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Example of different formats of stem cell-derived intestinal organoid cultures. Stem cells can be obtained from embryonic blastocysts or generated from adult tissue (biopsy specimens of diseased or healthy patients). aSCs (orange) can be used immediately to grow tissue-specific organoids. Embryonic or reprogrammed induced stem cells first need to develop into somatic cells (darker blue) and then the relevant germ layer (endoderm, mesoderm, or ectoderm; green) before being grown in tissuespecific organoids. Organoids then can be used directly to screen drugs or microbial metabolites in a patient-specific manner, or co-cultured with nonepithelial cells thought to interact with epithelial cells in vivo, either as 3D structures or as monolayers. To test the role of specific genetic determinants, organoids also can be genetically edited first and then used for compound screening or co-cultured with other cells containing different cell types (eg, secretory cells in green, yellow, and magenta, or absorptive in pink). KLF4, Kruppel-like factor 4; MYC, Protooncogene MYC protein; OCT4, (octamer-binding transcription factor 4; SOX2, SRY (sex determining region Y)-box 2.
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Homeostatic functions of a living tissue, such as the gastrointestinal tract (GIT), rely on highly sophisticated and finely tuned cell-to-cell interactions. These crosstalks evolve and are continuously refined as the tissue develops and give rise to specialized cells performing general and tissue-specific functions. To study these systems, stem cel...
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... Epigenetic signatures of iPSCs differ enormously from ePSCs because they can affect the reprogramming of fibroblasts into iPSCs. 58 Therefore, ePSC-and iPSC-derived organoids present some distinctions in their potential use to model human genetic disorders (Figure 1). 64 Although presenting fetal features, PSC-derived organoids can quickly gain adult maturation when first transplanted for kidney organoids, for example, 65,66 as well as intestine, liver, pancreas, and retina organoids, as recently discussed. ...
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... interaction with neighboring cells found in vivo also can be recapitulated, at least partially, in vitro, involving co-culturing organoids as monolayers or 3D structures with 2 or more different cell types (Figure 1). Various examples for such approaches are given in Table 1. ...
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... of organoids with nonepithelial cells such as peripheral blood mononuclear cell-derived dendritic cells, intestinal intraepithelial lymphocytes, or endothelial cells already has been performed by using existing systems that originally permit direct or indirect contact between different cell types. 83,94,144,145 For that, cells derived from organoids can be grown as monolayers on filter Transwell devices and exposed to signaling molecules secreted by other cells or directly to those cells (Figure 1). Alternatively, culture of organoids with other cell types into 150-to 400-mm diameter heterotypic 3D structures has proven useful in the case of hair follicle, intestinal, or kidney organoids. ...
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... Coculture systems reproducing in vitro the tissue-specific cell movement and migration within organoids also have been developed successfully. 94 These co-culture systems are highly relevant to investigate the interactions of infiltrating cell types with organoid cells 56,149 (eg, proinflammatory cells and homeostatic cells) (Figure 1). ...
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Self-organizing three-dimensional cellular models derived from human pluripotent stem cells or primary tissue have great potential to provide insights into how the human nervous system develops, what makes it unique and how disorders of the nervous system arise, progress and could be treated. Here, to facilitate progress and improve communication w...
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... Indeed, organoids can be maintained in culture and expanded for months at a time, preventing the need to obtain more tissue samples from patients. There are two major classes of organoids depending on the origin of the stem cells they were cultured from adult stem cell (aSC)-and pluripotent stem cell (PSC)-derived organoids [4,5]. ASC-derived organoids can be generated from healthy and patient tissue samples of fast-renewing tissues, e.g. ...
The intertwined interactions various immune cells have with epithelial cells in our body require sophisticated experimental approaches to be studied. Due to the limitations of immortalised cell lines and animal models, there is an increasing demand for human in vitro model systems to investigate the microenvironment of immune cells in normal and in pathological conditions. Organoids, which are self-renewing, 3D cellular structures that are derived from stem cells, have started to provide gap-filling tissue modelling solutions. In this review, we first demonstrate with some of the available examples how organoid-based immune cell co-culture experiments can advance disease modelling of cancer, inflammatory bowel disease and tissue regeneration. Then, we argue that to achieve both complexity and scale, organ-on-chip models combined with cutting-edge microfluidics-based technologies can provide more precise manipulation and readouts. Finally, we discuss how genome editing techniques and the use of patient-derived organoids and immune cells can improve disease modelling and facilitate precision medicine. To achieve maximum impact and efficiency, these efforts should be supported by novel infrastructures such as organoid biobanks, organoid facilities, as well as drug screening and host-microbe interaction testing platforms. All these together or in combination can allow researchers to shed more detailed, and often patient-specific, light on the crosstalk between immune cells and epithelial cells in health and disease.
... An alternative approach involves generating intestinal organoids from human pluripotent stem cells (PSC). The PSC could be established from either pluripotent embryonic stem cells or by inducing differentiated cells, such as fibroblasts, to convert into the pluripotent stage (92,93). Human intestinal organoids could be generated from PSC via a multistep process involving initial stem cell conversion into appropriate germ layers with subsequent differentiation into tissue-specific organoids (92,94). ...
... Inducible pluripotent stem cell-derived organoids. Since intestinal organoids generated from human inducible pluripotent stem cells (iPSC) represent more complex structures, retaining both epithelial and mesenchymal compartments, they should more closely recapitulate epithelial responses characteristic of the complex multicellular environment of the intestinal tissue (92)(93)(94). Depending on cultured conditions, iPSC-derived intestinal organoids developed either 3-D epithelial structures (103,110,129) or 2-D monolayers with well-defined AJs and TJs (114,115,130). The epithelial compartment of these organoid was shown to contain all major mucosal cell types, such as enterocytes, Goblet cells, Paneth cells, and enteroendocrine cells, identified by the presence of their lineage-specific protein markers. ...
... One of the most attractive applications of intestinal organoid research is to understand the role and mechanisms of intestinal barrier disruption during gut inflammation. There are, however, intrinsic problems and limitations of the organoid utilization, such as their significant heterogeneity, alterations in long-term culture, high data variability, the significant cost and labor intensity of the technology (81,92,93,173). Furthermore, the most frequently used adult stem cell-derived organoids still do not recapitulate the complexity of intestinal tissue due to lack of intraepithelial immune cells and the stromal compartment. Because of these limitations it is important to understand which unique aspect of gut barrier regulation should be investigated using the organoid technology and which could be addressed by working with conventional IEC lines. ...
Disruption of the intestinal epithelial barrier is a hallmark of mucosal inflammation. It increases exposure of the immune system to luminal microbes, triggering a perpetuating inflammatory response. For several decades, the inflammatory stimuli-induced breakdown of the human gut barrier was studied in vitro by using colon cancer derived epithelial cell lines. While providing a wealth of important data, these cell lines do not completely mimic the morphology and function of normal human intestinal epithelial cells (IEC) due to cancer-related chromosomal abnormalities and oncogenic mutations. The development of human intestinal organoids provided a physiologically-relevant experimental platform to study homeostatic regulation and disease-dependent dysfunctions of the intestinal epithelial barrier. There is need to align and integrate the emerging data obtained with intestinal organoids and classical studies that utilized colon cancer cell lines. This review discusses the utilization of human intestinal organoids to dissect the roles and mechanisms of gut barrier disruption during mucosal inflammation. We summarize available data generated with two major types of organoids derived from either intestinal crypts or induced pluripotent stem cells and compare them to the results of earlier studies with conventional cell lines. We identify research areas where the complementary use of colon cancer-derived cell lines and organoids advance our understanding of epithelial barrier dysfunctions in the inflamed gut and identify unique questions that could be addressed only by using the intestinal organoid platforms.
... An organoid is a three-dimensional multicellular in vitro tissue construct grown from stem cells on an extracellular matrix-like scaffold with specific niche factors (Ranga et al., 2014;Clevers, 2016;Sridhar et al., 2020;Hautefort et al., 2022) They can self-renew and aim to mimics their corresponding in vivo organ, generating in vitro functional structures containing the cell types present in the tissue they model (Hautefort et al., 2022). Therefore, organoids represent a valuable tool for studying aspects of an organ in the tissue culture dish. ...
... An organoid is a three-dimensional multicellular in vitro tissue construct grown from stem cells on an extracellular matrix-like scaffold with specific niche factors (Ranga et al., 2014;Clevers, 2016;Sridhar et al., 2020;Hautefort et al., 2022) They can self-renew and aim to mimics their corresponding in vivo organ, generating in vitro functional structures containing the cell types present in the tissue they model (Hautefort et al., 2022). Therefore, organoids represent a valuable tool for studying aspects of an organ in the tissue culture dish. ...
As new pathogens emerge, new challenges must be faced. This is no different in infectious disease research, where identifying the best tools available in laboratories to conduct an investigation can, at least initially, be particularly complicated. However, in the context of an emerging virus, such as SARS-CoV-2, which was recently detected in China and has become a global threat to healthcare systems, developing models of infection and pathogenesis is urgently required. Cell-based approaches are crucial to understanding coronavirus infection biology, growth kinetics, and tropism. Usually, laboratory cell lines are the first line in experimental models to study viral pathogenicity and perform assays aimed at screening antiviral compounds which are efficient at blocking the replication of emerging viruses, saving time and resources, reducing the use of experimental animals. However, determining the ideal cell type can be challenging, especially when several researchers have to adapt their studies to specific requirements. This review strives to guide scientists who are venturing into studying SARS-CoV-2 and help them choose the right cellular models. It revisits basic concepts of virology and presents the currently available in vitro models, their advantages and disadvantages, and the known consequences of each choice.
... Furthermore, Matrigel composition (60% laminin, 30% collagen IV, 8% entactin, and 2-3% perlecan) [92] does not reflect the composition of the lung connective tissue, which is mainly based on collagen I and III fibers [93]. A variety of natural and synthetic materials are being explored as alternatives to Matrigel for organoid cultures [8,92,[94][95][96]. Briefly, alternative scaffolds for organoid cultures can be divided in natural and synthetic substrates [92]. ...
Lung cancer is the leading cause of cancer death worldwide. Despite significant advances in research and therapy, a dismal 5-year survival rate of only 10–20% urges the development of reliable preclinical models and effective therapeutic tools. Lung cancer is characterized by a high degree of heterogeneity in its histology, a genomic landscape, and response to therapies that has been traditionally difficult to reproduce in preclinical models. However, the advent of three-dimensional culture technologies has opened new perspectives to recapitulate in vitro individualized tumor features and to anticipate treatment efficacy. The generation of lung cancer organoids (LCOs) has encountered greater challenges as compared to organoids derived from other tumors. In the last two years, many efforts have been dedicated to optimizing LCO-based platforms, resulting in improved rates of LCO production, purity, culture timing, and long-term expansion. However, due to the complexity of lung cancer, further advances are required in order to meet clinical needs. Here, we discuss the evolution of LCO technology and the use of LCOs in basic and translational lung cancer research. Although the field of LCOs is still in its infancy, its prospective development will likely lead to new strategies for drug testing and biomarker identification, thus allowing a more personalized therapeutic approach for lung cancer patients.
Bacterial enteritis has a substantial role in contributing to a large portion of the global disease burden and serves as a major cause of newborn mortality. Despite advancements gained from current animal and cell models in improving our understanding of pathogens, their widespread application is hindered by apparent drawbacks. Therefore, more precise models are imperatively required to develop more accurate studies on host-pathogen interactions and drug discovery. Since the emergence of intestinal organoids, massive studies utilizing organoids have been conducted to study the pathogenesis of bacterial enteritis, revealing new mechanisms and validating established ones. In this review, we focus on the advancements of several bacterial pathogenesis mechanisms observed in intestinal organoid/enteroid models, exploring the host response and bacterial effectors during the infection process. Finally, we address the features that warrant additional investigation or could be enhanced in existing organoid models in order to guide future research endeavors.
Cellular phenotypes and functional responses are modulated by the signals present in their microenvironment, including extracellular matrix (ECM) proteins, tissue mechanical properties, soluble signals and nutrients, and cell–cell interactions. To better recapitulate and analyze these complex signals within the framework of more physiologically relevant culture models, high throughput culture platforms can be transformative. High throughput methodologies enable scientists to extract increasingly robust and broad datasets from individual experiments, screen large numbers of conditions for potential hits, better qualify and predict responses for preclinical applications, and reduce reliance on animal studies. High throughput cell culture systems require uniformity, assay miniaturization, specific target identification, and process simplification. In this review, we detail the various techniques that researchers have used to face these challenges and explore cellular responses in a high throughput manner. We highlight several common approaches including two‐dimensional multiwell microplates, microarrays, and microfluidic cell culture systems as well as unencapsulated and encapsulated three‐dimensional high throughput cell culture systems, featuring multiwell microplates, micromolds, microwells, microarrays, granular hydrogels, and cell‐encapsulated microgels. We also discuss current applications of these high throughput technologies, namely stem cell sourcing, drug discovery and predictive toxicology, and personalized medicine, along with emerging opportunities and future impact areas.
Human intestinal organoids (HIOs) derived from pluripotent stem cells or adult stem cell biopsies represent a powerful platform to study human development, drug testing, and disease modeling in vitro, and serve as a cell source for tissue regeneration and therapeutic advances in vivo. Synthetic hydrogels can be engineered to serve as analogs of the extracellular matrix to support HIO growth and differentiation. These hydrogels allow for tuning the mechanical and biochemical properties of the matrix, offering an advantage over biologically derived hydrogels such as Matrigel TM . Human intestinal organoids have been used for repopulating transplantable intestinal grafts and for in vivo delivery to an injured intestinal site. The use of synthetic hydrogels for in vitro culture and for in vivo delivery is expected to significantly increase the relevance of human intestinal organoids for drug screening, disease modeling, and therapeutic applications.
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Inflammatory bowel disease causes a major burden to patients and healthcare systems, raising the need to develop effective therapies. Technological advances in cell culture, allied with ethical issues, have propelled in vitro models as essential tools to study disease aetiology, its progression, and possible therapies. Several cell‐based in vitro models of intestinal inflammation have been used, varying in their complexity and methodology to induce inflammation. Immortalized cell lines are extensively used due to their long‐term survival, in contrast to primary cultures that are short‐lived but patient‐specific. Recently, organoids and organ‐chips have demonstrated great potential by being physiologically more relevant. This review aims to shed light on the intricate nature of intestinal inflammation and cover recent works that report cell‐based in vitro models of human intestinal inflammation, encompassing diverse approaches and outcomes.
In vitro models of the human gut help compensate for the limitations of animal models in studying the human gut-microbiota interaction and are indispensable in the clarification the mechanism of microbial action or in the high-throughput screening and functional evaluation of probiotics. The development of these models constitutes a rapidly developing field of research. From 2D1 to 3D2 and from simple to complex, several in vitro cell and tissue models have been developed and continuously improved. In this review, we categorized and summarized these models and described their development, applications, advances, and limitations by using specific examples. We also highlighted the best ways to select an appropriate in vitro model, and we also discussed which variables to consider when imitating microbial and human gut epithelial interactions.
