of metastatic cancer-derived organoids.

Background & Aims: This review was developed to provide a thorough and effective update on models relevant to esophageal metaplasia, dysplasia, and carcinogenesis, focusing on the advantages and limitations of different models of Barrett’s esophagus (BE) and esophageal adenocarcinoma (EAC). Methods: This expert review was written on the basis of a thorough review of the literature combined with expert interpretation of the state of the field. We emphasized advances over the years 2012–2023 and provided detailed information related to the characterization of established human esophageal cell lines. Results: New insights have been gained into the pathogenesis of BE and EAC using patient-derived samples and single-cell approaches. Relevant animal models include genetic as well as surgical mouse models and emphasize the development of lesions at the squamocolumnar junction in the mouse stomach. Rat models are generated using surgical approaches that directly connect the small intestine and esophagus. Large animal models have the advantage of including features in human esophagus such as esophageal submucosal glands. Alternatively, cell culture approaches remain important in the field and allow for personalized approaches, and scientific rigor can be ensured by authentication of cell lines. Conclusions: Research in BE and EAC remains highly relevant given the morbidity and mortality associated with cancers of the tubular esophagus and gastroesophageal junction. Careful selection of models and inclusion of human samples whenever possible will ensure relevance to human health and disease.

Summary: Neurodevelopmental disorders are characterized by complex phenotypes that often result from concomitant dysregulation of cell proliferation, differentiation, or other crucial developmental processes. Here, we present a protocol to quantify differentiation of progenitor populations during early stages of neurogenesis in induced pluripotent stem cell (iPSC)-derived cerebral organoids. We describe steps for organoid differentiation and maturation, sample preparation, immunofluorescence, and imaging and analysis using epifluorescence microscopy. This protocol can be used to compare cerebral organoids from control and patient-derived iPSCs.For complete details on the use and execution of this protocol, please refer to Rakotomamonjy et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Postnatal development of the uterus involves the specification of undifferentiated epithelium into uterine-type epithelium. That specification is regulated by stromal-epithelial interactions as well as intrinsic cell-specific transcription factors and gene regulatory networks. Here, we present a co-culture system to study the effects of stromal-derived factors on epithelial cell growth and differentiation into organoids. First, we describe epithelial cell isolation and organoid growth characterization. Second, we detail a co-culture system that allows the study of stromal-derived paracrine factors on epithelial development.For complete details on the use and execution of this protocol, please refer to Rizo et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Functional precision oncology—a strategy based on perturbing primary tumor cells from cancer patients—could provide a road forward for personalized treatment. Here, we present a comprehensive protocol covering generation and culture of patient-derived colorectal organoids, isolation and expansion of tumor-infiltrating lymphocytes (TILs), and isolation and culture of peripheral blood mononuclear cells (PBMCs). With this protocol, samples fulfilling the demands for performing multi-omics analysis, e.g., RNA sequencing (RNA-seq), whole-exome sequencing (WES), single-cell RNA sequencing (scRNA-seq), and (phospho-)proteomics, can be generated.For complete details on the use and execution of this protocol, please refer to Plattner et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Organ-on-a-chip technology incorporating stem cell techniques represents a promising strategy to improve modeling of human organs. Here, we present a protocol for generating a standardized 3D placenta-on-a-chip model using trophoblast derived from human induced pluripotent stem cells (hiPSCs). We describe steps for seeding hiPSCs into multi-chip OrganoPlate devices and on-chip differentiation into trophoblasts against an extracellular matrix under perfused conditions. We then detail procedures for conducting a functional barrier integrity assay, immunostaining, and collecting protein or RNA for molecular analysis.For complete details on the use and execution of this protocol, please refer to Lermant et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Human papillomaviruses (HPVs) are commensal viruses with pathogenic potential. Their life cycle requires the proliferation and differentiation of keratinocytes (KCs) to form pluristratified epithelia. Based on the original organotypic epithelial raft cultures protocol, we provide an updated workflow to optimally generate pluristratified human epithelia supporting the complete HPV replicative life cycle, here called 3D full-thickness epithelial cultures (3Deps). We describe steps for HPV genome preparation, KC transfection, and dermal equivalent preparation. We then detail procedures for 3Deps culture, harvesting, and analysis. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Here, we present a protocol for rapidly isolating single cells from the mouse pancreas, minimizing damage caused by digestive enzymes in exocrine cells. We guide you through steps to optimize the dissection sequence, enzyme composition, and operational procedures, resulting in high yields of viable pancreatic single cells. This protocol can be applied across a wide range of research areas, including single-cell sequencing, gene expression profiling, primary cell culture, and even the development of spheroids or organoids.For complete details on the use and execution of this protocol, please refer to Jiang et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Force generation is an essential property of skeletal muscle models in vitro. We describe a versatile 1-step procedure to direct undifferentiated human pluripotent stem cells (PSCs) into contractile skeletal muscle organoids (SMOs). Our protocol provides detailed steps for 3D casting of PSCs using either collagen-I/Matrigel- or fibrin/Geltrex-based hydrogels, SMO differentiation, and application of different culture platforms for mechanical loading and contractility analysis. The SMO model may be particularly useful to study human muscle development and developmental skeletal muscle disorders in vitro.For complete details on the use and execution of this protocol, please refer to Shahriyari et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Here, we present a protocol for microinjection of bacteria into mouse small intestinal organoids that recapitulates the natural route of infection of intestinal epithelial cells from the intestinal lumen. We describe steps for visualizing bacteria-cell interactions by live imaging of infected organoids using light sheet microscopy. We then detail procedures for generating doxycycline-inducible expression of mutant proteins in organoids to study essential gene functions. The different techniques described in this protocol can be used independently as required.For complete details on the use and execution of this protocol, please refer to Kim et al. (2021).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Primary sclerosing cholangitis (PSC) is an autoimmune cholangiopathy characterized by chronic inflammation of the biliary epithelium and periductal fibrosis, with no curative treatment available, and liver transplantation is inevitable for end-stage patients. Human placental mesenchymal stem cell (hpMSC)-derived exosomes have demonstrated the ability to prevent fibrosis, inhibit collagen production and possess immunomodulatory properties in autoimmune liver disease. Here, we prepared hpMSC-derived exosomes (ExoMSC) and further investigated the anti-fibrotic effects and detailed mechanism on PSC based on Mdr2−/− mice and multicellular organoids established from PSC patients. The results showed that ExoMSC ameliorated liver fibrosis in Mdr2−/− mice with significant collagen reduction in the preductal area where Th17 differentiation was inhibited as demonstrated by RNAseq analysis, and the percentage of CD4+IL-17A+T cells was reduced both in ExoMSC-treated Mdr2−/− mice (Mdr2−/−-Exo) in vivo and ExoMSC-treated Th17 differentiation progressed in vitro. Furthermore, ExoMSC improved the hypersecretory phenotype and intercellular interactions in the hepatic Th17 microenvironment by regulating PERK/CHOP signaling as supported by multicellular organoids. Thus, our data demonstrate the anti-fibrosis effect of ExoMSC in PSC disease by inhibiting Th17 differentiation, and ameliorating the Th17-induced microenvironment, indicating the promising potential therapeutic role of ExoMSC in liver fibrosis of PSC or Th17-related diseases.

An increasing number of harmful environmental factors are causing serious impacts on human health, and there is an urgent need to accurately identify the toxic effects and mechanisms of these harmful environmental factors. However, traditional toxicity test methods (e.g., animal models and cell lines) often fail to provide accurate results. Fortunately, organoids differentiated from stem cells can more accurately, sensitively and specifically reflect the effects of harmful environmental factors on the human body. They are also suitable for specific studies and are frequently used in environmental toxicology nowadays. As a combination of organoids and organ-on-a-chip technology, organoids-on-a-chip has great potential in environmental toxicology. It is more controllable to the physicochemical microenvironment and is not easy to be contaminated. It has higher homogeneity in the size and shape of organoids. In addition, it can achieve vascularization and exchange the nutrients and metabolic wastes in time. Multi-organoids-chip can also simulate the interactions of different organs. These advantages can facilitate better function and maturity of organoids, which can also make up for the shortcomings of common organoids to a certain extent. This review firstly discussed the limitations of traditional toxicology testing platforms, leading to the introduction of new platforms: organoids and organoids-on-a-chip. Next, the applications of different organoids and organoids-on-a-chip in environmental toxicology were summarized and prospected. Since the advantages of the new platforms have not been sufficiently considered in previous literature, we particularly emphasized them. Finally, this review also summarized the opportunities and challenges faced by organoids and organoids-on-a-chip, with the expectation that readers will gain a deeper understanding of their value in the field of environmental toxicology.

Epidermal growth factor (EGF) is a known signaling cue essential towards the development and organoid biofabrication particularly for exocrine glands. This study developed an in vitro EGF delivery platform with Nicotiana benthamiana plant-produced EGF (P-EGF) encapsulated on hyaluronic acid/alginate (HA/Alg) hydrogel to improve the effectiveness of glandular organoid biofabrication in short-term culture systems. Primary submandibular gland epithelial cells were treated with 5 – 20 ng/mL of P-EGF and commercially available bacteria-derived EGF (B-EGF). Cell proliferation and metabolic activity were measured by MTT and luciferase-based ATP assays. P-EGF and B-EGF 5 – 20 ng/mL promoted glandular epithelial cell proliferation during 6 culture days on a comparable fashion. Organoid forming efficiency and cellular viability, ATP-dependent activity and expansion were evaluated using two EGF delivery systems, HA/Alg-based encapsulation and media supplementation. Phosphate buffered saline (PBS) was used as a control vehicle. Epithelial organoids fabricated from PBS-, B-EGF-, and P-EGF-encapsulated hydrogels were characterized genotypically, phenotypically and by functional assays. P-EGF-encapsulated hydrogel enhanced organoid formation efficiency and cellular viability and metabolism relative to P-EGF supplementation. At culture day 3, epithelial organoids developed from P-EGF-encapsulated HA/Alg platform contained functional cell clusters expressing specific glandular epithelial markers such as exocrine pro-acinar (AQP5, NKCC1, CHRM1, CHRM3, Mist1), ductal (K18, Krt19), and myoepithelial (α-SMA, Acta2), and possessed a high mitotic activity (38–62% Ki67 cells) with a large epithelial progenitor population (∼70% K14 cells). The P-EGF encapsulation strikingly upregulated the expression of pro-acinar AQP5 cells through culture time when compared to others (B-EGF, PBS). Thus, the utilization of Nicotiana benthamiana in molecular farming can produce EGF biologicals amenable to encapsulation in HA/Alg-based in vitro platforms, which can effectively and promptly induce the biofabrication of exocrine gland organoids.

Cancer treatment development is a complex process, with tumor heterogeneity and inter-patient variations limiting the success of therapeutic intervention. Traditional two-dimensional cell culture has been used to study cancer metabolism, but it fails to capture physiologically relevant cell-cell and cell-environment interactions required to mimic tumor-specific architecture. Over the past three decades, research efforts in the field of 3D cancer model fabrication using tissue engineering have addressed this unmet need. The self-organized and scaffold-based model has shown potential to study the cancer microenvironment and eventually bridge the gap between 2D cell culture and animal models. Recently, three-dimensional (3D) bioprinting has emerged as an exciting and novel biofabrication strategy aimed at developing a 3D compartmentalized hierarchical organization with the precise positioning of biomolecules, including living cells. In this review, we discuss the advancements in 3D culture techniques for the fabrication of cancer models, as well as their benefits and limitations. We also highlight future directions associated with technological advances, detailed applicative research, patient compliance, and regulatory challenges to achieve a successful bed-to-bench transition.

Aged cells have declined regenerative ability when subjected to environmental insult. Here we elucidate the mechanism by which mechanical stimulus induces hair regeneration at the microenvironmental regulation level using the hair plucking and organoid culture models. We observed that the skin cells harvested from post-plucking day 3 (PPD3) have the best self-organizing ability during skin organoid culture and have the highest hair regeneration upon transplantation. By bulk RNA sequencing (RNA-seq) and single-cell RNA-seq analysis and in situ hybridization, we identified that the chemokine signaling pathway genes including CCL2 are significantly increased in the skin at PPD3 and in skin organoid cultures. Immunostaining shows that the PPD3 skin epithelial cells have increased multipotency, which is verified by the ability to self-organize to form epidermal aggregates during organoid culture. By adding CCL2 recombinant protein to the organoid culture using an environmental reprogramming protocol, we observed the PPD0 adult skin cells, which lose their regenerative ability can self-organize in organoid culture and regenerate hair follicles robustly upon transplantation. Our study demonstrates that CCL2 functions in immune regulation of hair regeneration under mechanical stimulus, and enhances cell multipotency during organoid culture. This provides a therapeutic potential for future clinical application.

Epigallocatechin (EGC) is one of the main bioactive polyphenols in green tea. This study aims to determine the anti-tumor effect of EGC against colorectal cancer. Our results revealed that treatment of EGC led to significant suppression of cell proliferation and apoptosis in three colorectal cancer cells. Western blot confirmed that EGC activated Caspase-3 and PARP, down-regulated the phosphorylation of STAT3 and decreased Bcl-2 expression. Luciferase-reporter-activity assay further determined that EGC suppressed the promoter activity of STAT3 and downregulated the transcription of STAT3. Besides, in vivo study proved that EGC (100 mg/kg) resulted in significant suppression of tumor volume and tumor weight (decreased by 63.7 %) in mice bearing SW620 tumors. Tumor organoid assays demonstrated the inhibitory efficacy of EGC in colorectal organoids. In conclusion, our study present evidence that EGC inhibited colon cancer cell proliferation both in vitro and in vivo by down-regulation phosphorylated STAT3 and activated downstreams.

Organoid technology, a novel 3D cell culture system, can reproduce a patient’s cancer and may be a novel immunotherapy experimental model. However, currently no gastric cancer organoid (GCO) models in which the organoid and immune cells are in free contact and sufficiently react with each other exist. In this study, we aimed to create a coculture model in which immune cells can move freely and stay in contact with GCOs. We coated the bottom surface of the plate with Matrigel and adhered stem cells to the Matrigel surface, instead of completely embedding them in Matrigel to culture organoids. This method allowed GCOs to grow on the Matrigel surface while maintaining a three-dimensional structure and reproducing the characteristics of the patient’s cancer. We cocultured GCOs and immune cells. Using this model, immune cells could freely move and were in sufficient contact with the cultured GCOs. Our model allowed real-time observation of the immune response and tumor destruction with time. In addition, the GCO killing assay was assessed with natural killer cells from the same patient. This organoid culture model enabled repeated evaluation of the GCO killing assay with various immune cells in vitro. We established a new experimental model that allowed free movement of immune cells and sufficient contact with GCOs. Using this model, it may be possible to predict the effects of immune checkpoint inhibitors in vitro (using GCOs) before administering them to patients.

Summary: Primary human lung organoid-derived air-liquid interface (ALI) cultures serve as a physiologically relevant model to study human airway epithelium in vitro. Here, we present a protocol for establishing these cultures from cryopreserved human lung tissue. We describe steps for lung tissue cryostorage, tissue dissociation, lung epithelial organoid generation, and ALI culture differentiation. We also include quality control steps and technical readouts for monitoring virus response. This protocol demonstrates severe acute respiratory syndrome coronavirus 2 infection in these cultures as an example of their utility.For complete details on the use and execution of this protocol, please refer to Diana Cadena Castaneda et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Patient-derived organoids (PDOs) are ideal ex vivo model systems to study cancer progression and drug resistance mechanisms. Here, we present a protocol for measuring drug efficacy in three-dimensional (3D) high-grade serous ovarian cancer PDO cultures through quantification of cytotoxicity using propidium iodide incorporation in dead cells. We also provide detailed steps to analyze proliferation of PDOs using the Ki67 biomarker. We describe steps for sample processing, immunofluorescent staining, high-throughput confocal imaging, and image-based quantification for 3D cultures.For complete details on the use and execution of this protocol, please refer to Lahtinen et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: In response to the scarcity of advanced in vitro models dedicated to human CNS white matter research, we present a protocol to generate neuroectoderm-derived embedding-free human brain organoids enriched with oligodendrocytes. We describe steps for neuroectoderm differentiation, development of neural spheroids, and their transferal to Matrigel. We then detail procedures for the development, maturation, and application of oligodendrocyte-enriched brain organoids. The presence of myelin-producing cells makes these organoids useful for studying human white matter diseases, such as leukodystrophy. : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

This article presents a modified protocol for embedding and sectioning spheroids and organoids, which are increasingly used in research due to their ability to emulate living tissue. The modifications aim to reduce the distortion and damage of these fragile structures during the embedding and sectioning process. The new method involves using optimized embedding containers, a modified embedding protocol, and optimized temperatures for cryosectioning. A heat-induced antigen retrieval protocol was tested and found to significantly increase immunostaining intensity without compromising spheroid integrity. The combined approach allowed for the creation of thinner cryosections, leading to clearer and more detailed images. The results suggest that the modified protocol could be widely adopted to enhance the imaging of spheroids and organoids. • Paraformaldehyde fixation of spheroids • Antigen retrieval treatment of spheroids • Embedding in freezing medium and cryosectioning

Summary: Parasitic helminth worms frequently infect the gastrointestinal tract and interact with the intestinal epithelium and specialized cell types within it. Intestinal organoids derived from stem cells that line the intestine represent a transformational technology in the study of epithelial-parasite dialogue. Here, we present a protocol for establishing small intestine organoid cultures and administering parasite products of interest to these cultures. We then describe steps for evaluating their impact by microscopy, flow cytometry, immunohistology, and mRNA gene expression.For complete details on the use and execution of this protocol, please refer to Drurey et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Alpha-synuclein (α-syn) aggregation is a principal factor in Parkinson’s disease (PD) onset. Here, we present a protocol for optogenetic induction of α-syn aggregation in human midbrain dopaminergic (mDA) neurons, facilitating a detailed PD pathology study. We describe steps for nucleofection of the opto-α-syn construct, single colony selection and validation, alongside mDA neuron differentiation and rapid induction of toxic α-syn aggregates via blue light. This establishes a potent human induced pluripotent-stem-cell-based platform for PD drug testing and validation.For complete details on the use and execution of this protocol, please refer to Kim et al. (2023).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Summary: Here, we present a protocol for producing 3D pancreatic-like organoids from human pluripotent stem cells in suspension bioreactors. We describe scalable techniques for generating 10,000–100,000 organoids that further mature in 4–5 weeks into α- and β-like cells with glucose-responsive insulin and glucagon release. We detail procedures for culturing, passaging, and cryopreserving stem cells as suspended clusters and specify growth media and differentiation factors for differentiation. Finally, we discuss functional assays for research applications.For complete details on the use and execution of this protocol, please refer to Alvarez-Dominguez et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Establishment of drug testing of patient-derived cancer cells (PDCs) in physiologically relevant 3-dimensional (3D) culture is central for drug discovery and cancer research, as well as for functional precision medicine. Here, we describe the detailed protocol allowing the 3D drug testing of PDCs – or any type of cells of interest – in Matrigel in 384-well plate format using automation. We also provide an alternative protocol, which does not require supporting matrices. The cancer tissue is obtained directly from clinics (after surgery or biopsy) and processed into single cell suspension. Systematic drug sensitivity and resistance testing (DSRT) is carried out on the PDCs directly after cancer cell isolation from tissue or on cells expanded for a few passages. In the 3D-DSRT assay, the PDCs are plated in 384-well plates in Matrigel, grown as spheroids, and treated with compounds of interest for 72 h. The cell viability is directly measured using a luminescence-based assay. Alternatively, prior to the cell viability measurement, drug-treated cells can be directly subjected to automated high-content bright field imaging or stained for fluorescence (live) cell microscopy for further image analysis. This is followed by the quality control and data analysis. The 3D-DSRT can be performed within a 1–3-week timeframe of the clinical sampling of cancer tissue, depending on the amount of the obtained tissue, growth rate of cancer cells, and the number of drugs being tested. The 3D-DSRT method can be flexibly modified, e.g., to be carried out with or without supporting matrices with U-bottom 384-well plates when appropriate for the PDCs or other cell models used.