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BACKGROUND Recent advances in gastrointestinal (GI) organoids from PSCs have enabled modeling crosstalk among epithelial, mesenchymal, and immune cells. Moreover, by modulating Wnt, FGF, and BMP signals, GI organoids can be regionalized into Human Intestinal Organoids (HIO) with duodenal (duo-HIO) or ileal (ile-HIO) identity, or Human Colonic Organoids (HCO). These traits make them a key platform for studying the transmural and multiregional pathophysiology of Crohn’s disease (CD). Given experimental variability in the differentiation ability into different GI regions, as well as across different PSC lines, we aimed to optimize current HIO/HCO protocols by analyzing each differentiation step using multiple PSC lines. METHODS This study used iPSC lines from CD patients and hESC. PSCs were treated with Activin A for 3 days, followed by FGF4 and CHIR99021 for 4 days (duo-HIO/HCO) or 6 days (ile-HIO). Mid-hindgut (MHG) spheroids were cultured with EGF to induce duo/ile-HIO. For HCO, BMP2 was added for the first 3 days. Cells and organoids were analyzed by immunostaining and qPCR. Immunostaining with PDX1, CDX2, SATB2 and GATA3 confirmed duodenal, intestinal, ileal/colon, and urothelial identity, respectively. RESULTS Using the original protocols, we and others observed regional heterogeneity in organoids at the end of differentiation, approximately 4 weeks in culture. In duo-HIO conditions we observed organoids with duodenal and non-duodenal intestinal domains. In ile-HIO/HCO conditions, we observed co-emerging urothelium. These results suggest that changes in reagents since the original protocols were established is causing regional drift. We hypothesized that activities of Activin A and FGF4 may be higher. By optimizing these we observed more robust and reproducible production of uniformly regionalized organoids. For example, more uniform duo-HIOs can be generated by lowering FGF4. The second significant area of variability was in spheroid viability across iPSC lines and experiments. We observed that spheroids were less viable when the ratio of FOXF1+ mesodermal to CDX2+ cells was too low at the MHG stage. The main drivers of this ratio were the activities of Activin A and FGF4. Compared to the original protocols, we found that the recommended concentrations of Activin A and FGF4 were too high and negatively impacted mesoderm. By lowering both and adding ROCK inhibitor for the first 3 days of spheroid culture, we found that the growth of spheroids and production of HIOs and HCOs from even difficult iPSC lines were vastly improved. CONCLUSIONS Through methodical analysis of each stage of HIO/HCO differentiation across multiple PSC lines, we identified that changes in reagent activities over the years may be the driver of variability in GI organoid production. Our current optimized protocol yields a higher number of properly regionalized organoids.

Recently, many types of in vitro 3‐D culture systems have been developed to recapitulate the in vivo growth conditions of cancer. The cancer 3‐D culture methods aim to preserve the biological characteristics of original tumors better than conventional 2‐D monolayer cultures, and include tumor‐derived organoids, tumor‐derived spheroids, organotypic multicellular spheroids, and multicellular tumor spheroids. The 3‐D culture methods differ in terms of cancer cell sources, protocols for cell handling, and the required time intervals. Tumor‐derived spheroids are unique because they are purposed for the enrichment of cancer stem cells (CSCs) or cells with stem cell‐related characteristics. These spheroids are grown as floating spheres and have been used as surrogate systems to evaluate the CSC‐related characteristics of solid tumors in vitro. Because eradication of CSCs is likely to be of clinical importance due to their association with the malignant nature of cancer cells, such as tumorigenicity or chemoresistance, the investigation of tumor‐derived spheroids may provide invaluable clues to fight against cancer. Spheroid cultures have been established from cancers including glioma, breast, colon, ovary, and prostate cancers, and their biological and biochemical characteristics have been investigated by many research groups. In addition to the investigation of CSCs, tumor‐derived spheroids may prove to be instrumental for a high‐throughput screening platform or for the cultivation of CSC‐related tumor cells found in the circulation or body fluids. Tumor‐derived spheroid culture is one of the representative 3D culture methods in which cancer cells with stem cell‐like features are expanded in vitro as floating spheres. In this review, we summarize the major discoveries from studies using tumor‐derived spheroids and future clinical applications.

Tumour spheroids are common in vitro experimental models of avascular tumour growth. Compared with traditional two-dimensional culture, tumour spheroids more closely mimic the avascular tumour microenvironment where spatial differences in nutrient availability strongly influence growth. We show that spheroids initiated using significantly different numbers of cells grow to similar limiting sizes, suggesting that avascular tumours have a limiting structure; in agreement with untested predictions of classical mathematical models of tumour spheroids. We develop a novel mathematical and statistical framework to study the structure of tumour spheroids seeded from cells transduced with fluorescent cell cycle indicators, enabling us to discriminate between arrested and cycling cells and identify an arrested region. Our analysis shows that transient spheroid structure is independent of initial spheroid size, and the limiting structure can be independent of seeding density. Standard experimental protocols compare spheroid size as a function of time; however, our analysis suggests that comparing spheroid structure as a function of overall size produces results that are relatively insensitive to variability in spheroid size. Our experimental observations are made using two melanoma cell lines, but our modelling framework applies across a wide range of spheroid culture conditions and cell lines.

Background Most high-throughput screening (HTS) systems studying the cytotoxic effect of chimeric antigen receptor (CAR) T cells on tumor cells rely on two-dimensional cell culture that does not recapitulate the tumor microenvironment (TME). Tumor spheroids, however, can recapitulate the TME and have been used for cytotoxicity assays of CAR T cells. But a major obstacle to the use of tumor spheroids for cytotoxicity assays is the difficulty in separating unbound CAR T and dead tumor cells from spheroids. Here, we present a three-dimensional hanging spheroid plate (3DHSP), which facilitates the formation of spheroids and the separation of unbound and dead cells from spheroids during cytotoxicity assays. Results The 3DHSP is a 24-well plate, with each well composed of a hanging dripper, spheroid wells, and waste wells. In the dripper, a tumor spheroid was formed and mixed with CAR T cells. In the 3DHSP, droplets containing the spheroids were deposited into the spheroid separation well, where unbound and dead T and tumor cells were separated from the spheroid through a gap into the waste well by tilting the 3DHSP by more than 20°. Human epidermal growth factor receptor 2 (HER2)-positive tumor cells (BT474 and SKOV3) formed spheroids of approximately 300–350 μm in diameter after 2 days in the 3DHSP. The cytotoxic effects of T cells engineered to express CAR recognizing HER2 (HER2-CAR T cells) on these spheroids were directly measured by optical imaging, without the use of live/dead fluorescent staining of the cells. Our results suggest that the 3DHSP could be incorporated into a HTS system to screen for CARs that enable T cells to kill spheroids formed from a specific tumor type with high efficacy or for spheroids consisting of tumor types that can be killed efficiently by T cells bearing a specific CAR. Conclusions The results suggest that the 3DHSP could be incorporated into a HTS system for the cytotoxic effects of CAR T cells on tumor spheroids. Graphical Abstract

Spheroids are three-dimensional cellular models with widespread basic and translational application across academia and industry. However, methodological transparency and guidelines for spheroid research have not yet been established. The MISpheroID Consortium developed a crowdsourcing knowledgebase that assembles the experimental parameters of 3,058 published spheroid-related experiments. Interrogation of this knowledgebase identified heterogeneity in the methodological setup of spheroids. Empirical evaluation and interlaboratory validation of selected variations in spheroid methodology revealed diverse impacts on spheroid metrics. To facilitate interpretation, stimulate transparency and increase awareness, the Consortium defines the MISpheroID string, a minimum set of experimental parameters required to report spheroid research. Thus, MISpheroID combines a valuable resource and a tool for three-dimensional cellular models to mine experimental parameters and to improve reproducibility.A knowledgebase developed for increased the transparency of reporting in spheroid research.

Traditionally, the monolayer (two-dimensional) cell cultures are used for initial evaluation of the effectiveness of anticancer drugs. In particular, these experiments provide the IC50 curves that determine drug concentration that can inhibit growth of a tumor colony by half when compared to the cells grown with no exposure to the drug. Low IC50 value means that the drug is effective at low concentrations, and thus will show lower systemic toxicity when administered to the patient. However, in these experiments cells are grown in a monolayer, all well exposed to the drug, while in vivo tumors expand as three-dimensional multicellular masses, where inner cells have a limited access to the drug. Therefore, we performed computational studies to compare the IC50 curves for cells grown as a two-dimensional monolayer and a cross section through a three-dimensional spheroid. Our results identified conditions (drug diffusivity, drug action mechanisms and cell proliferation capabilities) under which these IC50 curves differ significantly. This will help experimentalists to better determine drug dosage for future in vivo experiments and clinical trials.

Cancer is a complex disease in which both genetic defects and microenvironmental components contribute to the development, progression, and metastasization of disease, representing major hurdles in the identification of more effective and safer treatment regimens for patients. Three-dimensional (3D) models are changing the paradigm of preclinical cancer research as they more closely resemble the complex tissue environment and architecture found in clinical tumors than in bidimensional (2D) cell cultures. Among 3D models, spheroids and organoids represent the most versatile and promising models in that they are capable of recapitulating the heterogeneity and pathophysiology of human cancers and of filling the gap between conventional 2D in vitro testing and animal models. Such 3D systems represent a powerful tool for studying cancer biology, enabling us to model the dynamic evolution of neoplastic disease from the early stages to metastatic dissemination and the interactions with the microenvironment. Spheroids and organoids have recently been used in the field of drug discovery and personalized medicine. The combined use of 3D models could potentially improve the robustness and reliability of preclinical research data, reducing the need for animal testing and favoring their transition to clinical practice. In this review, we summarize the recent advances in the use of these 3D systems for cancer modeling, focusing on their innovative translational applications, looking at future challenges, and comparing them with most widely used animal models.

Organogenesis is a complex and interconnected process that is orchestrated by multiple boundary tissue interactions 1 – 7 . However, it remains unclear how individual, neighbouring components coordinate to establish an integral multi-organ structure. Here we report the continuous patterning and dynamic morphogenesis of hepatic, biliary and pancreatic structures, invaginating from a three-dimensional culture of human pluripotent stem cells. The boundary interactions between anterior and posterior gut spheroids differentiated from human pluripotent stem cells enables retinoic acid-dependent emergence of hepato-biliary-pancreatic organ domains specified at the foregut–midgut boundary organoids in the absence of extrinsic factors. Whereas transplant-derived tissues are dominated by midgut derivatives, long-term-cultured microdissected hepato-biliary-pancreatic organoids develop into segregated multi-organ anlages, which then recapitulate early morphogenetic events including the invagination and branching of three different and interconnected organ structures, reminiscent of tissues derived from mouse explanted foregut–midgut culture. Mis-segregation of multi-organ domains caused by a genetic mutation in HES1 abolishes the biliary specification potential in culture, as seen in vivo 8 , 9 . In sum, we demonstrate that the experimental multi-organ integrated model can be established by the juxtapositioning of foregut and midgut tissues, and potentially serves as a tractable, manipulatable and easily accessible model for the study of complex human endoderm organogenesis. Juxtaposition of region-specific gut spheroids derived from human pluripotent stem cells in the absence of extrinsic factors results in development of segregated hepato-biliary-pancreatic anlages that recapitulate early morphogenetic events.

Tissue spheroids fuse to form larger tissue structures, a process controlled by their living material properties. However, how these properties emerge from the active behavior of individual cells is not well understood. Here, we studied fusion dynamics of spheroids from human periosteum-derived cells. Using confocal microscopy, we measured spheroid granularity and, with two-photon microscopy, we quantified active cell movements during fusion. Inhibiting cytoskeletal contractility with Y-27632 Rho kinase inhibitor produced more granular tissues with fewer cell rearrangements but faster fusion. Further reducing contractility with blebbistatin and Y-27632 increased granularity, reduced rearrangements, and slowed fusion. Across all conditions, complete fusion coincided with frequent cell rearrangements. We present an active foam model representing cells as viscous shells with interfacial tension and persistent motility to link fusion outcomes and tissue fluidity to measurable cell properties. This framework shows how cell activity regulates tissue mechanics and offers insights for tissue assembly in regenerative medicine. Combining microscopy and modeling, the authors reveal that tissue fluidity, driven by active cell motion and interfacial tension, governs how living spheroids merge into larger structures.

Recent breakthroughs in 3-dimensional (3D) organoid cultures for many organ systems have led to new physiologically complex in vitro models to study human development and disease. Here, we report the step-wise differentiation of human pluripotent stem cells (hPSCs) (embryonic and induced) into lung organoids. By manipulating developmental signaling pathways hPSCs generate ventral-anterior foregut spheroids, which are then expanded into human lung organoids (HLOs). HLOs consist of epithelial and mesenchymal compartments of the lung, organized with structural features similar to the native lung. HLOs possess upper airway-like epithelium with basal cells and immature ciliated cells surrounded by smooth muscle and myofibroblasts as well as an alveolar-like domain with appropriate cell types. Using RNA-sequencing, we show that HLOs are remarkably similar to human fetal lung based on global transcriptional profiles, suggesting that HLOs are an excellent model to study human lung development, maturation and disease. Cell behavior has traditionally been studied in the lab in two-dimensional situations, where cells are grown in thin layers on cell-culture dishes. However, most cells in the body exist in a three-dimensional environment as part of complex tissues and organs, and so researchers have been attempting to re-create these environments in the lab. To date, several such ‘organoids’ have been successfully generated, including models of the human intestine, stomach, brain and liver. These organoids can mimic the responses of real tissues and can be used to investigate how organs form, change with disease, and how they might respond to potential therapies. Here, Dye et al. developed a new three-dimensional model of the human lung by coaxing human stem cells to become specific types of cells that then formed complex tissues in a petri dish. To make these lung organoids, Dye et al. manipulated several of the signaling pathways that control the formation of organs during the development of animal embryos. First, the stem cells were instructed to form a type of tissue called endoderm, which is found in early embryos and gives rise to the lung, liver and other several other internal organs. Then, Dye et al. activated two important developmental pathways that are known to make endoderm form three-dimensional intestinal tissue. However, by inhibiting two other key developmental pathways at the same time, the endoderm became tissue that resembles the early lung found in embryos instead. This early lung-like tissue formed three-dimensional spherical structures as it developed. The next challenge was to make these structures develop into lung tissue. Dye et al. worked out a method to do this, which involved exposing the cells to additional proteins that are involved in lung development. The resulting lung organoids survived in laboratory cultures for over 100 days and developed into well-organized structures that contain many of the types of cells found in the lung. Further analysis revealed the gene activity in the lung organoids resembles that of the lung of a developing human fetus, suggesting that lung organoids grown in the dish are not fully mature. Dye et al.'s findings provide a new approach for creating human lung organoids in culture that may open up new avenues for investigating lung development and diseases.