Organoid Culture Method Research Articles

Brain organoids: A new tool for modelling of neurodevelopmental disorders.

Neurodevelopmental disorders are mostly studied using mice as models. However, the mouse brain lacks similar cell types and structures as those of the human brain. In recent years, emergence of three-dimensional brain organoids derived from human embryonic stem cells or induced pluripotent stem cells allows for controlled monitoring and evaluation of early neurodevelopmental processes and has opened a window for studying various aspects of human brain development. However, such organoids lack original anatomical structure of the brain during maturation, and neurodevelopmental maturation processes that rely on unique cellular interactions and neural network connections are limited. Consequently, organoids are difficult to be used extensively and effectively while modelling later stages of human brain development and disease progression. To address this problem, several methods and technologies have emerged that aim to enhance the sophisticated regulation of brain organoids developmental processes through bioengineering approaches, which may alleviate some of the current limitations. This review discusses recent advances and application areas of human brain organoid culture methods, aiming to generalize optimization strategies for organoid systems, improve the ability to mimic human brain development, and enhance the application value of organoids.

Oncofetal morphogenesis similar to embryonic gut formation by a subpopulation of DLD-1 human colon cancer cells

Cancer stem cells (CSC) are thought to be responsible for cancer phenotypes and cellular heterogeneity. Here we demonstrate that the human colon cancer cell line DLD1 contains two types of CSC-like cells that undergo distinct morphogenesis in the reconstituted basement membrane gel Matrigel. In our method with cancer cell spheroids, the parent cell line (DLD1-P) developed grape-like budding structures, whereas the other (DLD1-Wm) and its single-cell clones dynamically developed worm-like ones. Gene expression analysis suggested that the former mimicked intestinal crypt-villus morphogenesis, while the latter mimicked embryonic hindgut development. The organoids of DLD1-Wm cells rapidly extended in two opposite directions by expressing dipolar proteolytic activity. The invasive morphogenesis required the expression of MMP-2 and CD133 genes and ROCK activity. These cells also exhibited gastrula-like morphogenesis even in two-dimensional cultures without Matrigel. Moreover, the two DLD1 cell lines showed clear differences in cellular growth, tumor growth and susceptibility to paclitaxel. This study also provides a simple organoid culture method for human cancer cell lines. HT-29 and other cancer cell lines underwent characteristic morphogenesis in direct contact with normal fibroblasts. Such organoid cultures would be useful for investigating the nature of CSCs and for screening anti-cancer drugs. Our results lead to the hypothesis that CSC-like cells with both invasive activity and a fetal phenotype, i. e. oncofetal CSCs, are generated in some types of colon cancers.

Recent Progress in Generation of Inner Ear Organoid.

Inner ear organoids play a crucial role in hearing research. In comparison to other animal models and 2D cell culture systems, inner ear organoids offer significant advantages for studying the mechanisms of inner ear development and exploring novel approaches to disease treatment. Inner ear organoids derived from human cells are more closely resemble normal human organs in development and function. The 3D culture system of the inner ear organoid enhances cell-cell interactions and mimics the internal environment. In this review, the progress and limitations of organoid culture methods derived from tissue-specific progenitors and pluripotent stem cells (PSCs) are summarized, which may offer new insights into generating organoids that closely resemble the inner ear in terms of morphology and function.

Breast organoid suspension cultures maintain long-term estrogen receptor expression and responsiveness.

Organoid cultures offer a powerful technology to investigate many different aspects of development, physiology, and pathology of diverse tissues. Unlike standard tissue culture of primary breast epithelial cells, breast organoids preserve the epithelial lineages and architecture of the normal tissue. However, existing organoid culture methods are tedious, difficult to scale, and do not robustly retain estrogen receptor (ER) expression and responsiveness in long-term culture. Here, we describe a modified culture method to generate and maintain organoids as suspension cultures in reconstituted basement membrane (™Matrigel). This method improves organoid growth and uniformity compared to the conventional Matrigel dome embedding method, while maintaining the fidelity of the three major epithelial lineages. Using this adopted method, we are able to culture and passage purified hormone sensing (HS) cells that retain ER responsiveness upon estrogen stimulation in long-term culture. This culture system presents a valuable platform to study the events involved in initiation and evolution of ER-positive breast cancer.

Guidelines for Manufacturing and Application of Organoids: Brain.

This study offers a comprehensive overview of brain organoids for researchers. It combines expert opinions with technical summaries on organoid definitions, characteristics, culture methods, and quality control. This approach aims to enhance the utilization of brain organoids in research. Brain organoids, as three-dimensional human cell models mimicking the nervous system, hold immense promise for studying the human brain. They offer advantages over traditional methods, replicating anatomical structures, physiological features, and complex neuronal networks. Additionally, brain organoids can model nervous system development and interactions between cell types and the microenvironment. By providing a foundation for utilizing the most human-relevant tissue models, this work empowers researchers to overcome limitations of two-dimensional cultures and conduct advanced disease modeling research.

Abstract 4243: Revolutionizing cancer research, new drug screening approach with vascularized cancer organoid platform mimicking patient tumor microenvironment

Abstract Patient-derived organoids (PDOs) are improving our understanding of cancer heterogeneity and personalized medicine. In particular, patients' intrinsic and acquired treatment resistance is being evaluated with PDOs, which has become an essential factor in developing new anticancer drugs and designing successful clinical trials. However, there are obstacles to cancer heterogeneity and patient-specific cancer therapy due to lack of understanding of tumoral diversity resulting from the tumor microenvironment (TME). Complex direct and indirect interactions between various types of TME cells and the resulting tumoral diversity are challenges that must be addressed for customized cancer therapy. Standard cancer therapy targets tumor cells without considering possible damage on the tumor microenvironment that could impair therapy response. Therefore, the construction and analysis of a platform for co-culturing cancer organoids with other cell types that make up the TME over a long period of time is necessary. We generated and performed characterization of blood vessel organoid (BVO). The BVO expressed endothelial marker CD31 and pericyte marker PDGFRβ. We attempted to co-culture with patient-derived lung cancer organoid (LCO) on Matrigel by dissociating the constructed BVO, and it was confirmed that BVO and LCO were interconnected after 10 days. This new platform was treated with Gefitinib and Poziotinib, and after 5 days of drug treatment, the IC50 value increased compared to LCO-only culture. Additionally, flow cytometry analysis confirmed that co-culturing with BVO during drug treatment resulted in a lower percentage of dead LCO cells compared to LCO-only culture. Image-based analysis also verified that co-culturing with blood vessels enhances the viability of LCOs when treated with drugs. Taken together, the co-culture of cancer organoids and BVOs present a platform that closely mimics the in vivo TME compared to the existing cancer organoid culture method. This innovative platform demonstrates higher drug resistance than existing approaches, offering a novel method for drug screening. Citation Format: Mingyun Lee, Yeo-Joon Yoon, Yong-Soo Lee, Jinguen Rheey. Revolutionizing cancer research, new drug screening approach with vascularized cancer organoid platform mimicking patient tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4243.

Abstract A114: Robust establishment and expansion of multilineage human fallopian tube organoids in serum-free medium

Abstract High-grade serous ovarian cancer (HGSOC) is the most prevalent subtype of epithelial ovarian cancer. Despite its name, HGSOC originates in the fallopian tube (FT), but metastasizes quickly in the adnexal region and is the most lethal gynecological cancer worldwide. Organoid culture is emerging as a powerful model for studying normal FT cell biology and ovarian cancer. To standardize primary human FT and HGSOC organoid culture, we have developed GyneCult Fallopian Tube Organoid Medium (FTOM), an optimized serum-free medium and a workflow that supports robust and representative FT organoid culture from freshly isolated or cryopreserved primary human FT cells. FT cultures were initiated by seeding 4000 fresh or cryopreserved single cells (8 donors) directly into 20 µL Corning® Matrigel® domes and overlaying them with FT Organoid Medium. Cultures were maintained with full-medium changes every 3 - 4 days and were passaged every 8 - 14 days into new 20 µL Matrigel® droplets, either at a passage ratio of 1:3 or by seeding at 4000 cells per droplet. Cultures were analyzed by immunocytochemistry (ICC) to detect secretory markers keratin 7 (KRT7), oviductal glycoprotein 1 (OVGP1), and PAX8, as well as ciliated cell markers acetylated alpha tubulin (TUBA1A) and FOXJ1. Across all donors, 16 ± 8% (mean ± SD) of dissociated EpCAM+ FT cells formed 50 - 300 µm diameter cystic organoids within 14 days of seeding (n = 8). Cultures can be maintained for at least 5 passages with 14 - 20 cumulative population doublings, at 3 - 4 doublings per passage. ICC analysis confirmed that organoids contained both polarized secretory cells (i.e., KRT7+, OVGP1+, PAX8+) and ciliated cells (i.e. acetylated TUBA1A+, FOXJ1+), indicating multilineage capacity (n = 5). We also tested the compatibility of GyneCult FTOM with culturing HGSOC samples and observed that one of three tumor samples tested generated organoids. These tumour organoids had a solid and irregular shaped morphology and were composed of PAX8+ cells. These cells underwent ≥ 5 cumulative population doublings over 5 passages, with 0.5 - 2 doublings per passage; this performance is consistent with recently published HGSOC organoid culture methods. These results demonstrate that GyneCult Fallopian Tube Organoid Medium is a robust medium for initiating and culturing FT epithelium as organoids, can support HGSOC organoid culture, and is a valuable tool for studying FT biology. Citation Format: Victor Ho, Maggie Chan, Manreet Chehal, Alice Liang, Allen C Eaves, Sharon A Louis, John Stingl. Robust establishment and expansion of multilineage human fallopian tube organoids in serum-free medium [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr A114.

A human lymphoma organoid model for evaluating and targeting the follicular lymphoma tumor immune microenvironment

Heterogeneity in the tumor microenvironment (TME) of follicular lymphomas (FLs) can affect clinical outcomes. Current immunotherapeutic strategies, including antibody- and cell-based therapies, variably overcome pro-tumorigenic mechanisms for sustained disease control. Modeling the intact FL TME, with its native, syngeneic tumor-infiltrating leukocytes, is a major challenge. Here, we describe an organoid culture method for cultivating patient-derived lymphoma organoids (PDLOs), which include cells from the native FL TME. We define the robustness of this method by successfully culturing cryopreserved FL specimens from diverse patients and demonstrate the stability of TME cellular composition, tumor somatic mutations, gene expression profiles, and B/T cell receptor dynamics over 3weeks. PDLOs treated with CD3:CD19 and CD3:CD20 therapeutic bispecific antibodies showed B cell killing and Tcell activation. This stable system offers a robust platform for advancing precision medicine efforts in FL through patient-specific modeling, high-throughput screening, TME signature identification, and treatment response evaluation.

Uniform cerebral organoid culture on a pillar plate by simple and reproducible spheroid transfer from an ultralow attachment well plate

Human induced pluripotent stem cell (iPSC)-derived brain organoids have potential to recapitulate the earliest stages of brain development, serving as an effective in vitro model for studying both normal brain development and disorders. However, current brain organoid culture methods face several challenges, including low throughput, high variability in organoid generation, and time-consuming, multiple transfer and encapsulation of cells in hydrogels throughout the culture. These limitations hinder the widespread application of brain organoids including high-throughput assessment of compounds in clinical and industrial lab settings. In this study, we demonstrate a straightforward approach of generating multiple cerebral organoids from iPSCs on a pillar plate platform, eliminating the need for labor-intensive, multiple transfer and encapsulation steps to ensure the reproducible generation of cerebral organoids. We formed embryoid bodies in an ultra-low attachment 384-well plate and subsequently transferred them to the pillar plate containing Matrigel, using a straightforward sandwiching and inverting method. Each pillar on the pillar plate contains a single spheroid, and the success rate of spheroid transfer was in a range of 95%–100%. Using this approach, we robustly generated cerebral organoids on the pillar plate and demonstrated an intra-batch coefficient of variation below 9%–19% based on ATP-based cell viability and compound treatment. Notably, our spheroid transfer method in combination with the pillar plate allows miniaturized culture of cerebral organoids, alleviates the issue of organoid variability, and has potential to significantly enhance assay throughput by allowing in situ organoid assessment as compared to conventional organoid culture in 6-/24-well plates, petri dishes, and spinner flasks.

A bioactive calcium silicate nanowire-containing hydrogel for organoid formation and functionalization.

Organoids, which are 3D multicellular constructs, have garnered significant attention in recent years. Existing organoid culture methods predominantly utilize natural and synthetic polymeric hydrogels. This study explored the potential of a composite hydrogel mainly consisting of calcium silicate (CS) nanowires and methacrylated gelatin (GelMA) as a substrate for organoid formation and functionalization, specifically for intestinal and liver organoids. Furthermore, the research delved into the mechanisms by which CS nanowires promote the structure formation and development of organoids. It was discovered that CS nanowires can influence the stiffness of the hydrogel, thereby regulating the expression of the mechanosensory factor yes-associated protein (YAP). Additionally, the bioactive ions released by CS nanowires in the culture medium could accelerate Wnt/β-catenin signaling, further stimulating organoid development. Moreover, bioactive ions were found to enhance the nutrient absorption and ATP metabolic activity of intestinal organoids. Overall, the CS/GelMA composite hydrogel proves to be a promising substrate for organoid formation and development. This research suggested that inorganic biomaterials hold significant potential in organoid research, offering bioactivities, biosafety, and cost-effectiveness.

Culturing Cardiac Tissue Slices Under Continuous Physiological Mechanical Stretches.

Testing drugs in vivo and in vitro have been essential elements for the discovery of new therapeutics. Due to the recent advances in in vitro cell culture models, such as human-induced pluripotent stem cell-derived cardiomyocytes and 3D multicell type organoid culture methods, the detection of adverse cardiac events prior to human clinical trials has improved. However, there are still numerous therapeutics whose adverse cardiac effects are not detected until human trials due to the inability of these cell cultures to fully model the complex multicellular organization of an intact human myocardium. Cardiac tissue slices are a possible alternative solution. Myocardial slices are a 300-micron thin snapshot of the myocardium, capturing a section of the adult heart in a 1×1cm section. Using a culture method that incorporates essential nutrients and electrical stimulation, tissue slices can be maintained in culture for 6days with full viability and functionality. With the addition of mechanical stimulation and humoral cues, tissue slices can be cultured for 12days. Here we provide detailed methods for how to culture cardiac tissue slices under continuous mechanical stimulation in the cardiac tissue culture model (CTCM) device. The CTCM incorporates four essential factors for maintaining tissue slices in culture for 12days: mechanical stimulation, electrical stimulation, nutrients, and humoral cues. The CTCM can also be used to model disease conditions, such as overstretch-induced cardiac hypertrophy. The versatility of the CTCM illustrates its potential to be a medium-throughput screening platform for personalized drug testing.

Method for Two-Dimensional Epithelial Monolayer Formation Derived from Mouse Three-Dimensional Small Intestinal Organoids.

The intestinal epithelium is composed of two distinct structures, namely, the villi and crypts. The base of the crypts contains intestinal stem cells (ISCs), which support the high regenerative capacity of the intestinal epithelium. With the establishment of the three-dimensional (3D) organoid culture method, the cellular and molecular mechanisms of differentiation, proliferation, and maintenance of ISCs have been widely analyzed. However, the sphere-like morphology of the 3D organoids prevents access to the apical side of the epithelium. To overcome this limitation, two-dimensional (2D) monolayer cultures derived from 3D organoids have been attempted; however, 2D culture methods for the mouse small intestine have not been well established. In this study, we developed a simple method that uses only commercially available materials, for the formation of 2D epithelial monolayers from mouse 3D small intestinal organoids. Using this method, confluent 2D epithelial monolayers were established within 4days. This monolayer showed stable tight junction and included ISCs and differentiated intestinal cells. It also showed physiologically relevant transepithelial electrical resistance values. On the basis of these findings, this method opens a novel platform for analyzing the physiology of the intestinal epithelium, its interaction with microbes, and mechanisms of villus formation.

Alveolar Type 2 Epithelial Cell Organoids: Focus on Culture Methods.

Lung diseases rank third in terms of mortality and represent a significant economic burden globally. Scientists have been conducting research to better understand respiratory diseases and find treatments for them. An ideal in vitro model must mimic the in vivo organ structure, physiology, and pathology. Organoids are self-organizing, three-dimensional (3D) structures originating from adult stem cells, embryonic lung bud progenitors, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs). These 3D organoid cultures may provide a platform for exploring tissue development, the regulatory mechanisms related to the repair of lung epithelia, pathophysiological and immunomodulatory responses to different respiratory conditions, and screening compounds for new drugs. To create 3D lung organoids in vitro, both co-culture and feeder-free methods have been used. However, there exists substantial heterogeneity in the organoid culture methods, including the sources of AT2 cells, media composition, and feeder cell origins. This article highlights the currently available methods for growing AT2 organoids and prospective improvements to improve the available culture techniques/conditions. Further, we discuss various applications, particularly those aimed at modeling human distal lung diseases and cell therapy.

Application of organoids in precision immunotherapy of lung cancer (Review).

In immunotherapy, the immune system is modulated in order to treat cancer. Traditional two dimensional in vitro models and in vivo animal models are insufficient to simulate the complex tumor microenvironment (TME) in the original tumor. As tumor immunotherapy involves the immune system, additional tumor mimic models, such as patient-derived organoids, are required for the evaluation of the efficacy of immunotherapy. Furthermore, non-tumor components and host tumor cells in the TME may interact to promote cancer incidence, progression, drug resistance and metastasis. It is possible to produce organoid models for lung cancer by retaining endogenous stromal components (e.g., multiple immune cell types), supplying cancer-associated fibroblasts and exogenous immune cells, constructing tumor vasculature and adding other biological or chemical components that emulate the TME. Therefore, the lung cancer organoid culture platform may facilitate preclinical testing of immunotherapy drugs for lung cancer by mimicking immunotherapy responses. The present review summarizes current lung cancer organoid culture methods for TME modeling and discusses the use of lung cancer-derived organoids for the detection of lung cancer immunotherapy and individualized cancer immunotherapy.

Genetic characterization of a novel organoid from human malignant giant-cell tumor

Malignant giant-cell tumors are extremely rare bone sarcomas that transform from conventional giant-cell tumors during long periods of treatment. Owing to their rarity, no further analysis of their molecular pathogenesis exists, and thus, no standard treatment has been established. Recently, organoid culture methods have been highlighted for recapturing the tumor microenvironment, and we have applied the culture methods and succeeded in establishing patient-derived organoids (PDO) of rare sarcomas. This study aimed to investigate the genomic characteristics of our established novel organoids from human malignant giant-cell tumors. At our institute, we treated a patient with malignant giant-cell tumor. The remaining sarcoma specimens after surgical resection were cultured according to the air–liquid interface organoid-culture method. Organoids were xenografted into NOD-scid IL2Rgnull mice. The developed tumors were histologically and genomically analyzed to compare their characteristics with those of the original tumors. Genetic changes over time throughout treatment were analyzed, and the genomic status of the established organoid was confirmed. Organoids from malignant giant-cell tumors could be serially maintained using air–liquid interface organoid-culture methods. The tumors developed in xenografted NOD-scid IL2Rgnull mice. After several repetitions of the process, a patient-derived organoid line from the malignant giant-cell tumor was established. Immunohistochemical analyses and next-generation sequencing revealed that the established organoids lacked the H3-3A G34W mutation. The xenografted organoids of the malignant giant-cell tumor had phenotypes histologically and genetically similar to those of the original tumor. The established organoids were confirmed to be derived from human malignant giant-cell tumors. In summary, the present study demonstrated a novel organoid model of a malignant giant-cell tumor that was genetically confirmed to be a malignant transformed tumor. Our organoid model could be used to elucidate the molecular pathogenesis of a malignant giant-cell tumor and develop novel treatment modalities.

Prostate organoids: emerging experimental tools for translational research.

Organoid technology has provided new translational research opportunities in oncology, in part by enabling the development of patient-representative living biobanks. Prostate cancer research historically has been constrained to a small number of in vitro models, limiting the ability to translate experimental conclusions for contemporary, heterogeneous patient populations. The facility of organoid culture methods to maintain luminal prostate epithelia, the common lineage of prostate cancers, has greatly expanded the phenotypic and genotypic diversity of available tractable models, including luminal stem/progenitor cells and progressive patient-derived cancers. Biobanks of patient prostate cancer organoids enable increased accuracy in predicting therapeutic efficacy and informative clinical trial designs. Here, we discuss how prostate organoid technology is currently being used, the promising areas of future therapeutic applications, and the current obstacles to be overcome.

Standardization of organoid culture in cancer research.

Establishing a valid in vitro model to represent tumor heterogeneity and biology is critical but challenging. Tumor organoids are self-assembled three-dimensional cell clusters which are of great significance for recapitulating the histopathological, genetic, and phenotypic characteristics of primary tissues. The organoid has emerged as an attractive in vitro platform for tumor biology research and high-throughput drug screening in cancer medicine. Organoids offer unique advantages over cell lines and patient-derived xenograft models, but there are no standardized methods to guide the culture of organoids, leading to confusion in organoid studies that may affect accurate judgments of tumor biology. This review summarizes the shortcomings of current organoid culture methods, presents the latest research findings on organoid standardization, and proposes an outlook for organoid modeling.

Ciliated cell cultures for diagnosis of primary ciliary dyskinesia

Primary ciliary dyskinesia (PCD) is a hereditary autosomal recessive disease that results in a defect in the ultrastructure of epithelial cilia. To date, there is no single diagnostic test for PCD, so the diagnosis is based on the results of multiple tests, such as DNA diagnostics, assessment of nasal nitric oxide levels, ciliary beat frequency (CBF) in nasal biopsy, ciliary ultrastructure, etc. Diagnosis of PCD can be difficult due to secondary damage to the airway epithelium, leading to undiagnosed or false positive cases.The aim of this work was to review studies on the cultivation of human nasal epithelial cells and subsequent differentiation into ciliated cells for the diagnosis of PCD.Conclusion. In vitro ciliogenesis helps to make a correct diagnosis of PCD while avoiding false positives. There are three different methods of ciliogenesis in vitro: the suspension culture method, the ALI culture method, and the organoid culture method. Each method of ciliogenesis has its own advantages and disadvantages. The ALI culture method is the most widely used. It produces a sufficient number of ciliated cells for diagnosis, which can be maintained in culture for a long time. The obtained cultures of nasal epithelial ciliated cells allow to analyze the ultrastructure of cilia, to evaluate CBF and localization of ciliary proteins, which helps in the diagnosis of PCD.

Abstract 152: Robust establishment and expansion of human fallopian tube organoids in serum-free culture

Abstract High-grade serous ovarian cancer (HGSOC) is the most prevalent subtype of epithelial ovarian cancer. Despite its name, HGSOC originates in the fallopian tube (FT), but metastasizes quickly in the adnexal region and is the most lethal gynecological cancer worldwide. Organoid culture is emerging as a powerful model for studying normal FT cell biology and ovarian cancer. To standardize primary human FT and HGSOC organoid culture, we have developed GyneCult Fallopian Tube Organoid Medium (FTOM), an optimized serum-free medium and a workflow that supports robust and representative FT organoid culture from freshly isolated or cryopreserved primary human FT cells. FT cultures were initiated by seeding 4000 dissociated single cells directly into 20 µL Corning® Matrigel® domes and overlaying them with FT Organoid Medium. After seeding, cultures were maintained with full-medium changes every 3 - 4 days and were split either in a passage ratio of 1:3, or at 4,000 cells per 20 µL of Matrigel®, as single cells every 8 - 14 days. Cultures were analyzed by immunocytochemistry (ICC) to detect secretory markers keratin 7 (KRT7), oviductal glycoprotein 1 (OVGP1), and PAX8, as well as ciliated cell markers acetylated alpha tubulin (TUBA1A) and FOXJ1. Across all donor samples, approximately 16 ± 8% (mean ± SD) of dissociated EpCAM+ FT cells formed 50 - 300 µm diameter cystic organoids within 14 days (n = 8). Cultures can be maintained for at least 5 passages with 14 - 20 cumulative population doublings, at 3 - 4 doublings per passage. ICC analysis confirmed that organoids contain both polarized KRT7+ OVGP1+ PAX8+ secretory cells and acetylated TUBA1A+ FOXJ1+ ciliated cells, indicating multilineage capacity (n = 5). We also tested the compatibility of GyneCult FTOM for growing HGSOC samples, and we observed that 1 of the 3 tumor samples tested generated organoids with a solid and irregular shaped morphology. These cells could undergo a minimum of 5 cumulative population doublings over 5 passages, at 0.5 - 2 doublings per passage; this performance is in line with recently published HGSOC organoid culture methods. These results demonstrate that GyneCult Fallopian Tube Organoid Medium is a robust medium for initiating and culturing FT epithelium as organoids and is a valuable tool for studying FT biology, with the capacity to support HGSOC organoid culture. Citation Format: Victor Ho, Maggie Chan, Manreet Chehal, Alice Liang, Allen Eaves, Sharon Louis, John Stingl. Robust establishment and expansion of human fallopian tube organoids in serum-free culture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 152.

Bioengineering Liver Organoids for Diseases Modelling and Transplantation.

Organoids as three-dimension (3D) cellular organizations partially mimic the physiological functions and micro-architecture of native tissues and organs, holding great potential for clinical applications. Advances in the identification of essential factors including physical cues and biochemical signals for controlling organoid development have contributed to the success of growing liver organoids from liver tissue and stem/progenitor cells. However, to recapitulate the physiological properties and the architecture of a native liver, one has to generate liver organoids that contain all the major liver cell types in correct proportions and relative 3D locations as found in a native liver. Recent advances in stem-cell-, biomaterial- and engineering-based approaches have been incorporated into conventional organoid culture methods to facilitate the development of a more sophisticated liver organoid culture resembling a near to native mini-liver in a dish. However, a comprehensive review on the recent advancement in the bioengineering liver organoid is still lacking. Here, we review the current liver organoid systems, focusing on the construction of the liver organoid system with various cell sources, the roles of growth factors for engineering liver organoids, as well as the recent advances in the bioengineering liver organoid disease models and their biomedical applications.