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Hans Clevers - One of the best experts on this subject based on the ideXlab platform.
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Establishment of human fetal hepatocyte Organoids and CRISPR–Cas9-based gene knockin and knockout in Organoid cultures from human liver
Nature Protocols, 2020Co-Authors: Delilah Hendriks, Benedetta Artegiani, Susana Chuva De Sousa Lopes, Hans CleversAbstract:The liver is composed of two epithelial cell types: hepatocytes and liver ductal cells. Culture conditions for expansion of human liver ductal cells in vitro as Organoids were previously described in a protocol; however, primary human hepatocytes remained hard to expand, until recently. In this protocol, we provide full details of how we overcame this limitation, establishing culture conditions that facilitate long-term expansion of human fetal hepatocytes as Organoids. In addition, we describe how to generate (multi) gene knockouts using CRISPR–Cas9 in both human fetal hepatocyte and adult liver ductal Organoid systems. Using a CRISPR–Cas9 and homology-independent Organoid transgenesis (CRISPR-HOT) approach, efficient gene knockin can be achieved in these systems. These gene knockin and knockout approaches, and their multiplexing, should be useful for a variety of applications, such as disease modeling, investigating gene functions and studying processes, such as cellular differentiation and cell division. The protocol to establish human fetal hepatocyte Organoid cultures takes ~1–2 months. The protocols to genome engineer human liver ductal Organoids and human fetal hepatocyte Organoids take 2–3 months. Culture conditions are described for long-term expansion of human fetal hepatocytes as 3D Organoids. Gene knockin and knockout approaches are also described for Organoids derived from human fetal hepatocytes and human adult liver ductal cells.
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Establishment of patient-derived cancer Organoids for drug-screening applications
Nature Protocols, 2020Co-Authors: Else Driehuis, Kai Kretzschmar, Hans CleversAbstract:Adult stem cell–based Organoid technology is a versatile tool for the generation and long-term maintenance of near-native 3D epithelial tissues in vitro. The generation of cancer Organoids from primary patient material enables a range of therapeutic agents to be tested in the resulting Organoid cultures. Patient-derived cancer Organoids therefore hold great promise for personalized medicine. Here, we provide an overview of the protocols used by different groups to establish Organoids from various epithelial tissues and cancers, plus the different protocols subsequently used to test the in vitro therapy sensitivity of these patient-derived Organoids. We also provide an in-depth protocol for the generation of head and neck squamous cell carcinoma Organoids and their subsequent use in semi-automated therapy screens. Establishment of Organoids and subsequent screening can be performed within 3 months, although this timeline is highly dependent on a.o. starting material and the number of therapies tested. The protocol provided may serve as a reference to successfully establish Organoids from other cancer types and perform drug screenings thereof. This protocol summarizes the various approaches available to derive Organoids from cancer patients and use these for screening of possible treatments. An optimized protocol for using head and neck cancer Organoids is also described.
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Cancer modeling meets human Organoid technology.
Science (New York N.Y.), 2019Co-Authors: David A. Tuveson, Hans CleversAbstract:Organoids are microscopic self-organizing, three-dimensional structures that are grown from stem cells in vitro. They recapitulate many structural and functional aspects of their in vivo counterpart organs. This versatile technology has led to the development of many novel human cancer models. It is now possible to create indefinitely expanding Organoids starting from tumor tissue of individuals suffering from a range of carcinomas. Alternatively, CRISPR-based gene modification allows the engineering of Organoid models of cancer through the introduction of any combination of cancer gene alterations to normal Organoids. When combined with immune cells and fibroblasts, tumor Organoids become models for the cancer microenvironment enabling immune-oncology applications. Emerging evidence indicates that Organoids can be used to accurately predict drug responses in a personalized treatment setting. Here, we review the current state and future prospects of the rapidly evolving tumor Organoid field.
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Stem cell-derived Organoids and their application for medical research and patient treatment
Journal of Molecular Medicine, 2017Co-Authors: Sina Bartfeld, Hans CleversAbstract:3D culture has allowed the initiation and expansion of organ-like structures, called Organoids, from either tissue-resident adult stem cells or pluripotent stem cells. Today, Organoids can be grown to resemble a wide variety of organs, exhibiting remarkable similarity to their in vivo counterparts. As successful Organoid generation is possible from virtually every patient, Organoids hold a great promise for medical research and the development of new treatments. They have already found their way into the clinic, enabling personalized medicine in small patient trials. In this review, we provide an update on current Organoid technology and summarize their application in basic research, disease modelling, drug development, personalized treatment and regenerative medicine.
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designer matrices for intestinal stem cell and Organoid culture
Nature, 2016Co-Authors: Nikolce Gjorevski, Hans Clevers, Norman Sachs, Andrea Manfrin, Sonja Giger, Maiia E Bragina, Paloma Ordonezmoran, Matthias P LutolfAbstract:Epithelial Organoids recapitulate multiple aspects of real organs, making them promising models of organ development, function and disease. However, the full potential of Organoids in research and therapy has remained unrealized, owing to the poorly defined animal-derived matrices in which they are grown. Here we used modular synthetic hydrogel networks to define the key extracellular matrix (ECM) parameters that govern intestinal stem cell (ISC) expansion and Organoid formation, and show that separate stages of the process require different mechanical environments and ECM components. In particular, fibronectin-based adhesion was sufficient for ISC survival and proliferation. High matrix stiffness significantly enhanced ISC expansion through a yes-associated protein 1 (YAP)-dependent mechanism. ISC differentiation and Organoid formation, on the other hand, required a soft matrix and laminin-based adhesion. We used these insights to build a fully defined culture system for the expansion of mouse and human ISCs. We also produced mechanically dynamic matrices that were initially optimal for ISC expansion and subsequently permissive to differentiation and intestinal Organoid formation, thus creating well-defined alternatives to animal-derived matrices for the culture of mouse and human stem-cell-derived Organoids. Our approach overcomes multiple limitations of current Organoid cultures and greatly expands their applicability in basic and clinical research. The principles presented here can be extended to identify designer matrices that are optimal for long-term culture of other types of stem cells and Organoids.
Hugo J. Snippert - One of the best experts on this subject based on the ideXlab platform.
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High-resolution 3D imaging of fixed and cleared Organoids
Nature Protocols, 2019Co-Authors: Johanna F. Dekkers, Gimano D Amatngalim, Koen C. Oost, Maria Alieva, Lianne M Wellens, Hendrikus C R Ariese, Paul R. Jamieson, Annelotte M. Vonk, Huili Hu, Hugo J. SnippertAbstract:In vitro 3D Organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of Organoids and demonstrated Organoids’ phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire Organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of Organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor Organoids, as well as on mouse mammary gland Organoids. It includes a simple clearing method utilizing a homemade fructose–glycerol clearing agent that captures 3D Organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From Organoid harvest to image analysis, the protocol takes 3 d. This protocol for clearing and high-resolution 3D imaging of entire Organoids expressing fluorescence reporters or following immunolabeling enables confocal, super-resolution confocal, multiphoton and light-sheet microscopy to be performed.
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High-resolution 3D imaging of fixed and cleared Organoids
Nature protocols, 2019Co-Authors: Johanna F. Dekkers, Gimano D Amatngalim, Koen C. Oost, Maria Alieva, Lianne M Wellens, Hendrikus C R Ariese, Paul R. Jamieson, Annelotte M. Vonk, Hugo J. SnippertAbstract:In vitro 3D Organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of Organoids and demonstrated Organoids' phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire Organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of Organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor Organoids, as well as on mouse mammary gland Organoids. It includes a simple clearing method utilizing a homemade fructose-glycerol clearing agent that captures 3D Organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From Organoid harvest to image analysis, the protocol takes 3 d.
Aleksander Skardal - One of the best experts on this subject based on the ideXlab platform.
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Model of Patient-Specific Immune-Enhanced Organoids for Immunotherapy Screening: Feasibility Study
Annals of Surgical Oncology, 2019Co-Authors: Konstantinos I. Votanopoulos, Steven Forsythe, Hemamylammal Sivakumar, Andrea Mazzocchi, Julio Aleman, Lance Miller, Edward Levine, Pierre Triozzi, Aleksander SkardalAbstract:Introduction We hypothesized that engineering a combined lymph node/melanoma Organoid from the same patient would allow tumor, stroma, and immune system to remain viable for personalized immunotherapy screening. Methods Surgically obtained matched melanoma and lymph node biospecimens from the same patient were transferred to the laboratory and washed with saline, antibiotic, and red blood cell lysis buffer. Biospecimens were dissociated, incorporated into an extracellular matrix (ECM)-based hydrogel system, and biofabricated into three dimensional (3D) mixed melanoma/node Organoids. Cells were not sorted, so as to preserve tumor heterogeneity, including stroma and immune cell components, resulting in immune-enhanced patient tumor Organoids (iPTOs). Organoid sets were screened in parallel with nivolumab, pembrolizumab, ipilimumab, and dabrafenib/trametinib for 72 h. LIVE/DEAD staining and quantitative metabolism assays recorded relative drug efficacy. Histology and immunohistochemistry were used to compare tumor melanoma cells with Organoid melanoma cells. Lastly, node-enhanced iPTOs were employed to activate patient-matched peripheral blood T cells for killing of tumor cells in naïve PTOs. Results Ten biospecimen sets obtained from eight stage III and IV melanoma patients were reconstructed as symbiotic immune/tumor Organoids between September 2017 and June 2018. Successful establishment of viable Organoid sets was 90% (9/10), although Organoid yield varied with biospecimen size. Average time from Organoid development to initiation of immunotherapy testing was 7 days. In three patients for whom a node was not available, it was substituted with peripheral blood mononuclear cells. iPTO response to immunotherapy was similar to specimen clinical response in 85% (6/7) patients. In an additional pilot study, peripheral T cells were circulated through iPTOs, and subsequently transferred to naïve PTOs from the same patient, resulting in tumor killing, suggesting a possible role of iPTOs in generating adaptive immunity. Conclusion Development of 3D mixed immune-enhanced tumor/node Organoids is a feasible platform, allowing individual patient immune system and tumor cells to remain viable for studying of personalized immunotherapy response.
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Applications of Bioengineered 3D Tissue and Tumor Organoids in Drug Development and Precision Medicine: Current and Future
BioDrugs, 2018Co-Authors: Mahesh Devarasetty, Andrea Mazzocchi, Aleksander SkardalAbstract:Over the past decade, advances in biomedical and tissue engineering technologies, such as cell culture techniques, biomaterials, and biofabrication, have driven increasingly widespread use of three-dimensional (3D) cell culture platforms and, subsequently, the use of Organoids in a variety of research endeavors. Given the 3D nature of these Organoid systems, and the frequent inclusion of extracellular matrix components, these constructs typically have more physiologically accurate cell–cell and cell–matrix interactions than traditional 2D cell cultures. As a result, 3D Organoids can serve as better model systems than their 2D counterparts. Moreover, as Organoids can be biofabricated from highly functional human cells, they have certain advantages over animal models, being human in nature and more easily manipulated in the laboratory. In this review, we describe such Organoid technologies and their deployment in drug development and precision medicine efforts. Organoid technologies are rapidly being developed for these applications and now represent a wide variety of tissue types and diseases. Evidence is emerging that Organoids are poised for widespread adoption, not only in academia but also in the pharmaceutical industry and in clinical diagnostic applications, positioning them as indispensable tools in medicine.
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Applications of Bioengineered 3D Tissue and Tumor Organoids in Drug Development and Precision Medicine: Current and Future
BioDrugs, 2018Co-Authors: Mahesh Devarasetty, Andrea Mazzocchi, Aleksander SkardalAbstract:Over the past decade, advances in biomedical and tissue engineering technologies, such as cell culture techniques, biomaterials, and biofabrication, have driven increasingly widespread use of three-dimensional (3D) cell culture platforms and, subsequently, the use of Organoids in a variety of research endeavors. Given the 3D nature of these Organoid systems, and the frequent inclusion of extracellular matrix components, these constructs typically have more physiologically accurate cell–cell and cell–matrix interactions than traditional 2D cell cultures. As a result, 3D Organoids can serve as better model systems than their 2D counterparts. Moreover, as Organoids can be biofabricated from highly functional human cells, they have certain advantages over animal models, being human in nature and more easily manipulated in the laboratory. In this review, we describe such Organoid technologies and their deployment in drug development and precision medicine efforts. Organoid technologies are rapidly being developed for these applications and now represent a wide variety of tissue types and diseases. Evidence is emerging that Organoids are poised for widespread adoption, not only in academia but also in the pharmaceutical industry and in clinical diagnostic applications, positioning them as indispensable tools in medicine.
Gimano D Amatngalim - One of the best experts on this subject based on the ideXlab platform.
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High-resolution 3D imaging of fixed and cleared Organoids
Nature Protocols, 2019Co-Authors: Johanna F. Dekkers, Gimano D Amatngalim, Koen C. Oost, Maria Alieva, Lianne M Wellens, Hendrikus C R Ariese, Paul R. Jamieson, Annelotte M. Vonk, Huili Hu, Hugo J. SnippertAbstract:In vitro 3D Organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of Organoids and demonstrated Organoids’ phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire Organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of Organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor Organoids, as well as on mouse mammary gland Organoids. It includes a simple clearing method utilizing a homemade fructose–glycerol clearing agent that captures 3D Organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From Organoid harvest to image analysis, the protocol takes 3 d. This protocol for clearing and high-resolution 3D imaging of entire Organoids expressing fluorescence reporters or following immunolabeling enables confocal, super-resolution confocal, multiphoton and light-sheet microscopy to be performed.
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High-resolution 3D imaging of fixed and cleared Organoids
Nature protocols, 2019Co-Authors: Johanna F. Dekkers, Gimano D Amatngalim, Koen C. Oost, Maria Alieva, Lianne M Wellens, Hendrikus C R Ariese, Paul R. Jamieson, Annelotte M. Vonk, Hugo J. SnippertAbstract:In vitro 3D Organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of Organoids and demonstrated Organoids' phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire Organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of Organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor Organoids, as well as on mouse mammary gland Organoids. It includes a simple clearing method utilizing a homemade fructose-glycerol clearing agent that captures 3D Organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From Organoid harvest to image analysis, the protocol takes 3 d.
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long term expanding human airway Organoids for disease modeling
The EMBO Journal, 2019Co-Authors: Norman Sachs, Angelos Papaspyropoulos, Domenique Zomervan D Ommen, Inha Heo, Lena Bottinger, Dymph Klay, Fleur Weeber, Guizela Huelszprince, Nino Iakobachvili, Gimano D AmatngalimAbstract:Organoids are self-organizing 3D structures grown from stem cells that recapitulate essential aspects of organ structure and function. Here, we describe a method to establish long-term-expanding human airway Organoids from broncho-alveolar resections or lavage material. The pseudostratified airway Organoids consist of basal cells, functional multi-ciliated cells, mucus-producing secretory cells, and CC10-secreting club cells. Airway Organoids derived from cystic fibrosis (CF) patients allow assessment of CFTR function in an Organoid swelling assay. Organoids established from lung cancer resections and metastasis biopsies retain tumor histopathology as well as cancer gene mutations and are amenable to drug screening. Respiratory syncytial virus (RSV) infection recapitulates central disease features, dramatically increases Organoid cell motility via the non-structural viral NS2 protein, and preferentially recruits neutrophils upon co-culturing. We conclude that human airway Organoids represent versatile models for the in vitro study of hereditary, malignant, and infectious pulmonary disease.
Koen C. Oost - One of the best experts on this subject based on the ideXlab platform.
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Engineering human knock-in Organoids
Nature Cell Biology, 2020Co-Authors: Qiutan Yang, Koen C. Oost, Prisca LiberaliAbstract:The lack of endogenous reporter lines is a bottleneck in the study of subcellular dynamics in human adult stem cell (ASC)-derived Organoids. An approach using CRISPR–Cas9-mediated homology-independent Organoid transgenesis (CRISPR–HOT) in ASC-derived Organoids now narrows the gap between basic research and translational studies in human Organoids.
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High-resolution 3D imaging of fixed and cleared Organoids
Nature Protocols, 2019Co-Authors: Johanna F. Dekkers, Gimano D Amatngalim, Koen C. Oost, Maria Alieva, Lianne M Wellens, Hendrikus C R Ariese, Paul R. Jamieson, Annelotte M. Vonk, Huili Hu, Hugo J. SnippertAbstract:In vitro 3D Organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of Organoids and demonstrated Organoids’ phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire Organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of Organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor Organoids, as well as on mouse mammary gland Organoids. It includes a simple clearing method utilizing a homemade fructose–glycerol clearing agent that captures 3D Organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From Organoid harvest to image analysis, the protocol takes 3 d. This protocol for clearing and high-resolution 3D imaging of entire Organoids expressing fluorescence reporters or following immunolabeling enables confocal, super-resolution confocal, multiphoton and light-sheet microscopy to be performed.
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High-resolution 3D imaging of fixed and cleared Organoids
Nature protocols, 2019Co-Authors: Johanna F. Dekkers, Gimano D Amatngalim, Koen C. Oost, Maria Alieva, Lianne M Wellens, Hendrikus C R Ariese, Paul R. Jamieson, Annelotte M. Vonk, Hugo J. SnippertAbstract:In vitro 3D Organoid systems have revolutionized the modeling of organ development and diseases in a dish. Fluorescence microscopy has contributed to the characterization of the cellular composition of Organoids and demonstrated Organoids' phenotypic resemblance to their original tissues. Here, we provide a detailed protocol for performing high-resolution 3D imaging of entire Organoids harboring fluorescence reporters and upon immunolabeling. This method is applicable to a wide range of Organoids of differing origins and of various sizes and shapes. We have successfully used it on human airway, colon, kidney, liver and breast tumor Organoids, as well as on mouse mammary gland Organoids. It includes a simple clearing method utilizing a homemade fructose-glycerol clearing agent that captures 3D Organoids in full and enables marker quantification on a cell-by-cell basis. Sample preparation has been optimized for 3D imaging by confocal, super-resolution confocal, multiphoton and light-sheet microscopy. From Organoid harvest to image analysis, the protocol takes 3 d.