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Moskal K, Khurana N, Siegert L, Lee YS, Clevers H, Elinav E, Puschhof J. Modeling cancer-microbiome interactions in vitro: A guide to co-culture platforms. Int J Cancer 2025; 156:2053-2067. [PMID: 39716471 PMCID: PMC11970552 DOI: 10.1002/ijc.35298] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/15/2024] [Revised: 10/10/2024] [Accepted: 10/29/2024] [Indexed: 12/25/2024]
Abstract
The biology of cancer is characterized by an intricate interplay of cells originating not only from the tumor mass, but also its surrounding environment. Different microbial species have been suggested to be enriched in tumors and the impacts of these on tumor phenotypes is subject to intensive investigation. For these efforts, model systems that accurately reflect human-microbe interactions are rapidly gaining importance. Here we present a guide for selecting a suitable in vitro co-culture platform used to model different cancer-microbiome interactions. Our discussion spans a variety of in vitro models, including 2D cultures, tumor spheroids, organoids, and organ-on-a-chip platforms, where we delineate their respective advantages, limitations, and applicability in cancer microbiome research. Particular focus is placed on methodologies that facilitate the exposure of cancer cells to microbes, such as organoid microinjections and co-culture on microfluidic devices. We highlight studies offering critical insights into possible cancer-microbe interactions and underscore the importance of in vitro models in those discoveries. We anticipate the integration of more complex microbial communities and the inclusion of immune cells into co-culture systems to more accurately simulate the tumor microenvironment. The advent of ever more sophisticated co-culture models will aid in unraveling the mechanisms of cancer-microbiome interplay and contribute to exploiting their potential in novel diagnostic and therapeutic strategies.
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Affiliation(s)
- Kamil Moskal
- Junior Research Group Epithelium Microbiome Interactions (EMIL), German Cancer Research CenterHeidelbergGermany
- Microbiome and Cancer Division, German Cancer Research CenterHeidelbergGermany
- Faculty of BiosciencesHeidelberg UniversityHeidelbergGermany
- DKFZ Hector Cancer Institute at the University Medical CenterMannheimGermany
| | - Nimisha Khurana
- Junior Research Group Epithelium Microbiome Interactions (EMIL), German Cancer Research CenterHeidelbergGermany
- Microbiome and Cancer Division, German Cancer Research CenterHeidelbergGermany
- Faculty of BiosciencesHeidelberg UniversityHeidelbergGermany
| | - Luisa Siegert
- Junior Research Group Epithelium Microbiome Interactions (EMIL), German Cancer Research CenterHeidelbergGermany
- Microbiome and Cancer Division, German Cancer Research CenterHeidelbergGermany
| | - Ye Seul Lee
- Junior Research Group Epithelium Microbiome Interactions (EMIL), German Cancer Research CenterHeidelbergGermany
- Microbiome and Cancer Division, German Cancer Research CenterHeidelbergGermany
- Faculty of BiosciencesHeidelberg UniversityHeidelbergGermany
| | - Hans Clevers
- Royal Netherlands Academy of Arts and Sciences (KNAW) and UMC UtrechtHubrecht InstituteUtrechtThe Netherlands
- Present address:
Roche Pharmaceutical Research and Early DevelopmentBaselSwitzerland
| | - Eran Elinav
- Microbiome and Cancer Division, German Cancer Research CenterHeidelbergGermany
- Systems Immunology DepartmentWeizmann Institute of ScienceRehovotIsrael
| | - Jens Puschhof
- Junior Research Group Epithelium Microbiome Interactions (EMIL), German Cancer Research CenterHeidelbergGermany
- Microbiome and Cancer Division, German Cancer Research CenterHeidelbergGermany
- Faculty of BiosciencesHeidelberg UniversityHeidelbergGermany
- DKFZ Hector Cancer Institute at the University Medical CenterMannheimGermany
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2
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Zook HN, Quijano JC, Ortiz JA, Donohue C, Erdem N, Ku HT. Protocol to study ductal progenitor-like cells from the adult human pancreas using 3D suspension and methylcellulose-based culture systems. STAR Protoc 2025; 6:103847. [PMID: 40424137 DOI: 10.1016/j.xpro.2025.103847] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2025] [Revised: 04/15/2025] [Accepted: 05/08/2025] [Indexed: 05/29/2025] Open
Abstract
Primary human ductal progenitor-like cells derived from donated pancreas have the potential to serve as a source of therapeutic insulin-producing beta cells for the treatment of diabetes. Here, we present a protocol for studying ductal progenitor-like cells using a good manufacturing practice (GMP)-compatible 3D suspension culture system and a methylcellulose- and Matrigel-based 3D colony assay. We describe steps for dispersing and cryopreserving human pancreatic cells and initiating and maintaining cultures. We then detail how to prepare ductal spheroids or colonies for downstream applications. For complete details on the use and execution of this protocol, please refer to Zook et al.1 and Quijano et al.2.
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Affiliation(s)
- Heather N Zook
- Department of Translational Research and Cellular Therapeutics, Arthur Riggs Diabetes and Metabolism Research Institute, City of Hope, Duarte, CA 91010, USA.
| | - Janine C Quijano
- Department of Translational Research and Cellular Therapeutics, Arthur Riggs Diabetes and Metabolism Research Institute, City of Hope, Duarte, CA 91010, USA
| | - Jose A Ortiz
- Department of Translational Research and Cellular Therapeutics, Arthur Riggs Diabetes and Metabolism Research Institute, City of Hope, Duarte, CA 91010, USA
| | - Cecile Donohue
- Department of Translational Research and Cellular Therapeutics, Arthur Riggs Diabetes and Metabolism Research Institute, City of Hope, Duarte, CA 91010, USA
| | - Neslihan Erdem
- Department of Translational Research and Cellular Therapeutics, Arthur Riggs Diabetes and Metabolism Research Institute, City of Hope, Duarte, CA 91010, USA; Irell & Manella Graduate School of Biological Sciences, Beckman Research Institute, City of Hope, Duarte, CA 91010, USA
| | - Hsun Teresa Ku
- Department of Translational Research and Cellular Therapeutics, Arthur Riggs Diabetes and Metabolism Research Institute, City of Hope, Duarte, CA 91010, USA; Irell & Manella Graduate School of Biological Sciences, Beckman Research Institute, City of Hope, Duarte, CA 91010, USA.
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3
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O'Mahony ET, Arian CM, Aryeh KS, Wang K, Thummel KE, Kelly EJ. Human intestinal enteroids: Nonclinical applications for predicting oral drug disposition, toxicity, and efficacy. Pharmacol Ther 2025:108879. [PMID: 40398537 DOI: 10.1016/j.pharmthera.2025.108879] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/12/2024] [Revised: 02/19/2025] [Accepted: 05/15/2025] [Indexed: 05/23/2025]
Abstract
The application of human enteroid systems presents a significant opportunity within the drug development pipeline, highlighting considerable potential for advancements in the characterization and evaluation of new molecular entities. Derived from LGR5+ crypt-based columnar cells, enteroid systems more accurately recapitulate the microanatomy and physiological processes of the human intestinal mucosa compared to traditionally used systems. They contain the complement of major mucosal epithelial cell types, maintain the genetic identity of the donor and intestinal segment they were derived from, and exhibit biological functions and specific activities that are seen in vivo. In this review, we examine the applications of human enteroid systems in nonclinical drug development and compare findings to existing and emerging in vitro models of the small intestine. Specifically, we explore enteroid systems in the context of predicting oral drug disposition, focusing on apparent permeability, intestinal first-pass metabolism, and drug interactions, as well as their utility in assessing drug-induced gastrointestinal toxicity and screening therapeutic efficacy against enteric diseases. Additionally, we highlight aspects of enteroid systems that warrant further study.
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Affiliation(s)
- Eimear T O'Mahony
- Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle, WA, United States of America
| | - Christopher M Arian
- Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle, WA, United States of America
| | - Kayenat S Aryeh
- Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle, WA, United States of America
| | - Kai Wang
- Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle, WA, United States of America
| | - Kenneth E Thummel
- Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle, WA, United States of America; Center of Excellence for Natural Product Drug Interaction Research, Spokane, WA, United States of America
| | - Edward J Kelly
- Department of Pharmaceutics, School of Pharmacy, University of Washington, Seattle, WA, United States of America; Kidney Research Institute, University of Washington, Seattle, WA, United States of America.
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4
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Adelfio M, Callen GE, He X, Paster BJ, Hasturk H, Ghezzi CE. Engineered Tissue Models to Decode Host-Microbiota Interactions. ADVANCED SCIENCE (WEINHEIM, BADEN-WURTTEMBERG, GERMANY) 2025:e2417687. [PMID: 40364768 DOI: 10.1002/advs.202417687] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/28/2024] [Revised: 04/13/2025] [Indexed: 05/15/2025]
Abstract
A mutualistic co-evolution exists between the host and its associated microbiota in the human body. Bacteria establish ecological niches in various tissues of the body, locally influencing their physiology and functions, but also contributing to the well-being of the whole organism through systemic communication with other distant niches (axis). Emerging evidence indicates that when the composition of the microbiota inhabiting the niche changes toward a pathogenic state (dysbiosis) and interactions with the host become unbalanced, diseases may present. In addition, imbalances within a single niche can cause dysbiosis in distant organs. Current research efforts are focused on elucidating the mechanisms leading to dysbiosis, with the goal of restoring tissue homeostasis. In vitro models can provide critical experimental platforms to address this need, by reproducing the niche cyto-architecture and physiology with high fidelity. This review surveys current in in vitro host-microbiota research strategies and provides a roadmap that can guide the field in further developing physiologically relevant in vitro models of ecological niches, thus enabling investigation of the role of the microbiota in human health and diseases. Lastly, given the Food and Drug Administration Modernization Act 2.0, this review highlights emerging in vitro strategies to support the development and validation of new therapies on the market.
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Affiliation(s)
- Miryam Adelfio
- Department of Biomedical Engineering, University of Massachusetts-Lowell, Lowell, MA, 01854, USA
| | - Grace E Callen
- Department of Biomedical Engineering, University of Massachusetts-Lowell, Lowell, MA, 01854, USA
| | - Xuesong He
- ADA Forsyth Institute, 245 First St, Cambridge, MA, 02142, USA
| | - Bruce J Paster
- ADA Forsyth Institute, 245 First St, Cambridge, MA, 02142, USA
| | - Hatice Hasturk
- ADA Forsyth Institute, 245 First St, Cambridge, MA, 02142, USA
| | - Chiara E Ghezzi
- Department of Biomedical Engineering, University of Massachusetts-Lowell, Lowell, MA, 01854, USA
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5
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Dykstra GD, Kawasaki M, Burbick CR, McConnel CS, Ambrosini YM. From in vitro development to accessible luminal interface of neonatal bovine-derived intestinal organoids. BMC Vet Res 2025; 21:319. [PMID: 40325425 PMCID: PMC12054211 DOI: 10.1186/s12917-025-04773-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2025] [Accepted: 04/21/2025] [Indexed: 05/07/2025] Open
Abstract
BACKGROUND Intestinal organoids provide physiologically relevant in vitro models that bridge the gap between conventional cell culture and animal studies. Although these systems have been developed for adult cattle, their use in neonatal calves-who are particularly vulnerable to enteric disease-has not been well established. Neonatal diarrhea remains a major health concern in modern agriculture, yet age-appropriate models for studying its pathogenesis are lacking. Given that host-pathogen interactions vary with developmental stage, there is a need for culture systems that reflect the distinct biology of the neonatal gut. In this study, we developed intestinal organoids and organoid-derived monolayers from 14-day-old dairy calves to enable research on early-life intestinal function and disease. RESULTS Organoids were successfully established from five intestinal sections of 14-day-old dairy calves using customized growth media and characterized by immunofluorescence and gene expression analyses. They remained viable for over 300 days of cryopreservation and were serially passaged at least 15 times. Rectal organoid-derived monolayers were further assessed by electron microscopy and barrier function assays, demonstrating stable transepithelial electrical resistance and controlled paracellular permeability. CONCLUSIONS Optimized methods for adult bovine intestinal organoids and rectal organoid-derived monolayers are applicable to neonatal intestinal epithelial stem cells. Organoids cultured from 14-day-old calves captured key aspects of the multicellularity and functionality of the native epithelium. Future work should focus on adapting monolayer culture methods for additional gut regions, particularly the proximal gastrointestinal tract. Neonatal rectal monolayers represent a promising platform for advancing veterinary research, agricultural innovation, and studies of zoonotic disease.
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Affiliation(s)
- Gerald D Dykstra
- Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, Washington, United States of America
| | - Minae Kawasaki
- Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, Washington, United States of America
| | - Claire R Burbick
- Department of Veterinary Microbiology and Pathology, College of Veterinary Medicine, Washington State University, Pullman, Washington, United States of America
| | - Craig S McConnel
- Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, Washington, United States of America
| | - Yoko M Ambrosini
- Department of Veterinary Clinical Sciences, College of Veterinary Medicine, Washington State University, Pullman, Washington, United States of America.
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6
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Han Y, Liu X, Qu S, Duan X, Xiang Y, Jiang N, Yang S, Fang X, Xu L, Wen H, Yu Y, Huang S, Huang J, Zhu K. Tissue geometry spatiotemporally drives bacterial infections. Cell 2025:S0092-8674(25)00394-0. [PMID: 40262607 DOI: 10.1016/j.cell.2025.03.042] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2023] [Revised: 02/28/2025] [Accepted: 03/25/2025] [Indexed: 04/24/2025]
Abstract
Epithelial tissues serve as the first line of host against bacterial infections. The self-organization of epithelial tissues continuously adapts to the architecture and mechanics of microenvironments, thereby dynamically impacting the initial niche of infections. However, the mechanism by which tissue geometry regulates bacterial infection remains poorly understood. Here, we showed geometry-guided infection patterns of bacteria in epithelial tissues using bioengineering strategies. We discovered that cellular traction forces play a crucial role in the regulation of bacterial invasive sites and marginal infection patterns in epithelial monolayers through triggering co-localization of mechanosensitive ion channel protein Piezo1 with bacteria. Further, we developed precise mechanobiology-based strategies to potentiate the antibacterial efficacy in animal models of wound and intestinal infection. Our findings demonstrate that tissue geometry exerts a key impact on mediating spatiotemporal infections of bacteria, which has important implications for the discovery and development of alternative strategies against bacterial infections.
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Affiliation(s)
- Yiming Han
- Department of Mechanics and Engineering Science, College of Engineering, Peking University, Beijing 100871, China; National Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China
| | - Xiaoye Liu
- Department of Mechanics and Engineering Science, College of Engineering, Peking University, Beijing 100871, China; Beijing Traditional Chinese Veterinary Engineering Center and Beijing Key Laboratory of Traditional Chinese Veterinary Medicine, Beijing University of Agriculture, Beijing 102206, China
| | - Shaoqi Qu
- National Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China; Animal-Derived Food Safety Innovation Team, College of Veterinary Medicine, Anhui Agricultural University, Hefei 230036, China
| | - Xiaocen Duan
- Department of Mechanics and Engineering Science, College of Engineering, Peking University, Beijing 100871, China
| | - Yunqing Xiang
- State Key Laboratory of Quantitative Synthetic Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, Guangdong, China
| | - Nan Jiang
- Department of Mechanics and Engineering Science, College of Engineering, Peking University, Beijing 100871, China
| | - Shuyu Yang
- National Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China
| | - Xu Fang
- Department of Mechanics and Engineering Science, College of Engineering, Peking University, Beijing 100871, China
| | - Liang Xu
- Department of Mechanics and Engineering Science, College of Engineering, Peking University, Beijing 100871, China
| | - Hui Wen
- State Key Laboratory of Quantitative Synthetic Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, Guangdong, China
| | - Yue Yu
- State Key Laboratory of Quantitative Synthetic Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, Guangdong, China
| | - Shuqiang Huang
- State Key Laboratory of Quantitative Synthetic Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, Guangdong, China.
| | - Jianyong Huang
- Department of Mechanics and Engineering Science, College of Engineering, Peking University, Beijing 100871, China.
| | - Kui Zhu
- National Key Laboratory of Veterinary Public Health and Safety, College of Veterinary Medicine, China Agricultural University, Beijing 100193, China.
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7
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Hsieh HC, Han Q, Brenes D, Bishop KW, Wang R, Wang Y, Poudel C, Glaser AK, Freedman BS, Vaughan JC, Allbritton NL, Liu JTC. Imaging 3D cell cultures with optical microscopy. Nat Methods 2025:10.1038/s41592-025-02647-w. [PMID: 40247123 DOI: 10.1038/s41592-025-02647-w] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2024] [Accepted: 01/16/2025] [Indexed: 04/19/2025]
Abstract
Three-dimensional (3D) cell cultures have gained popularity in recent years due to their ability to represent complex tissues or organs more faithfully than conventional two-dimensional (2D) cell culture. This article reviews the application of both 2D and 3D microscopy approaches for monitoring and studying 3D cell cultures. We first summarize the most popular optical microscopy methods that have been used with 3D cell cultures. We then discuss the general advantages and disadvantages of various microscopy techniques for several broad categories of investigation involving 3D cell cultures. Finally, we provide perspectives on key areas of technical need in which there are clear opportunities for innovation. Our goal is to guide microscope engineers and biomedical end users toward optimal imaging methods for specific investigational scenarios and to identify use cases in which additional innovations in high-resolution imaging could be helpful.
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Affiliation(s)
- Huai-Ching Hsieh
- Department of Bioengineering, University of Washington, Seattle, WA, USA
- Department of Mechanical Engineering, University of Washington, Seattle, WA, USA
| | - Qinghua Han
- Department of Bioengineering, University of Washington, Seattle, WA, USA
- Department of Mechanical Engineering, University of Washington, Seattle, WA, USA
| | - David Brenes
- Department of Mechanical Engineering, University of Washington, Seattle, WA, USA
| | - Kevin W Bishop
- Department of Bioengineering, University of Washington, Seattle, WA, USA
- Department of Mechanical Engineering, University of Washington, Seattle, WA, USA
| | - Rui Wang
- Department of Mechanical Engineering, University of Washington, Seattle, WA, USA
| | - Yuli Wang
- Department of Bioengineering, University of Washington, Seattle, WA, USA
| | - Chetan Poudel
- Department of Chemistry, University of Washington, Seattle, WA, USA
| | - Adam K Glaser
- Allen Institute for Neural Dynamics, Seattle, WA, USA
| | - Benjamin S Freedman
- Department of Bioengineering, University of Washington, Seattle, WA, USA
- Department of Medicine, Division of Nephrology, Kidney Research Institute and Institute for Stem Cell and Regenerative Medicine, Seattle, WA, USA
- Plurexa LLC, Seattle, WA, USA
- Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA
| | - Joshua C Vaughan
- Department of Chemistry, University of Washington, Seattle, WA, USA
- Department of Physiology and Biophysics, University of Washington, Seattle, WA, USA
| | - Nancy L Allbritton
- Department of Bioengineering, University of Washington, Seattle, WA, USA
| | - Jonathan T C Liu
- Department of Bioengineering, University of Washington, Seattle, WA, USA.
- Department of Mechanical Engineering, University of Washington, Seattle, WA, USA.
- Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA.
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8
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Galli G, Melcón-Fernández E, de Garnica García MG, Martínez-Fernández B, Dehnavi M, Andrés S, Pérez-Pertejo Y, Reguera RM, García-Estrada C, Martínez-Valladares M, Balaña-Fouce R. Development of Sheep Duodenum Intestinal Organoids and Implementation of High-Throughput Screening Platform for Veterinary Applications. Int J Mol Sci 2025; 26:3452. [PMID: 40244396 PMCID: PMC11989482 DOI: 10.3390/ijms26073452] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2025] [Revised: 03/25/2025] [Accepted: 04/04/2025] [Indexed: 04/18/2025] Open
Abstract
New therapeutic molecules for farm animals are needed to address worldwide problems in the food industry, like the rise of resistance among ruminant parasites and pathogenic microbes. Since in vivo testing would involve an excessive number of animals, with consequent ethical and economic issues, the generation of sheep intestinal organoids represents a promising close-to-reality in vitro model for veterinary drug development; however, the characterization and application of such organoids remain limited. In this study, ovine intestinal organoids were generated from adult LGR5+ stem cells from the intestinal crypts of freshly slaughtered lambs, and developed in an in vitro culture system. Morphological analysis via brightfield microscopy and immunocytochemical staining revealed a pseudostratified epithelium with multiple cell types, and distinct apical-basal polarity, while RNA sequencing validated the preservation of the physiological characteristics of the original organ. The development and characterization of a robust and reproducible protocol for culturing sheep duodenum intestinal organoids in a high-throughput screening (HTS) compatible format demonstrated reliability in HTS applications, with Z'-factor tests indicating robust assay performance. Dose-response studies using pre-identified compounds showed comparable pharmacodynamic profiles between mouse and sheep organoids. These findings establish sheep intestinal organoids as an innovative tool for veterinary pharmacology and toxicology, offering a cost-effective and sustainable platform to address challenges such as drug resistance and improve livestock health.
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Affiliation(s)
- Giulio Galli
- Departamento de Ciencias Biomédicas, Facultad de Veterinaria, Universidad de León, Campus de Vegazana s/n, 24007 León, Spain; (G.G.); (E.M.-F.); (Y.P.-P.); (R.M.R.); (C.G.-E.)
| | - Estela Melcón-Fernández
- Departamento de Ciencias Biomédicas, Facultad de Veterinaria, Universidad de León, Campus de Vegazana s/n, 24007 León, Spain; (G.G.); (E.M.-F.); (Y.P.-P.); (R.M.R.); (C.G.-E.)
| | | | | | - Mahsa Dehnavi
- Instituto de Ganadería de Montaña, CSIC-Universidad de León, Finca Marzanas s/n, Grulleros, 24346 León, Spain; (M.D.); (S.A.); (M.M.-V.)
| | - Sonia Andrés
- Instituto de Ganadería de Montaña, CSIC-Universidad de León, Finca Marzanas s/n, Grulleros, 24346 León, Spain; (M.D.); (S.A.); (M.M.-V.)
| | - Yolanda Pérez-Pertejo
- Departamento de Ciencias Biomédicas, Facultad de Veterinaria, Universidad de León, Campus de Vegazana s/n, 24007 León, Spain; (G.G.); (E.M.-F.); (Y.P.-P.); (R.M.R.); (C.G.-E.)
- Instituto de Biomedicina (IBIOMED), Universidad de León, Campus de Vegazana s/n, 24007 León, Spain
| | - Rosa M. Reguera
- Departamento de Ciencias Biomédicas, Facultad de Veterinaria, Universidad de León, Campus de Vegazana s/n, 24007 León, Spain; (G.G.); (E.M.-F.); (Y.P.-P.); (R.M.R.); (C.G.-E.)
- Instituto de Biomedicina (IBIOMED), Universidad de León, Campus de Vegazana s/n, 24007 León, Spain
| | - Carlos García-Estrada
- Departamento de Ciencias Biomédicas, Facultad de Veterinaria, Universidad de León, Campus de Vegazana s/n, 24007 León, Spain; (G.G.); (E.M.-F.); (Y.P.-P.); (R.M.R.); (C.G.-E.)
- Instituto de Biomedicina (IBIOMED), Universidad de León, Campus de Vegazana s/n, 24007 León, Spain
| | - María Martínez-Valladares
- Instituto de Ganadería de Montaña, CSIC-Universidad de León, Finca Marzanas s/n, Grulleros, 24346 León, Spain; (M.D.); (S.A.); (M.M.-V.)
| | - Rafael Balaña-Fouce
- Departamento de Ciencias Biomédicas, Facultad de Veterinaria, Universidad de León, Campus de Vegazana s/n, 24007 León, Spain; (G.G.); (E.M.-F.); (Y.P.-P.); (R.M.R.); (C.G.-E.)
- Instituto de Biomedicina (IBIOMED), Universidad de León, Campus de Vegazana s/n, 24007 León, Spain
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9
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Farrell L, Bonnet C, Tang A, Peneva S, Williams NG, Dolwani S, Parry L, Dyson P. Organoids with a Type 1 Collagen Scaffold to Model Bacterial Cancer Therapy. Cells 2025; 14:524. [PMID: 40214478 PMCID: PMC11989105 DOI: 10.3390/cells14070524] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2025] [Revised: 03/26/2025] [Accepted: 03/27/2025] [Indexed: 04/14/2025] Open
Abstract
Bacterial cancer therapy (BCT) is emerging as an important option for the treatment of solid tumours, with promising outcomes in preclinical trials. Further progress is hampered by an incomplete understanding of how oncotropic bacteria, such as attenuated strains of Salmonella enterica serovar Typhimurium, colonise tumours and the responses of both the bacteria and tumour cells to this colonisation. To model this, we developed organoids that are permissive for bacterial colonisation, replacing the conventional commercially available extracellular matrix (e.g., Matrigel) with a type I collagen matrix scaffold. A comparison of the two extracellular matrices indicated that type 1 collagen permitted an initial infection efficiency more than 5-times greater than with Matrigel. In addition, subsequent growth within type 1 collagen expanded bacterial cell numbers by over 10-fold within 4 days of infection. These organoids allow for the visualisation of bacterial chemoattraction, cell invasion and subsequent population of the interior lumen, and will permit the future optimisation of BCT. In addition, by establishing patient-derived organoids, we demonstrate a platform for developing future personalised treatments exploiting BCT.
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Affiliation(s)
- Lydia Farrell
- Institute of Life Science, Swansea University Medical School, Singleton Park, Swansea SA2 8PP, UK; (L.F.); (C.B.); (A.T.); (S.P.)
| | - Cleo Bonnet
- Institute of Life Science, Swansea University Medical School, Singleton Park, Swansea SA2 8PP, UK; (L.F.); (C.B.); (A.T.); (S.P.)
| | - Alethea Tang
- Institute of Life Science, Swansea University Medical School, Singleton Park, Swansea SA2 8PP, UK; (L.F.); (C.B.); (A.T.); (S.P.)
| | - Severina Peneva
- Institute of Life Science, Swansea University Medical School, Singleton Park, Swansea SA2 8PP, UK; (L.F.); (C.B.); (A.T.); (S.P.)
| | - Non G. Williams
- European Cancer Stem Cell Research Institute, School of Biosciences, Hadyn Ellis Building, Cardiff University, Maindy Road, Cardiff CF24 4HQ, UK;
| | - Sunil Dolwani
- School of Medicine, Cardiff University, Cardiff and Vale University Health Board, Cardiff CF14 4XN, UK;
| | - Lee Parry
- European Cancer Stem Cell Research Institute, School of Biosciences, Hadyn Ellis Building, Cardiff University, Maindy Road, Cardiff CF24 4HQ, UK;
| | - Paul Dyson
- Institute of Life Science, Swansea University Medical School, Singleton Park, Swansea SA2 8PP, UK; (L.F.); (C.B.); (A.T.); (S.P.)
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10
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Ribes Martinez E, Franko Y, Franko R, Ferronato GA, Viana AES, Windenbach E, Stoeckl JB, Fröhlich T, Ferraz MAMM. Developing and characterising bovine decellularized extracellular matrix hydrogels to biofabricate female reproductive tissues. Acta Biomater 2025; 196:152-170. [PMID: 40058619 DOI: 10.1016/j.actbio.2025.03.009] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2024] [Revised: 02/16/2025] [Accepted: 03/05/2025] [Indexed: 03/15/2025]
Abstract
This study investigated the development and characterization of decellularized extracellular matrix (dECM) hydrogels tailored for the biofabrication of female reproductive tissues, specifically targeting ovarian cortex, endometrium, ovarian medulla, and oviduct tissues. We aimed to evaluate the cytocompatibility, biomechanical properties, and overall efficacy of these dECMs in promoting cell viability, proliferation, and morphology using the bovine model. Bovine species provide a valuable model due to their accessibility from slaughterhouse tissues, offering a practical alternative to human samples, which are often limited in availability. Additionally, bovine tissue closely mirrors certain physiological and biological characteristics of humans, making it a relevant model for translational research. Our findings revealed that these dECMs exhibited high biocompatibility with embryo development and cell viability, supporting micro vascularization and cellular morphology without the need for external growth factors. It is important to note that the addition of alginate was crucial for maintaining the structural integrity of the hydrogel during long-term cultures. These hydrogels displayed biomechanical properties that closely mimicked native tissues, which was vital for maintaining their functional integrity and supporting cellular activities. The printability assessments showed that dECMs, particularly those from cortex tissues, achieved high precision in replicating the intended structures, though challenges such as low porosity remained. The bioprinted constructs demonstrated robust cell growth, with over 97% viability observed by day 7, indicating their suitability for cell culture. This work represented a significant advancement in reproductive tissue biofabrication, demonstrating the potential of dECM-based hydrogels in creating structurally and viable tissue constructs. By tailoring each dECM to match the unique biomechanical properties of different tissues, we paved the way for more effective and reliable applications in reproductive medicine and tissue engineering. STATEMENT OF SIGNIFICANCE: This research explores the use of decellularized extracellular matrix (dECM) hydrogels as bio-inks for creating reproductive tissues. Ovarian cortex and medulla, oviduct and endometrium dECMs demonstrated biomechanical properties that mimicked native tissues, which is essential for maintaining functional integrity and supporting cellular processes. Notably, these hydrogels exhibited high biocompatibility with embryo development and cell viability, promoting microvascularization and cell differentiation without the need for supplemental growth factors. The successful bioprinting of these bio-inks underscores their potential for creating more complex models. This work represents a significant advancement in tissue engineering, offering promising new avenues for reproductive medicine.
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Affiliation(s)
- E Ribes Martinez
- Clinic of Ruminants, Faculty of Veterinary Medicine, Ludwig-Maximilians-Universität München, Sonnenstr. 16, Oberschleißheim, 85764, Germany; Gene Center, Ludwig-Maximilians-Universität München, Feodor-Lynen Str. 25, Munich, 81377, Germany
| | - Y Franko
- Clinic of Ruminants, Faculty of Veterinary Medicine, Ludwig-Maximilians-Universität München, Sonnenstr. 16, Oberschleißheim, 85764, Germany; Gene Center, Ludwig-Maximilians-Universität München, Feodor-Lynen Str. 25, Munich, 81377, Germany
| | - R Franko
- Clinic of Ruminants, Faculty of Veterinary Medicine, Ludwig-Maximilians-Universität München, Sonnenstr. 16, Oberschleißheim, 85764, Germany; Gene Center, Ludwig-Maximilians-Universität München, Feodor-Lynen Str. 25, Munich, 81377, Germany
| | - G A Ferronato
- Clinic of Ruminants, Faculty of Veterinary Medicine, Ludwig-Maximilians-Universität München, Sonnenstr. 16, Oberschleißheim, 85764, Germany; Gene Center, Ludwig-Maximilians-Universität München, Feodor-Lynen Str. 25, Munich, 81377, Germany
| | - A E S Viana
- Department of Veterinary Medicine, Faculty of Zootechnic and Food Engineering, University of São Paulo, Duque de Caxias Norte, 225, Jardim Elite, Pirassununga, São Paulo, 13635-900, Brazil
| | - E Windenbach
- Laboratory for Functional Genome Analysis, Gene Center, Ludwig-Maximilians-Universität München, Feodor-Lynen Str. 25, Munich, 81377, Germany
| | - J B Stoeckl
- Laboratory for Functional Genome Analysis, Gene Center, Ludwig-Maximilians-Universität München, Feodor-Lynen Str. 25, Munich, 81377, Germany
| | - T Fröhlich
- Laboratory for Functional Genome Analysis, Gene Center, Ludwig-Maximilians-Universität München, Feodor-Lynen Str. 25, Munich, 81377, Germany
| | - M A M M Ferraz
- Clinic of Ruminants, Faculty of Veterinary Medicine, Ludwig-Maximilians-Universität München, Sonnenstr. 16, Oberschleißheim, 85764, Germany; Gene Center, Ludwig-Maximilians-Universität München, Feodor-Lynen Str. 25, Munich, 81377, Germany.
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11
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Xu X, Zhang Y, Huang G, Perekatt A, Wang Y, Chen L. Advances and applications of gut organoids: modeling intestinal diseases and therapeutic development. LIFE MEDICINE 2025; 4:lnaf012. [PMID: 40276096 PMCID: PMC12018802 DOI: 10.1093/lifemedi/lnaf012] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 09/21/2024] [Accepted: 03/04/2025] [Indexed: 04/26/2025]
Abstract
Gut organoids are 3D cellular structures derived from adult or pluripotent stem cells, capable of closely replicating the physiological properties of the gut. These organoids serve as powerful tools for studying gut development and modeling the pathogenesis of intestinal diseases. This review provides an in-depth exploration of technological advancements and applications of gut organoids, with a focus on their construction methods. Additionally, the potential applications of gut organoids in disease modeling, microenvironmental simulation, and personalized medicine are summarized. This review aims to offer perspectives and directions for understanding the mechanisms of intestinal health and disease as well as for developing innovative therapeutic strategies.
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Affiliation(s)
- Xiaoting Xu
- School of Life Science and Technology, Key Laboratory of Developmental Genes and Human Disease, Southeast University, Nanjing 210031, China
| | - Yuping Zhang
- School of Life Science and Technology, Key Laboratory of Developmental Genes and Human Disease, Southeast University, Nanjing 210031, China
| | - Guoxin Huang
- Clinical Research Center, Shantou Key Laboratory of Basic and Translational Research of Malignant Tumor, Shantou Central Hospital, Shantou 515041, China
| | - Ansu Perekatt
- Department of Chemistry and Chemical Biology, Stevens Institute of Technology, Hoboken, NJ 07030, United States
| | - Yan Wang
- Center for Translation Medicine Research and Development, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China
| | - Lei Chen
- School of Life Science and Technology, Key Laboratory of Developmental Genes and Human Disease, Southeast University, Nanjing 210031, China
- Institute of Microphysiological Systems, Southeast University, Nanjing 211189, China
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12
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Liu X, Zhou Z, Zhang Y, Zhong H, Cai X, Guan R. Recent progress on the organoids: Techniques, advantages and applications. Biomed Pharmacother 2025; 185:117942. [PMID: 40043462 DOI: 10.1016/j.biopha.2025.117942] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2024] [Revised: 01/30/2025] [Accepted: 02/24/2025] [Indexed: 03/23/2025] Open
Abstract
Organoids are a cutting-edge technology in the life sciences field, with applications in precision medicine, bionic organs, and toxicological evaluations of chemicals. Their 3D structure closely resembles that of real organs, allowing more accurate functional mimicry. The 3D organoid culture system can simulate the growth state of cells in vivo and establish a suspension culture system for organoid 3D culture by using scaffold-less or scaffold technology to avoid direct contact between cells and plastic culture vessels. Furthermore, organoids can simulate the pathophysiological state of tissues and organs in vitro. This paper primarily discusses the construction methodologies, as well as the advantages and disadvantages of 3D culture systems for both scaffold-free organoids and scaffolded organoids. This review also summarizes the application of organoid models in chemical toxicology evaluation, drug screening and functional evaluation, establishment of in vitro disease models, and research on disease occurrence and potential mechanisms. The aim is to provide a reference for the research and practical applications of organoid-related scientific fields.
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Affiliation(s)
- Xiaofeng Liu
- College of Food Science and Technology, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, China
| | - Zhiyuan Zhou
- College of Food Science and Technology, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, China
| | - Yao Zhang
- Zhejiang Provincial Key Lab for Chem and Bio Processing Technology of Farm Produces, School of Biological and Chemical Engineering, Zhejiang University of Science and Technology, Hangzhou, Zhejiang 310023, China
| | - Hao Zhong
- College of Food Science and Technology, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, China
| | - Xiulei Cai
- College of Food Science and Technology, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, China
| | - Rongfa Guan
- College of Food Science and Technology, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, China; Moganshan Institute ZJUT, Kangqian District, Deqing 313200, China.
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13
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Alnasser SM. From gut to liver: organoids as platforms for next-generation toxicology assessment vehicles for xenobiotics. Stem Cell Res Ther 2025; 16:150. [PMID: 40140938 PMCID: PMC11948905 DOI: 10.1186/s13287-025-04264-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/15/2024] [Accepted: 03/04/2025] [Indexed: 03/28/2025] Open
Abstract
Traditional toxicological assessment relied heavily on 2D cell cultures and animal models of study, which were inadequate for the precise prediction of human response to chemicals. Researchers have now shifted focus on organoids for toxicological assessment. Organoids are 3D structures produced from stem cells that mimic the shape and functionality of human organs and have a number of advantages compared to traditional models of study. They have the capacity to replicate the intricate cellular microenvironment and in vivo interactions. They offer a physiologically pertinent platform that is useful for the researchers to monitor cellular responses in a more realistic manner and evaluate drug toxicity. Additionally, organoids can be created from cells unique to a patient, allowing for individualized toxicological research and providing understanding of the inter-individual heterogeneity in drug responses. Recent developments in the use of gut and liver organoids for assessment of the xenobiotics (environmental toxins and drugs) is reviewed in this article. Gut organoids can reveal potential damage to the digestive system and how xenobiotics affect nutrient absorption and barrier function. Liver is the primary site of detoxification and metabolism of xenobiotics, usually routed from the gut. Hence, these are linked and crucial for evaluating chemical or pollutant induced organ toxicity, forecasting their metabolism and pharmacokinetics. When incorporated into the drug development process, organoid models have the potential to improve the accuracy and efficiency of drug safety assessments, leading to safer and more effective treatments. We also discuss the limitations of using organoid-based toxicological assays, and future prospects, including the need for standardized protocols for overcoming reproducibility issues.
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Affiliation(s)
- Sulaiman Mohammed Alnasser
- Department of Pharmacology and Toxicology, College of Pharmacy, Qassim University, 51452, Buraydah, Qassim, Saudi Arabia.
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14
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Vanhoeijen R, Okkelman IA, Rogier N, Sedlačík T, Stöbener DD, Devriendt B, Dmitriev RI, Hoogenboom R. Poly(2-alkyl-2-oxazoline) Hydrogels as Synthetic Matrices for Multicellular Spheroid and Intestinal Organoid Cultures. Biomacromolecules 2025; 26:1860-1872. [PMID: 39898884 DOI: 10.1021/acs.biomac.4c01627] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/04/2025]
Abstract
The extracellular matrix (ECM) plays a crucial role in organoid cultures by supporting cell proliferation and differentiation. A key feature of the ECM is its mechanical influence on the surrounding cells, directly affecting their behavior. Matrigel, the most commonly used ECM, is limited by its animal-derived origin, batch variability, and uncontrollable mechanical properties, restricting its use in 3D cell-model-based mechanobiological studies. Poly(2-alkyl-2-oxazoline) (PAOx) synthetic hydrogels represent an appealing alternative because of their reproducibility and versatile chemistry, enabling tuning of hydrogel stiffness and functionalization. Here, we studied PAOx hydrogels with differing compressive moduli for their potential to support 3D cell growth. PAOx hydrogels support spheroid and organoid growth over several days without the addition of ECM components. Furthermore, we discovered intestinal organoid epithelial polarity reversion in PAOx hydrogels and demonstrate how the tunable mechanical properties of PAOx can be used to study effects on the morphology and oxygenation of live multicellular spheroids.
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Affiliation(s)
- Robin Vanhoeijen
- Supramolecular Chemistry Group, Centre of Macromolecular Chemistry (CMaC), Department of Organic and Macromolecular Chemistry, Faculty of Sciences, Ghent University, Krijgslaan 281-S4, Ghent 9000, Belgium
- Tissue Engineering and Biomaterials Group, Department of Human Structure and Repair, Faculty of Medicine and Health Sciences, Ghent University, C. Heymanslaan 10, Ghent 9000, Belgium
| | - Irina A Okkelman
- Tissue Engineering and Biomaterials Group, Department of Human Structure and Repair, Faculty of Medicine and Health Sciences, Ghent University, C. Heymanslaan 10, Ghent 9000, Belgium
| | - Nette Rogier
- Supramolecular Chemistry Group, Centre of Macromolecular Chemistry (CMaC), Department of Organic and Macromolecular Chemistry, Faculty of Sciences, Ghent University, Krijgslaan 281-S4, Ghent 9000, Belgium
- Tissue Engineering and Biomaterials Group, Department of Human Structure and Repair, Faculty of Medicine and Health Sciences, Ghent University, C. Heymanslaan 10, Ghent 9000, Belgium
| | - Tomáš Sedlačík
- Supramolecular Chemistry Group, Centre of Macromolecular Chemistry (CMaC), Department of Organic and Macromolecular Chemistry, Faculty of Sciences, Ghent University, Krijgslaan 281-S4, Ghent 9000, Belgium
- Hydrogel Lab, Department of Polymers, Faculty of Chemical Technology, University of Chemistry and Technology, Technicka 1903/5, Prague 6 166 28, Czech Republic
| | - Daniel D Stöbener
- Supramolecular Chemistry Group, Centre of Macromolecular Chemistry (CMaC), Department of Organic and Macromolecular Chemistry, Faculty of Sciences, Ghent University, Krijgslaan 281-S4, Ghent 9000, Belgium
| | - Bert Devriendt
- Laboratory of Immunology, Department of Translational Physiology, Infectiology and Public Health, Faculty of Veterinary Medicine, Ghent University, Merelbeke 9820, Belgium
| | - Ruslan I Dmitriev
- Tissue Engineering and Biomaterials Group, Department of Human Structure and Repair, Faculty of Medicine and Health Sciences, Ghent University, C. Heymanslaan 10, Ghent 9000, Belgium
| | - Richard Hoogenboom
- Supramolecular Chemistry Group, Centre of Macromolecular Chemistry (CMaC), Department of Organic and Macromolecular Chemistry, Faculty of Sciences, Ghent University, Krijgslaan 281-S4, Ghent 9000, Belgium
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15
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Al-Qadami G, Raposo A, Chien CC, Ma C, Priebe I, Hor M, Fung K. Intestinal organoid coculture systems: current approaches, challenges, and future directions. Am J Physiol Gastrointest Liver Physiol 2025; 328:G252-G276. [PMID: 39716040 DOI: 10.1152/ajpgi.00203.2024] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/08/2024] [Revised: 12/12/2024] [Accepted: 12/12/2024] [Indexed: 12/25/2024]
Abstract
The intestinal microenvironment represents a complex and dynamic ecosystem, comprising a diverse range of epithelial and nonepithelial cells, a protective mucus layer, and a diverse community of gut microbiota. Understanding the intricate interplay between these components is essential for uncovering the mechanisms underlying intestinal health and disease. The development of intestinal organoids, three-dimensional (3-D) mini-intestines that closely mimic the architecture, cellular diversity, and functionality of the intestine, offers a powerful platform for investigating different aspects of intestinal physiology and pathology. However, current intestinal organoid models, mainly adult stem cell-derived organoids, lack the nonepithelial and microbial components of the intestinal microenvironment. As such, several coculture systems have been developed to coculture intestinal organoids with other intestinal elements including microbes (bacteria and viruses) and immune, stromal, and neural cells. These coculture models allow researchers to recreate the complex intestinal environment and study the intricate cross talk between different components of the intestinal ecosystem under healthy and pathological conditions. Currently, there are several approaches and methodologies to establish intestinal organoid cocultures, and each approach has its own strengths and limitations. This review discusses the existing methods for coculturing intestinal organoids with different intestinal elements, focusing on the methodological approaches, strengths and limitations, and future directions.
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Affiliation(s)
| | - Anita Raposo
- Health and Biosecurity, CSIRO, Sydney, New South Wales, Australia
| | - Chia-Chi Chien
- Australian Animal Health Laboratory, Australian Centre for Disease Preparedness, CSIRO, Geelong, Victoria, Australia
| | - Chenkai Ma
- Health and Biosecurity, CSIRO, Sydney, New South Wales, Australia
| | - Ilka Priebe
- Health and Biosecurity, CSIRO, Adelaide, South Australia, Australia
| | - Maryam Hor
- Health and Biosecurity, CSIRO, Adelaide, South Australia, Australia
| | - Kim Fung
- Health and Biosecurity, CSIRO, Sydney, New South Wales, Australia
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16
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Meirelles LA, Persat A. Decoding host-microbe interactions with engineered human organoids. EMBO J 2025; 44:1569-1573. [PMID: 39984757 PMCID: PMC11914615 DOI: 10.1038/s44318-025-00387-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2025] [Revised: 02/05/2025] [Accepted: 02/06/2025] [Indexed: 02/23/2025] Open
Affiliation(s)
- Lucas A Meirelles
- Global Health Institute, School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland
- Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland
| | - Alexandre Persat
- Global Health Institute, School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.
- Institute of Bioengineering, School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.
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17
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Tsukamoto M, Akutsu H. Comparative gastrointestinal organoid models across species: A Zoobiquity approach for precision medicine. Regen Ther 2025; 28:314-320. [PMID: 39885871 PMCID: PMC11779682 DOI: 10.1016/j.reth.2024.12.013] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2024] [Accepted: 12/20/2024] [Indexed: 02/01/2025] Open
Abstract
Gastrointestinal (GI) health underpins systemic well-being, yet the complexity of gut physiology poses significant challenges to understanding disease mechanisms and developing effective, personalized therapies. Traditional models often fail to capture the intricate interplay between epithelial, mesenchymal, immune, and neuronal cells that govern gut homeostasis and disease. Over the past five years, advances in organoid technology have created physiologically relevant, three-dimensional GI models that replicate native tissue architecture and function. These models have revolutionized the study of autoimmune disorders, homeostatic dysfunction, and pathogen infections, such as norovirus and Salmonella, which affect millions of humans and animals globally. In this review, we explore how organoids, derived from intestinal and pluripotent stem cells, are transforming our understanding of GI development, disease etiology, and therapeutic innovation. Through the "Zoobiquity" paradigm and "One Health" framework, we highlight the integration of companion animal organoids, which provide invaluable insights into shared disease mechanisms and preclinical therapeutic development. Despite their promise, challenges remain in achieving organoid maturation, expanding immune and neuronal integration, and bridging the gap between organoid responses and in vivo outcomes. By refining these cutting-edge platforms, we can advance human and veterinary medicine alike, fostering a holistic approach to health and disease.
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Affiliation(s)
- Masaya Tsukamoto
- Center for Regenerative Medicine, National Center for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo 157-8535, Japan
- Department of Advanced Pathobiology, Graduate School of Veterinary Science, Osaka Metropolitan University, Izumisano, Osaka 598-8531, Japan
| | - Hidenori Akutsu
- Center for Regenerative Medicine, National Center for Child Health and Development, 2-10-1 Okura, Setagaya, Tokyo 157-8535, Japan
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18
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De Martinis ECP, Alves VF, Pereira MG, Andrade LN, Abichabki N, Abramova A, Dannborg M, Bengtsson-Palme J. Applying 3D cultures and high-throughput technologies to study host-pathogen interactions. Front Immunol 2025; 16:1488699. [PMID: 40051624 PMCID: PMC11882522 DOI: 10.3389/fimmu.2025.1488699] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2024] [Accepted: 02/04/2025] [Indexed: 03/09/2025] Open
Abstract
Recent advances in cell culturing and DNA sequencing have dramatically altered the field of human microbiome research. Three-dimensional (3D) cell culture is an important tool in cell biology, in cancer research, and for studying host-microbe interactions, as it mimics the in vivo characteristics of the host environment in an in vitro system, providing reliable and reproducible models. This work provides an overview of the main 3D culture techniques applied to study interactions between host cells and pathogenic microorganisms, how these systems can be integrated with high-throughput molecular methods, and how multi-species model systems may pave the way forward to pinpoint interactions among host, beneficial microbes and pathogens.
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Affiliation(s)
| | | | - Marita Gimenez Pereira
- Ribeirão Preto School of Pharmaceutical Sciences, University of São Paulo, Ribeirão Preto, São Paulo, Brazil
| | - Leonardo Neves Andrade
- Ribeirão Preto School of Pharmaceutical Sciences, University of São Paulo, Ribeirão Preto, São Paulo, Brazil
| | - Nathália Abichabki
- Ribeirão Preto School of Pharmaceutical Sciences, University of São Paulo, Ribeirão Preto, São Paulo, Brazil
- Division of Systems and Synthetic Biology, Department of Life Sciences, SciLifeLab, Chalmers University of Technology, Gothenburg, Sweden
| | - Anna Abramova
- Division of Systems and Synthetic Biology, Department of Life Sciences, SciLifeLab, Chalmers University of Technology, Gothenburg, Sweden
- Centre for Antibiotic Resistance Research (CARe), Gothenburg, Sweden
| | - Mirjam Dannborg
- Division of Systems and Synthetic Biology, Department of Life Sciences, SciLifeLab, Chalmers University of Technology, Gothenburg, Sweden
- Centre for Antibiotic Resistance Research (CARe), Gothenburg, Sweden
- Department of Infectious Diseases, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden
| | - Johan Bengtsson-Palme
- Division of Systems and Synthetic Biology, Department of Life Sciences, SciLifeLab, Chalmers University of Technology, Gothenburg, Sweden
- Centre for Antibiotic Resistance Research (CARe), Gothenburg, Sweden
- Department of Infectious Diseases, Institute of Biomedicine, University of Gothenburg, Gothenburg, Sweden
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19
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Ma X, Li M, Zhang Y, Xu T, Zhou X, Qian M, Yang Z, Han X. Akkermansia muciniphila identified as key strain to alleviate gut barrier injury through Wnt signaling pathway. eLife 2025; 12:RP92906. [PMID: 39912727 PMCID: PMC11801796 DOI: 10.7554/elife.92906] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2025] Open
Abstract
As the largest mucosal surface, the gut has built a physical, chemical, microbial, and immune barrier to protect the body against pathogen invasion. The disturbance of gut microbiota aggravates pathogenic bacteria invasion and gut barrier injury. Fecal microbiota transplantation (FMT) is a promising treatment for microbiome-related disorders, where beneficial strain engraftment is a significant factor influencing FMT outcomes. The aim of this research was to explore the effect of FMT on antibiotic-induced microbiome-disordered (AIMD) models infected with enterotoxigenic Escherichia coli (ETEC). We used piglet, mouse, and intestinal organoid models to explore the protective effects and mechanisms of FMT on ETEC infection. The results showed that FMT regulated gut microbiota and enhanced the protection of AIMD piglets against ETEC K88 challenge, as demonstrated by reduced intestinal pathogen colonization and alleviated gut barrier injury. Akkermansia muciniphila (A. muciniphila) and Bacteroides fragilis (B. fragilis) were identified as two strains that may play key roles in FMT. We further investigated the alleviatory effects of these two strains on ETEC infection in the AIMD mice model, which revealed that A. muciniphila and B. fragilis relieved ETEC-induced intestinal inflammation by maintaining the proportion of Treg/Th17 cells and epithelial damage by moderately activating the Wnt/β-catenin signaling pathway, while the effect of A. muciniphila was better than B. fragilis. We, therefore, identified whether A. muciniphila protected against ETEC infection using basal-out and apical-out intestinal organoid models. A. muciniphila did protect the intestinal stem cells and stimulate the proliferation and differentiation of intestinal epithelium, and the protective effects of A. muciniphila were reversed by Wnt inhibitor. FMT alleviated ETEC-induced gut barrier injury and intestinal inflammation in the AIMD model. A. muciniphila was identified as a key strain in FMT to promote the proliferation and differentiation of intestinal stem cells by mediating the Wnt/β-catenin signaling pathway.
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Affiliation(s)
- Xin Ma
- Key Laboratory of Animal Nutrition and Feed Science in East China, Ministry of Agriculture, College of Animal Sciences, Zhejiang UniversityHangzhouChina
- Hainan Institute of Zhejiang University, Yongyou Industry Park, Yazhou Bay Sci-Tech CitySanyaChina
| | - Meng Li
- Key Laboratory of Animal Nutrition and Feed Science in East China, Ministry of Agriculture, College of Animal Sciences, Zhejiang UniversityHangzhouChina
- Hainan Institute of Zhejiang University, Yongyou Industry Park, Yazhou Bay Sci-Tech CitySanyaChina
| | - Yuanyuan Zhang
- Key Laboratory of Animal Nutrition and Feed Science in East China, Ministry of Agriculture, College of Animal Sciences, Zhejiang UniversityHangzhouChina
| | - Tingting Xu
- Key Laboratory of Animal Nutrition and Feed Science in East China, Ministry of Agriculture, College of Animal Sciences, Zhejiang UniversityHangzhouChina
| | - Xinchen Zhou
- Key Laboratory of Animal Nutrition and Feed Science in East China, Ministry of Agriculture, College of Animal Sciences, Zhejiang UniversityHangzhouChina
- Hainan Institute of Zhejiang University, Yongyou Industry Park, Yazhou Bay Sci-Tech CitySanyaChina
| | - Mengqi Qian
- Key Laboratory of Animal Nutrition and Feed Science in East China, Ministry of Agriculture, College of Animal Sciences, Zhejiang UniversityHangzhouChina
| | - Zhiren Yang
- Key Laboratory of Animal Nutrition and Feed Science in East China, Ministry of Agriculture, College of Animal Sciences, Zhejiang UniversityHangzhouChina
- Hainan Institute of Zhejiang University, Yongyou Industry Park, Yazhou Bay Sci-Tech CitySanyaChina
| | - Xinyan Han
- Key Laboratory of Animal Nutrition and Feed Science in East China, Ministry of Agriculture, College of Animal Sciences, Zhejiang UniversityHangzhouChina
- Hainan Institute of Zhejiang University, Yongyou Industry Park, Yazhou Bay Sci-Tech CitySanyaChina
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20
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Quacquarelli F, Davila S, Taelman J, Guiu J, Antfolk M. Enhanced small intestinal organoid-derived epithelial cell adhesion and growth in organ-on-a-chip devices. RSC Adv 2025; 15:3693-3703. [PMID: 39911548 PMCID: PMC11795259 DOI: 10.1039/d4ra08290g] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/22/2024] [Accepted: 01/29/2025] [Indexed: 02/07/2025] Open
Abstract
Organ-on-a-chip devices are predominately made of the polymer polymethylsiloxane (PDMS), exhibiting several attractive properties e.g., transparency, gas permeability, and biocompatibility. However, the attachment of cells to this polymer has proven challenging, especially for delicate primary cells e.g., small intestinal organoid-derived epithelial cells. Hence, a need to functionalize and coat the surface has arisen to render it more hydrophilic and improve its ability to support cell adhesion and growth. While previous research has demonstrated some successful results in culturing primary cells, no comprehensive and comparative protocol has been proposed. Here, we provide a protocol for enhanced small intestinal organoid-derived epithelial cell adhesion and growth on PDMS and plastics, assessing both PDMS surface functionalization, adhesion protein coating as well as medium selection. We assess PDMS functionalization using (3-aminopropyl)trimethoxysilane (APTMS) or polyethyleneimine-glutaraldehyde (PEIGA), and adhesion protein coating using various Laminins, Collagen I, Matrigel, or mixtures thereof. Finally, we assess the use of two different medium compositions including growth factors EGF, Noggin and R-spondin1 (ENR medium) alone or combined with the two small molecules CHIR99021 and valproic acid (CV medium). We envision that our results will be useful for further attempts in emulating the small intestine using plastic- or PDMS-based devices for organs-on-a-chip development.
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Affiliation(s)
| | - Sergio Davila
- Department of Biomedical Engineering, Lund University Lund Sweden
| | - Jasin Taelman
- Cell Plasticity and Regeneration Group, Regenerative Medicine Program, Institut d'Investigació Biomèdica de Bellvitge-IDIBELL L'Hospitalet de Llobregat Spain
- Program for Advancing the Clinical Translation of Regenerative Medicine of Catalonia, P-CMR[C] L'Hospitalet de Llobregat Spain
| | - Jordi Guiu
- Cell Plasticity and Regeneration Group, Regenerative Medicine Program, Institut d'Investigació Biomèdica de Bellvitge-IDIBELL L'Hospitalet de Llobregat Spain
- Program for Advancing the Clinical Translation of Regenerative Medicine of Catalonia, P-CMR[C] L'Hospitalet de Llobregat Spain
| | - Maria Antfolk
- Department of Biomedical Engineering, Lund University Lund Sweden
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21
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Vitale A, De Musis C, Bimonte M, Rubert J, Fogliano V. In vitro cellular model systems provide a promising alternative to animal experiments for studying the intestine-organ axis. Crit Rev Biotechnol 2025:1-18. [PMID: 39848642 DOI: 10.1080/07388551.2025.2452620] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2024] [Revised: 10/30/2024] [Accepted: 12/17/2024] [Indexed: 01/25/2025]
Abstract
Limiting animal experiments is essential for ethical issues and also because scientific evidence highlights the discrepancies between human and animal metabolism. This review aims to provide a critical discussion of the strengths and limitations of the most appropriate in vitro intestine model to answer complex research questions in pharmaceutical and nutraceutical fields. This review describes the components contributing to the definition of the gut barrier structure, from the outer mucus layer to the inner part of lamina propria, including endothelial and neuronal networks. We conclude that the main advantage of these co-culture models is their versatility since they are modulable systems in which each component can be added, changed, or removed to reproduce a specific physiological condition each time. Additionally, we compare intestinal organoid models and microfluidic systems with well-established co-culture models.
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Affiliation(s)
| | | | | | - Josep Rubert
- Food Quality and Design group, Wageningen University, Wageningen, The Netherlands
| | - Vincenzo Fogliano
- Arterra Biosciences.P.A, Naples, Italy
- Food Quality and Design group, Wageningen University, Wageningen, The Netherlands
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22
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Lakemeyer M, Latorre R, Blazkova K, Jensen D, Wood HM, Shakil N, Thomas SC, Saxena D, Mulpuri Y, Poolman D, de Haro PD, Keller LJ, Reed DE, Schmidt BL, Lomax AE, Bunnett NW, Bogyo M. Identification of a secreted protease from Bacteroides fragilis that induces intestinal pain and inflammation by cleavage of PAR 2. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2025:2025.01.15.633241. [PMID: 39868234 PMCID: PMC11761754 DOI: 10.1101/2025.01.15.633241] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 01/28/2025]
Abstract
Protease-activated receptor 2 (PAR2) is a central regulator of intestinal barrier function, inflammation and pain. Upregulated intestinal proteolysis and PAR2-signaling are implicated in inflammatory bowel diseases (IBDs) and irritable bowel syndrome (IBS). To identify potential bacterial regulators of PAR2 activity, we developed a functional assay for PAR2 processing and used it to screen conditioned media from a library of diverse gut commensal microbes. We found that multiple bacteria secrete proteases that cleave host PAR2. Using chemoproteomic profiling with a covalent irreversible inhibitor, we identified a previously uncharacterized Bacteroides fragilis serine protease Bfp1, and showed that it cleaves and activates PAR2 in multicellular and murine models. PAR2 cleavage by Bfp1 disrupts the intestinal barrier, sensitizes nociceptors, and triggers colonic inflammation and abdominal pain. Collectively, our findings uncover Bfp1-mediated PAR2-processing as a new axis of host-commensal-interaction in the gut that has the potential to be targeted for therapeutic intervention in IBD or IBS.
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Affiliation(s)
- Markus Lakemeyer
- Institute for Organic Chemistry and Macromolecular Chemistry, Friedrich-Schiller-University Jena; Jena, Germany
- Cluster of Excellence Balance of the Microverse, Friedrich Schiller University Jena; Jena, Germany
- Department of Pathology, Stanford University School of Medicine; Stanford, CA, USA
| | - Rocco Latorre
- Department of Molecular Pathobiology, College of Dentistry, New York University; New York, USA
- Pain Research Center, New York University; New York, USA
| | - Kristyna Blazkova
- Department of Pathology, Stanford University School of Medicine; Stanford, CA, USA
| | - Dane Jensen
- Department of Molecular Pathobiology, College of Dentistry, New York University; New York, USA
- Pain Research Center, New York University; New York, USA
- Translational Research Center, College of Dentistry, New York University; New York, USA
- Department of Oral and Maxillofacial Surgery, New York University College of Dentistry, Bluestone Center for Clinical Research; New York, USA
| | - Hannah M Wood
- Gastrointestinal Diseases Research Unit, Queen's University; Kingston, Ontario, Canada
| | - Nayab Shakil
- Institute for Organic Chemistry and Macromolecular Chemistry, Friedrich-Schiller-University Jena; Jena, Germany
| | - Scott C Thomas
- Department of Molecular Pathobiology, College of Dentistry, New York University; New York, USA
| | - Deepak Saxena
- Department of Molecular Pathobiology, College of Dentistry, New York University; New York, USA
| | - Yatendra Mulpuri
- Department of Molecular Pathobiology, College of Dentistry, New York University; New York, USA
| | - David Poolman
- Department of Molecular Pathobiology, College of Dentistry, New York University; New York, USA
- Pain Research Center, New York University; New York, USA
| | - Paz Duran de Haro
- Department of Molecular Pathobiology, College of Dentistry, New York University; New York, USA
- Pain Research Center, New York University; New York, USA
- Translational Research Center, College of Dentistry, New York University; New York, USA
- Department of Oral and Maxillofacial Surgery, New York University College of Dentistry, Bluestone Center for Clinical Research; New York, USA
| | - Laura J Keller
- Department of Pathology, Stanford University School of Medicine; Stanford, CA, USA
| | - David E Reed
- Gastrointestinal Diseases Research Unit, Queen's University; Kingston, Ontario, Canada
| | - Brian L Schmidt
- Department of Molecular Pathobiology, College of Dentistry, New York University; New York, USA
- Pain Research Center, New York University; New York, USA
- Translational Research Center, College of Dentistry, New York University; New York, USA
- Department of Oral and Maxillofacial Surgery, New York University College of Dentistry, Bluestone Center for Clinical Research; New York, USA
| | - Alan E Lomax
- Gastrointestinal Diseases Research Unit, Queen's University; Kingston, Ontario, Canada
| | - Nigel W Bunnett
- Department of Molecular Pathobiology, College of Dentistry, New York University; New York, USA
- Pain Research Center, New York University; New York, USA
| | - Matthew Bogyo
- Department of Pathology, Stanford University School of Medicine; Stanford, CA, USA
- Department of Microbiology and Immunology, Stanford University School of Medicine; Stanford, CA, USA
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23
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Eiken MK, Childs CJ, Brastrom LK, Frum T, Plaster EM, Ahmed DW, Spencer RC, Shachaf O, Pfeiffer S, Levine JE, Alysandratos KD, Kotton DN, Spence JR, Loebel C. Nascent matrix deposition supports alveolar organoid formation from aggregates in synthetic hydrogels. Stem Cell Reports 2025; 20:102376. [PMID: 39672155 PMCID: PMC11784465 DOI: 10.1016/j.stemcr.2024.11.006] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2024] [Revised: 11/12/2024] [Accepted: 11/14/2024] [Indexed: 12/15/2024] Open
Abstract
Human induced pluripotent stem cell (iPSC)-derived alveolar organoids have emerged as a system to model the alveolar epithelium in homeostasis and disease. However, alveolar organoids are typically grown in Matrigel, a mouse sarcoma-derived basement membrane matrix that offers poor control over matrix properties, prompting the development of synthetic hydrogels as a Matrigel alternative. Here, we develop a two-step culture method that involves pre-aggregation of organoids in hydrogel-based microwells followed by embedding in a synthetic hydrogel that supports alveolar organoid growth, while also offering considerable control over organoid and hydrogel properties. We find that the aggregated organoids secrete their own nascent extracellular matrix (ECM) both in the microwells and upon embedding in synthetic hydrogels, which supports their growth. Thus, the synthetic hydrogels described here allow us to de-couple exogenous and nascent ECM to interrogate the role of ECM in organoid formation.
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Affiliation(s)
- Madeline K Eiken
- Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA
| | - Charlie J Childs
- Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA
| | - Lindy K Brastrom
- Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA
| | - Tristan Frum
- Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA
| | - Eleanor M Plaster
- Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA
| | - Donia W Ahmed
- Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA
| | - Ryan C Spencer
- Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA
| | - Orren Shachaf
- Department of Biomedical Engineering, University of Texas, Austin, TX, USA
| | - Suzanne Pfeiffer
- Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA
| | - Justin E Levine
- Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA
| | - Konstantinos-Dionysios Alysandratos
- Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA 02118, USA; The Pulmonary Center and Department of Medicine, Boston University Chobanian & Avedisian School of Medicine, Boston, MA 02118, USA
| | - Darrell N Kotton
- Center for Regenerative Medicine, Boston University and Boston Medical Center, Boston, MA 02118, USA; The Pulmonary Center and Department of Medicine, Boston University Chobanian & Avedisian School of Medicine, Boston, MA 02118, USA
| | - Jason R Spence
- Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA; Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA; Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI, USA; Program in Cell and Molecular Biology, University of Michigan Medical School, Ann Arbor, MI, USA
| | - Claudia Loebel
- Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, USA; Department of Materials Science and Engineering, University of Michigan, Ann Arbor, MI, USA.
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24
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Yoneyama Y, Wu Y, Mori K, Takebe T. In toto biological framework: Modeling interconnectedness during human development. Dev Cell 2025; 60:8-20. [PMID: 39765224 DOI: 10.1016/j.devcel.2024.09.027] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/30/2023] [Revised: 04/21/2024] [Accepted: 09/20/2024] [Indexed: 05/24/2025]
Abstract
Recent advancements in pluripotent stem cell and synthetic tissue technology have brought significant breakthroughs in studying early embryonic development, particularly within the first trimester of development in humans. However, during fetal stage development, investigating further biological events represents a major challenge, partly due to the evolving complexity and continued interaction across multiple organ systems. To bridge this gap, we propose an "in toto" biological framework that leverages a triad of technologies: synthetic tissues, intravital microscopy, and computer vision to capture in vivo cellular morphodynamics, conceptualized as single-cell choreography. This perspective will discuss the inherent challenges in capturing such complexities and explore engineering technologies to delve into the less-explored phase of human development. We also propose reframing the organ-centric to a system-centric paradigm, as such a framework broadens the value of the in vivo-embedded synthetic-tissue-based approach for interrogating the multifaceted interplay of human developmental processes during this crucial stage.
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Affiliation(s)
- Yosuke Yoneyama
- Premium Research Institute for Human Metaverse Medicine (WPI-PRIMe), Graduate School of Medicine, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita-shi, Osaka 565-0871, Japan; Human Biology Research Unit, Institute of Integrated Research, Institute of Science Tokyo, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan
| | - Yunheng Wu
- Graduate School of Informatics, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan
| | - Kensaku Mori
- Graduate School of Informatics, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan; Information Technology Center, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan; Research Center for Medical Bigdata, National Institute of Informatics, Tokyo 100-0003, Japan
| | - Takanori Takebe
- Premium Research Institute for Human Metaverse Medicine (WPI-PRIMe), Graduate School of Medicine, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita-shi, Osaka 565-0871, Japan; Human Biology Research Unit, Institute of Integrated Research, Institute of Science Tokyo, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan; Divisions of Gastroenterology, Hepatology & Nutrition, and Developmental Biology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA; Department of Pediatrics, University of Cincinnati College of Medicine, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA; Center for Stem Cell and Organoid Medicine (CuSTOM), Cincinnati Children's Hospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229-3039, USA.
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25
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Micati D, Hlavca S, Chan WH, Abud HE. Harnessing 3D models to uncover the mechanisms driving infectious and inflammatory disease in the intestine. BMC Biol 2024; 22:300. [PMID: 39736603 DOI: 10.1186/s12915-024-02092-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2024] [Accepted: 12/10/2024] [Indexed: 01/01/2025] Open
Abstract
Representative models of intestinal diseases are transforming our knowledge of the molecular mechanisms of disease, facilitating effective drug screening and avenues for personalised medicine. Despite the emergence of 3D in vitro intestinal organoid culture systems that replicate the genetic and functional characteristics of the epithelial tissue of origin, there are still challenges in reproducing the human physiological tissue environment in a format that enables functional readouts. Here, we describe the latest platforms engineered to investigate environmental tissue impacts, host-microbe interactions and enable drug discovery. This highlights the potential to revolutionise knowledge on the impact of intestinal infection and inflammation and enable personalised disease modelling and clinical translation.
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Affiliation(s)
- Diana Micati
- Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, 3800, Australia
- Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, 3800, Australia
| | - Sara Hlavca
- Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, 3800, Australia
- Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, 3800, Australia
| | - Wing Hei Chan
- Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, 3800, Australia
- Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, 3800, Australia
| | - Helen E Abud
- Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, 3800, Australia.
- Development and Stem Cells Program, Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, 3800, Australia.
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26
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Lee MG, Lee BR, Lee P, Choi S, Kim JH, Oh MH, Yoo JG. Apical-out intestinal organoids as an alternative model for evaluating deoxynivalenol toxicity and Lactobacillus detoxification in bovine. Sci Rep 2024; 14:31373. [PMID: 39733018 PMCID: PMC11682149 DOI: 10.1038/s41598-024-82928-0] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2024] [Accepted: 12/10/2024] [Indexed: 12/30/2024] Open
Abstract
Small intestinal organoids are similar to actual small intestines in structure and function and can be used in various fields, such as nutrition, disease, and toxicity research. However, the basal-out type is difficult to homogenize because of the diversity of cell sizes and types, and the Matrigel-based culture conditions. Contrastingly, the apical-out form of small intestinal organoids is relatively uniform and easy to manipulate without Matrigel. Therefore, we sought to investigate the possibility of replacing animal testing with bovine apical-out small intestinal organoids (Apo-IOs) by confirming the toxicity of mycotoxins and effectiveness of L. plantarum as mycotoxin-reducing agents. The characteristics and functions of Apo-IOs were first confirmed. The gene and protein expression of stem cell, proliferation, mucous, and adherence markers were detected, and the absorption capacity of amino and fatty acids was also confirmed. FITC-4 kDa dextran, a marker of intestinal barrier function, did not penetrate the Apo-IOs, confirming the role of the organoids as a barrier. However, when co-treated with deoxynivalenol (DON), FITC-4 kDa dextran was detected deep within the organoids. Moreover, qPCR and immunofluorescence staining confirmed a decrease in the expression of key markers, such as LGR5, Ki67, Mucin2, Villin2, and E-cadherin. In addition, when Apo-IOs were treated with Lactobacillus plantarum ATCC14917 culture supernatant (LCS) and DON together, cell death was reduced compared to when treated with DON alone, and FITC-4 kDa dextran was confirmed to flow only to the peripheral part of the organoid. The qPCR and immunofluorescence staining results of LCS and DON co-treatment group showed that LGR5, Ki67, Mucin2, Villin2, and E-cadherin were expressed at significant higher levels than those in the DON treatment group alone. In this study, we found that the characteristics and functions of bovine Apo-IOs were similar to those of the intestinal structure in vivo. Additionally, the effects of mycotoxins and effectiveness of L. plantarum as mycotoxin-reducing agents were confirmed using bovine Apo-IOs. Therefore, bovine Apo-IOs could be applied in toxicity studies of mycotoxins and could also be used as in vitro models to replace animal testing and improve animal welfare.
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Affiliation(s)
- Min Gook Lee
- Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju, Republic of Korea
| | - Bo Ram Lee
- Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju, Republic of Korea
| | - Poongyeon Lee
- Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju, Republic of Korea
| | - Soyoung Choi
- Animal Genomics and Bioinformatics Division, National Institute of Animal Science, Rural Development Administration, Wanju, Republic of Korea
| | - Jong-Hui Kim
- Animal Products Research and Development Division, National Institute of Animal Science, Rural Development Administration, Wanju, Republic of Korea
| | - Mi-Hwa Oh
- Animal Products Research and Development Division, National Institute of Animal Science, Rural Development Administration, Wanju, Republic of Korea
| | - Jae Gyu Yoo
- Animal Biotechnology Division, National Institute of Animal Science, Rural Development Administration, Wanju, Republic of Korea.
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27
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Hofer M, Duque-Correa MA, Lutolf MP. Patterned gastrointestinal monolayers with bilateral access as observable models of parasite gut infection. Nat Biomed Eng 2024:10.1038/s41551-024-01313-4. [PMID: 39633029 PMCID: PMC7617323 DOI: 10.1038/s41551-024-01313-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2023] [Accepted: 11/01/2024] [Indexed: 12/07/2024]
Abstract
Organoids for modelling the physiology and pathology of gastrointestinal tissues are constrained by a poorly accessible lumen. Here we report the development and applicability of bilaterally accessible organoid-derived patterned epithelial monolayers that allow the independent manipulation of their apical and basal sides. We constructed gastric, small-intestinal, caecal and colonic epithelial models that faithfully reproduced their respective tissue geometries and that exhibited stem cell regionalization and transcriptional resemblance to in vivo epithelia. The models' enhanced observability allowed single-cell tracking and studies of the motility of cells in immersion culture and at the air-liquid interface. Models mimicking infection of the caecal epithelium by the parasite Trichuris muris allowed us to live image syncytial tunnel formation. The enhanced observability of bilaterally accessible organoid-derived gastrointestinal tissue will facilitate the study of the dynamics of epithelial cells and their interactions with pathogens.
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Affiliation(s)
- Moritz Hofer
- Laboratory of Stem Cell Bioengineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland
| | - Maria A Duque-Correa
- Wellcome-MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK.
| | - Matthias P Lutolf
- Laboratory of Stem Cell Bioengineering, École Polytechnique Fédérale de Lausanne, Lausanne, Switzerland.
- Institute of Human Biology (IHB), Roche Pharma Research and Early Development, Roche Innovation Center Basel, Basel, Switzerland.
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28
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Kim MH, Lee Y, Seo GM, Park S. Advancements in Kidney-on-Chip: Antibiotic-Induced Kidney Injury and Future Directions. BIOCHIP JOURNAL 2024; 18:535-545. [DOI: 10.1007/s13206-024-00160-4] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/10/2024] [Revised: 06/17/2024] [Accepted: 06/20/2024] [Indexed: 01/06/2025]
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29
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Wang Z, Ye S, van der Laan LJ, Schneeberger K, Masereeuw R, Spee B. Chemically Defined Organoid Culture System for Cholangiocyte Differentiation. Adv Healthc Mater 2024; 13:e2401511. [PMID: 39044566 PMCID: PMC11616262 DOI: 10.1002/adhm.202401511] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/24/2024] [Revised: 07/15/2024] [Indexed: 07/25/2024]
Abstract
Cholangiocyte organoids provide a powerful platform for applications ranging from in vitro modeling to tissue engineering for regenerative medicine. However, their expansion and differentiation are typically conducted in animal-derived hydrogels, which impede the full maturation of organoids into functional cholangiocytes. In addition, these hydrogels are poorly defined and complex, limiting the clinical applicability of organoids. In this study, a novel medium composition combined with synthetic polyisocyanopeptide (PIC) hydrogels to enhance the maturation of intrahepatic cholangiocyte organoids (ICOs) into functional cholangiocytes is utilized. ICOs cultured in the presence of sodium butyrate and valproic acid, a histone deacetylase inhibitor, and a Notch signaling activator, respectively, in PIC hydrogel exhibit a more mature phenotype, as evidenced by increased expression of key cholangiocyte markers, crucial for biliary function. Notably, mature cholangiocyte organoids in PIC hydrogel display apical-out polarity, in contrast to the traditional basal-out polarization of ICOs cultured in Matrigel. Moreover, these mature cholangiocyte organoids effectively model the biliary pro-fibrotic response induced by transforming growth factor beta. Taken together, an animal-free, chemically defined culture system that promotes the ICOs into mature cholangiocytes with apical-out polarity, facilitating regenerative medicine applications and in vitro studies that require access to the apical membrane, is developed.
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Affiliation(s)
- Zhenguo Wang
- Division of PharmacologyUtrecht Institute for Pharmaceutical SciencesFaculty of ScienceUtrecht UniversityUniversiteitsweg 99Utrecht3584 CGThe Netherlands
- Department of Clinical SciencesFaculty of Veterinary MedicineUtrecht UniversityUppsalalaan 8Utrecht3584 CTThe Netherlands
| | - Shicheng Ye
- Department of Clinical SciencesFaculty of Veterinary MedicineUtrecht UniversityUppsalalaan 8Utrecht3584 CTThe Netherlands
| | - Luc J.W. van der Laan
- Department of SurgeryErasmus MC Transplant InstituteUniversity Medical Center RotterdamDoctor Molewaterplein 40Rotterdam3015 GDThe Netherlands
| | - Kerstin Schneeberger
- Department of Clinical SciencesFaculty of Veterinary MedicineUtrecht UniversityUppsalalaan 8Utrecht3584 CTThe Netherlands
| | - Rosalinde Masereeuw
- Division of PharmacologyUtrecht Institute for Pharmaceutical SciencesFaculty of ScienceUtrecht UniversityUniversiteitsweg 99Utrecht3584 CGThe Netherlands
| | - Bart Spee
- Department of Clinical SciencesFaculty of Veterinary MedicineUtrecht UniversityUppsalalaan 8Utrecht3584 CTThe Netherlands
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30
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Randall-Demllo S, Al-Qadami G, Raposo AE, Ma C, Priebe IK, Hor M, Singh R, Fung KYC. Ex Vivo Intestinal Organoid Models: Current State-of-the-Art and Challenges in Disease Modelling and Therapeutic Testing for Colorectal Cancer. Cancers (Basel) 2024; 16:3664. [PMID: 39518102 PMCID: PMC11544769 DOI: 10.3390/cancers16213664] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/20/2024] [Revised: 10/24/2024] [Accepted: 10/25/2024] [Indexed: 11/16/2024] Open
Abstract
Despite improvements in participation in population-based screening programme, colorectal cancer remains a major cause of cancer-related mortality worldwide. Targeted interventions are desirable to reduce the health and economic burden of this disease. Two-dimensional monolayers of colorectal cancer cell lines represent the traditional in vitro models for disease and are often used for diverse purposes, including the delineation of molecular pathways associated with disease aetiology or the gauging of drug efficacy. The lack of complexity in such models, chiefly the limited epithelial cell diversity and differentiation, attenuated mucus production, lack of microbial interactions and mechanical stresses, has driven interest in the development of more holistic and physiologically relevant in vitro model systems. In particular, established ex vivo patient-derived explant and patient-derived tumour xenograft models have been supplemented by progress in organoid and microfluidic organ-on-a-chip cultures. Here, we discuss the applicability of advanced culturing technologies, such as organoid systems, as models for colorectal cancer and for testing chemotherapeutic drug sensitivity and efficacy. We highlight current challenges associated with organoid technologies and discuss their future for more accurate disease modelling and personalized medicine.
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Affiliation(s)
- Sarron Randall-Demllo
- Health and Biosecurity, Commonwealth Scientific and Industrial Research Organisation, Adelaide 5000, Australia; (S.R.-D.); (G.A.-Q.)
| | - Ghanyah Al-Qadami
- Health and Biosecurity, Commonwealth Scientific and Industrial Research Organisation, Adelaide 5000, Australia; (S.R.-D.); (G.A.-Q.)
| | - Anita E. Raposo
- Health and Biosecurity, Commonwealth Scientific and Industrial Research Organisation, Westmead 2145, Australia; (A.E.R.); (C.M.)
| | - Chenkai Ma
- Health and Biosecurity, Commonwealth Scientific and Industrial Research Organisation, Westmead 2145, Australia; (A.E.R.); (C.M.)
| | - Ilka K. Priebe
- Health and Biosecurity, Commonwealth Scientific and Industrial Research Organisation, Adelaide 5000, Australia; (S.R.-D.); (G.A.-Q.)
| | - Maryam Hor
- Health and Biosecurity, Commonwealth Scientific and Industrial Research Organisation, Adelaide 5000, Australia; (S.R.-D.); (G.A.-Q.)
| | - Rajvinder Singh
- Division of Gastroenterology, Lyell McEwin Hospital, Adelaide 5112, Australia
| | - Kim Y. C. Fung
- Health and Biosecurity, Commonwealth Scientific and Industrial Research Organisation, Westmead 2145, Australia; (A.E.R.); (C.M.)
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31
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Sousa M, Magalhães R, Ferreira V, Teixeira P. Current methodologies available to evaluate the virulence potential among Listeria monocytogenes clonal complexes. Front Microbiol 2024; 15:1425437. [PMID: 39493856 PMCID: PMC11528214 DOI: 10.3389/fmicb.2024.1425437] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2024] [Accepted: 09/26/2024] [Indexed: 11/05/2024] Open
Abstract
Listeria monocytogenes is a foodborne pathogen that causes listeriosis in humans, the severity of which depends on multiple factors, including intrinsic characteristics of the affected individuals and the pathogen itself. Additionally, emerging evidence suggests that epigenetic modifications may also modulate host susceptibility to infection. Therefore, different clinical outcomes can be expected, ranging from self-limiting gastroenteritis to severe central nervous system and maternal-neonatal infections, and bacteremia. Furthermore, L. monocytogenes is a genetically and phenotypically diverse species, resulting in a large variation in virulence potential between strains. Multilocus sequence typing (MLST) has been widely used to categorize the clonal structure of bacterial species and to define clonal complexes (CCs) of genetically related isolates. The combination of MLST and epidemiological data allows to distinguish hypervirulent CCs, which are notably more prevalent in clinical cases and typically associated with severe forms of the disease. Conversely, other CCs, termed hypovirulent, are predominantly isolated from food and food processing environments and are associated with the occurrence of listeriosis in immunosuppressed individuals. Reports of genetic traits associated with this diversity have been described. The Food and Agriculture Organization (FAO) is encouraging the search for virulence biomarkers to rapidly identify the main strains of concern to reduce food waste and economical losses. The aim of this review is to comprehensively collect, describe and discuss the methodologies used to discriminate the virulence potential of L. monocytogenes CCs. From the exploration of in vitro and in vivo models to the study of expression of virulence genes, each approach is critically explored to better understand its applicability and efficiency in distinguishing the virulence potential of the pathogen.
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Affiliation(s)
| | | | | | - Paula Teixeira
- Universidade Católica Portuguesa, CBQF - Centro de Biotecnologia e Química Fina – Laboratório Associado, Escola Superior de Biotecnologia, Rua Diogo Botelho 1327, Porto, Portugal
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Rijns L, Hagelaars MJ, van der Tol JJB, Loerakker S, Bouten CVC, Dankers PYW. The Importance of Effective Ligand Concentration to Direct Epithelial Cell Polarity in Dynamic Hydrogels. ADVANCED MATERIALS (DEERFIELD BEACH, FLA.) 2024; 36:e2300873. [PMID: 37264535 DOI: 10.1002/adma.202300873] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 01/29/2023] [Revised: 05/26/2023] [Indexed: 06/03/2023]
Abstract
Epithelial cysts and organoids are multicellular hollow structures formed by correctly polarized epithelial cells. Important in steering these cysts from single cells is the dynamic regulation of extracellular matrix presented ligands, and matrix dynamics. Here, control over the effective ligand concentration is introduced, decoupled from bulk and local mechanical properties, in synthetic dynamic supramolecular hydrogels formed through noncovalent crosslinking of supramolecular fibers. Control over the effective ligand concentration is realized by 1) keeping the ligand concentration constant, but changing the concentration of nonfunctionalized molecules or by 2) varying the ligand concentration, while keeping the concentration of non-functionalized molecules constant. The results show that in 2D, the effective ligand concentration within the supramolecular fibers rather than gel stiffness (from 0.1 to 8 kPa) regulates epithelial polarity. In 3D, increasing the effective ligand concentration from 0.5 × 10-3 to 2 × 10-3 m strengthens the effect of increased gel stiffness from 0.1 to 2 kPa, to synergistically yield more correctly polarized cysts. Through integrin manipulation, it is shown that epithelial polarity is regulated by tension-based homeostasis between cells and matrix. The results reveal the effective ligand concentration as influential factor in regulating epithelial polarity and provide insights on engineering of synthetic biomaterials for cell and organoid culture.
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Affiliation(s)
- Laura Rijns
- Institute for Complex Molecular Systems, Eindhoven University of Technology, PO Box 513, Eindhoven, The Netherlands
- Department of Biomedical Engineering, Laboratory of Chemical Biology, Eindhoven University of Technology, PO Box 513, Eindhoven, The Netherlands
| | - Maria J Hagelaars
- Institute for Complex Molecular Systems, Eindhoven University of Technology, PO Box 513, Eindhoven, The Netherlands
- Department of Biomedical Engineering, Laboratory for Cell and Tissue Engineering, Eindhoven University of Technology, PO Box 513, Eindhoven, The Netherlands
| | - Joost J B van der Tol
- Institute for Complex Molecular Systems, Eindhoven University of Technology, PO Box 513, Eindhoven, The Netherlands
- Department of Chemical Engineering and Chemistry, Laboratory of Macromolecular and Organic Chemistry, Eindhoven University of Technology, PO Box 513, Eindhoven, The Netherlands
| | - Sandra Loerakker
- Institute for Complex Molecular Systems, Eindhoven University of Technology, PO Box 513, Eindhoven, The Netherlands
- Department of Biomedical Engineering, Laboratory for Cell and Tissue Engineering, Eindhoven University of Technology, PO Box 513, Eindhoven, The Netherlands
| | - Carlijn V C Bouten
- Institute for Complex Molecular Systems, Eindhoven University of Technology, PO Box 513, Eindhoven, The Netherlands
- Department of Biomedical Engineering, Laboratory for Cell and Tissue Engineering, Eindhoven University of Technology, PO Box 513, Eindhoven, The Netherlands
| | - Patricia Y W Dankers
- Institute for Complex Molecular Systems, Eindhoven University of Technology, PO Box 513, Eindhoven, The Netherlands
- Department of Biomedical Engineering, Laboratory of Chemical Biology, Eindhoven University of Technology, PO Box 513, Eindhoven, The Netherlands
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33
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Wu J, Gupta G, Buerki-Thurnherr T, Nowack B, Wick P. Bridging the gap: Innovative human-based in vitro approaches for nanomaterials hazard assessment and their role in safe and sustainable by design, risk assessment, and life cycle assessment. NANOIMPACT 2024; 36:100533. [PMID: 39454678 DOI: 10.1016/j.impact.2024.100533] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/05/2024] [Revised: 10/22/2024] [Accepted: 10/22/2024] [Indexed: 10/28/2024]
Abstract
The application of nanomaterials in industry and consumer products is growing exponentially, which has pressed the development and use of predictive human in vitro models in pre-clinical analysis to closely extrapolate potential toxic effects in vivo. The conventional cytotoxicity investigation of nanomaterials using cell lines from cancer origin and culturing them two-dimensionally in a monolayer without mimicking the proper pathophysiological microenvironment may affect a precise prediction of in vitro effects at in vivo level. In recent years, complex in vitro models (also belonging to the new approach methodologies, NAMs) have been established in unicellular to multicellular cultures either by using cell lines, primary cells or induced pluripotent stem cells (iPSCs), and reconstituted into relevant biological dimensions mimicking in vivo conditions. These advanced in vitro models retain physiologically reliant exposure scenarios particularly appropriate for oral, dermal, respiratory, and intravenous administration of nanomaterials, which have the potential to improve the in vivo predictability and lead to reliable outcomes. In this perspective, we discuss recent developments and breakthroughs in using advanced human in vitro models for hazard assessment of nanomaterials. We identified fit-for-purpose requirements and remaining challenges for the successful implementation of in vitro data into nanomaterials Safe and Sustainable by Design (SSbD), Risk Assessment (RA), and Life Cycle Assessment (LCA). By addressing the gap between in vitro data generation and the utility of in vitro data for nanomaterial safety assessments, a prerequisite for SSbD approaches, we outlined potential key areas for future development.
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Affiliation(s)
- Jimeng Wu
- Empa, Swiss Federal Laboratories for Materials Science and Technology, Particles-Biology Interactions Laboratory, Lerchenfeldstrasse 5, 9014 St. Gallen, Switzerland; Empa, Swiss Federal Laboratories for Materials Science and Technology, Technology and Society Laboratory, Lerchenfeldstrasse 5, 9014 St. Gallen, Switzerland
| | - Govind Gupta
- Empa, Swiss Federal Laboratories for Materials Science and Technology, Particles-Biology Interactions Laboratory, Lerchenfeldstrasse 5, 9014 St. Gallen, Switzerland
| | - Tina Buerki-Thurnherr
- Empa, Swiss Federal Laboratories for Materials Science and Technology, Particles-Biology Interactions Laboratory, Lerchenfeldstrasse 5, 9014 St. Gallen, Switzerland
| | - Bernd Nowack
- Empa, Swiss Federal Laboratories for Materials Science and Technology, Technology and Society Laboratory, Lerchenfeldstrasse 5, 9014 St. Gallen, Switzerland
| | - Peter Wick
- Empa, Swiss Federal Laboratories for Materials Science and Technology, Particles-Biology Interactions Laboratory, Lerchenfeldstrasse 5, 9014 St. Gallen, Switzerland.
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Burton TD, Carrera Montoya J, Frota T, Mackenzie JM. Human norovirus cultivation models, immune response and vaccine landscape. Adv Virus Res 2024; 120:1-37. [PMID: 39455167 DOI: 10.1016/bs.aivir.2024.09.001] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/28/2024]
Abstract
Norovirus infections are a leading cause of gastroenteritis worldwide. Despite the substantial global health burden and economic impact, there are currently no approved antiviral therapeutics or vaccines. Additionally, much of our knowledge of norovirus comes from experiments using surrogate viruses, such as murine norovirus and feline calicivirus. The challenge surrounding human norovirus research arises from a lack of robust cell culture systems and efficient animal models. In this review, we explore recent advances in the in vitro cultivation of human norovirus and reverse genetics systems and discuss commonly used in vivo models. We summarize the current understanding of both innate and adaptive immune responses to norovirus infection and provide an overview of vaccine strategies and the current clinical trial landscape, with a focus on the only vaccine candidate that has reached phase III clinical development stage.
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Affiliation(s)
- Thomas D Burton
- Department of Microbiology and Immunology, University of Melbourne, within the Peter Doherty Institute for Infection and Immunity, Parkville, Melbourne, VIC, Australia
| | - Julio Carrera Montoya
- Department of Microbiology and Immunology, University of Melbourne, within the Peter Doherty Institute for Infection and Immunity, Parkville, Melbourne, VIC, Australia
| | - Thalia Frota
- Department of Microbiology and Immunology, University of Melbourne, within the Peter Doherty Institute for Infection and Immunity, Parkville, Melbourne, VIC, Australia
| | - Jason M Mackenzie
- Department of Microbiology and Immunology, University of Melbourne, within the Peter Doherty Institute for Infection and Immunity, Parkville, Melbourne, VIC, Australia.
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35
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van Dinteren S, Araya-Cloutier C, Bastiaan-Net S, Boudewijn A, van Heek T, Vincken JP, Witkamp R, Meijerink J. Biotransformation and Epithelial Toxicity of Prenylated Phenolics from Licorice Roots ( Glycyrrhiza spp.) in 3D Apical-Out Mucus-Producing Human Enteroids. JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY 2024; 72:20396-20409. [PMID: 39240776 PMCID: PMC11421016 DOI: 10.1021/acs.jafc.4c03120] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 04/10/2024] [Revised: 08/06/2024] [Accepted: 08/24/2024] [Indexed: 09/08/2024]
Abstract
Apical-out enteroids mimic the in vivo environment well due to their accessible apical surface and mucus layer, making them an ideal model for studying the impact of (bioactive) food compounds. Generated human ileal apical-out enteroids showed a fucose-containing mucus layer surrounding the apical brush border on their exposure side, indicating their physiological relevance. Effects on the mucosal epithelium of antibacterial prenylated phenolics (glabridin, licochalcone A, and glycycoumarin) from licorice roots were investigated for cytotoxicity, cell viability, barrier integrity, and biotransformation. At concentrations up to 500 μg mL-1, licochalcone A and glycycoumarin did not significantly affect apical-out enteroids, with cytotoxicities of -6 ± 2 and -2 ± 2% and cell viabilities of 77 ± 22 and 77 ± 13%, respectively (p > 0.05). Conversely, 500 μg mL-1 glabridin induced significant cytotoxicity (31 ± 25%, p < 0.05) and reduced cell viability (21 ± 14%, p < 0.01). Apical-out enteroids revealed differential sensitivities to prenylated phenolics not observed in apical-in enteroids and Caco-2 cells. Both enteroid models showed phase II biotransformation but differed in the extent of glucuronide conversion. The apical mucus layer of apical-out enteroids likely contributed to these differential interactions, potentially due to differences in electrostatic repulsion. This study underscores the relevance of 3D apical-out enteroid models and highlights the promise of prenylated phenolics for antimicrobial applications.
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Affiliation(s)
- Sarah van Dinteren
- Division
of Human Nutrition and Health, Wageningen
University, P.O. Box 17, Wageningen 6700 AA, The Netherlands
- Laboratory
of Food Chemistry, Wageningen University, P.O. Box 17, Wageningen 6700 AA, The Netherlands
| | - Carla Araya-Cloutier
- Laboratory
of Food Chemistry, Wageningen University, P.O. Box 17, Wageningen 6700 AA, The Netherlands
| | - Shanna Bastiaan-Net
- Wageningen
Food & Biobased Research, Wageningen
University & Research, P.O. Box 17, Wageningen 6700 AA, The Netherlands
| | - Anouk Boudewijn
- Wageningen
Food & Biobased Research, Wageningen
University & Research, P.O. Box 17, Wageningen 6700 AA, The Netherlands
| | - Tjarda van Heek
- Department
of Abdominal Surgery, Hospital Gelderse
Vallei, Willy Brandtlaan 10, Ede 6716 RP, The Netherlands
| | - Jean-Paul Vincken
- Laboratory
of Food Chemistry, Wageningen University, P.O. Box 17, Wageningen 6700 AA, The Netherlands
| | - Renger Witkamp
- Division
of Human Nutrition and Health, Wageningen
University, P.O. Box 17, Wageningen 6700 AA, The Netherlands
| | - Jocelijn Meijerink
- Division
of Human Nutrition and Health, Wageningen
University, P.O. Box 17, Wageningen 6700 AA, The Netherlands
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36
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Kraski A, Migdał P, Klopfleisch R, Räckel C, Sharbati J, Heimesaat MM, Alter T, Hanisch C, Gölz G, Einspanier R, Sharbati S. Structured multicellular intestinal spheroids (SMIS) as a standardized model for infection biology. Gut Pathog 2024; 16:47. [PMID: 39289703 PMCID: PMC11406839 DOI: 10.1186/s13099-024-00644-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/21/2024] [Accepted: 09/12/2024] [Indexed: 09/19/2024] Open
Abstract
BACKGROUND 3D cell culture models have recently garnered increasing attention for replicating organ microarchitecture and eliciting in vivo-like responses, holding significant promise across various biological disciplines. Broadly, 3D cell culture encompasses organoids as well as single- and multicellular spheroids. While the latter have found successful applications in tumor research, there is a notable scarcity of standardized intestinal models for infection biology that mimic the microarchitecture of the intestine. Hence, this study aimed to develop structured multicellular intestinal spheroids (SMIS) specifically tailored for studying molecular basis of infection by intestinal pathogens. RESULTS We have successfully engineered human SMIS comprising four relevant cell types, featuring a fibroblast core enveloped by an outer monolayer of enterocytes and goblet cells along with monocytic cells. These SMIS effectively emulate the in vivo architecture of the intestinal mucosal surface and manifest differentiated morphological characteristics, including the presence of microvilli, within a mere two days of culture. Through analysis of various differentiation factors, we have illustrated that these spheroids attain heightened levels of differentiation compared to 2D monolayers. Moreover, SMIS serve as an optimized intestinal infection model, surpassing the capabilities of traditional 2D cultures, and exhibit a regulatory pattern of immunological markers similar to in vivo infections after Campylobacter jejuni infection. Notably, our protocol extends beyond human spheroids, demonstrating adaptability to other species such as mice and pigs. CONCLUSION Based on the rapid attainment of enhanced differentiation states, coupled with the emergence of functional brush border features, increased cellular complexity, and replication of the intestinal mucosal microarchitecture, which allows for exposure studies via the medium, we are confident that our innovative SMIS model surpasses conventional cell culture methods as a superior model. Moreover, it offers advantages over stem cell-derived organoids due to scalability and standardization capabilities of the protocol. By showcasing differentiated morphological attributes, our model provides an optimal platform for diverse applications. Furthermore, the investigated differences of several immunological factors compared to monotypic monolayers after Campylobacter jejuni infection underline the refinement of our spheroid model, which closely mimics important features of in vivo infections.
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Affiliation(s)
- Angelina Kraski
- Institute of Veterinary Biochemistry, Freie Universität Berlin, Berlin, Germany
| | - Paweł Migdał
- Institute of Animal Husbandry and Breeding, Wrocław University of Environmental and Life Sciences, Wrocław, Poland
| | - Robert Klopfleisch
- Institute of Veterinary Pathology, Freie Universität Berlin, Berlin, Germany
| | - Clara Räckel
- Institute of Veterinary Biochemistry, Freie Universität Berlin, Berlin, Germany
| | | | - Markus M Heimesaat
- Institute of Microbiology, Infectious Diseases and Immunology, Charité, Universitätsmedizin Berlin, Corporate Member of Freie Universität Berlin, Humboldt-Universität Zu Berlin, and Berlin Institute of Health, Berlin, Germany
| | - Thomas Alter
- Institute of Food Safety and Food Hygiene, Freie Universität Berlin, Berlin, Germany
| | | | - Greta Gölz
- Institute of Food Safety and Food Hygiene, Freie Universität Berlin, Berlin, Germany
| | - Ralf Einspanier
- Institute of Veterinary Biochemistry, Freie Universität Berlin, Berlin, Germany
| | - Soroush Sharbati
- Institute of Veterinary Biochemistry, Freie Universität Berlin, Berlin, Germany.
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37
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Kromann EH, Cearra AP, Neves JF. Organoids as a tool to study homeostatic and pathological immune-epithelial interactions in the gut. Clin Exp Immunol 2024; 218:28-39. [PMID: 38551817 PMCID: PMC11404120 DOI: 10.1093/cei/uxad118] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/10/2023] [Revised: 05/28/2023] [Accepted: 11/07/2023] [Indexed: 09/17/2024] Open
Abstract
The intestine hosts the largest immune cell compartment in the body as a result of its continuous exposure to exogenous antigens. The intestinal barrier is formed by a single layer of epithelial cells which separate immune cells from the gut lumen. Bidirectional interactions between the epithelium and the immune compartment are critical for maintaining intestinal homeostasis by limiting infection, preventing excessive immune activation, and promoting tissue repair processes. However, our understanding of epithelial-immune interactions incomplete as the complexity of in vivo models can hinder mechanistic studies, cell culture models lack the cellular heterogeneity of the intestine and when established from primary cell can be difficult to maintain. In the last decade, organoids have emerged as a reliable model of the intestine, recapitulating key cellular and architectural features of native tissues. Herein, we provide an overview of how intestinal organoids are being co-cultured with immune cells leading to substantial advances in our understanding of immune-epithelial interactions in the gut. This has enabled new discoveries of the immune contribution to epithelial maintenance and regeneration both in homeostasis and in disease such as chronic inflammation, infection and cancer. Organoids can additionally be used to generate immune cells with a tissue-specific phenotype and to investigate the impact of disease associated risk genes on the intestinal immune environment. Accordingly, this review demonstrates the multitude of applications for intestinal organoids in immunological research and their potential for translational approaches.
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Affiliation(s)
- Emma Højmose Kromann
- Centre for Host Microbiome Interactions, King's College London, London, United Kingdom
| | - Ainize Peña Cearra
- Centre for Host Microbiome Interactions, King's College London, London, United Kingdom
- Department of Immunology, Microbiology and Parasitology, Faculty of Medicine and Nursing, University of the Basque Country (UPV/EHU), Bilbao, Spain
| | - Joana F Neves
- Centre for Host Microbiome Interactions, King's College London, London, United Kingdom
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38
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Paužuolis M, Samperio Ventayol P, Neyazi M, Bartfeld S. Organoids as a tool to study the impact of heterogeneity in gastrointestinal epithelium on host-pathogen interactions. Clin Exp Immunol 2024; 218:16-27. [PMID: 38245816 PMCID: PMC11404121 DOI: 10.1093/cei/uxae002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2023] [Revised: 11/17/2023] [Accepted: 01/19/2024] [Indexed: 01/22/2024] Open
Abstract
The epithelium of the gastrointestinal (GI) tract has been extensively characterized using advanced histological and RNA sequencing techniques, which has revealed great cellular diversity. Pathogens, such as viruses and bacteria, are highly adapted to their host and often exhibit not only species-specificity but also a preference or tropism for specific GI segments or even cell types-some of these preferences are so specific, that these pathogens still cannot be cultured invitro. Organoid technology now provides a tool to generate human cell types, which enables the study of host cell tropism. Focussing on the GI tract, we provide an overview about cellular differentiation in vivo and in organoids and how differentiation in organoids and their derived models is used to advance our understanding of viral, bacterial, and parasitic infection. We emphasize that it is central to understand the composition of the model, as the alteration of culture conditions yields different cell types which affects infection. We examine future directions for wider application of cellular heterogeneity and potential advanced model systems for GI tract infection studies.
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Affiliation(s)
- Mindaugas Paužuolis
- Research Centre for Infectious Diseases, Institute for Molecular Infection Biology, Julius Maximilians Universität Würzburg, Würzburg, Germany
| | | | - Mastura Neyazi
- Research Centre for Infectious Diseases, Institute for Molecular Infection Biology, Julius Maximilians Universität Würzburg, Würzburg, Germany
| | - Sina Bartfeld
- Research Centre for Infectious Diseases, Institute for Molecular Infection Biology, Julius Maximilians Universität Würzburg, Würzburg, Germany
- Institute of Biotechnology, Technische Universität Berlin, Berlin, Germany
- Si-M/‘Der Simulierte Mensch’, Technische Universität Berlin and Charité–Universitätsmedizin Berlin, Berlin, Germany
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39
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Parente IA, Chiara L, Bertoni S. Exploring the potential of human intestinal organoids: Applications, challenges, and future directions. Life Sci 2024; 352:122875. [PMID: 38942359 DOI: 10.1016/j.lfs.2024.122875] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2024] [Revised: 06/13/2024] [Accepted: 06/25/2024] [Indexed: 06/30/2024]
Abstract
The complex and dynamic environment of the gastrointestinal tract shapes one of the fastest renewing tissues in the human body, the intestinal epithelium. Considering the lack of human preclinical studies, reliable models that mimic the intestinal environment are increasingly explored. Patient-derived intestinal organoids are powerful tools that recapitulate in vitro many pathophysiological features of the human intestine. In this review, the possible applications of human intestinal organoids in different research fields are highlighted. From physiologically relevant to intestinal disease modeling, regenerative medicine, and toxicology studies, the potential of intestinal organoids will be here presented and discussed. Despite the remarkable opportunities offered, limitations related to ethical concerns, tissue collection, reproducibility, and methodologies may hinder the full exploitation of this cell-based model into high throughput studies and clinical practice. Currently, distinct approaches can be used to overcome the numerous challenges found along the way and to allow the full implementation of this ground-breaking technology.
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Affiliation(s)
- Inês A Parente
- Department of Food and Drug, University of Parma, Parma, Italy
| | - Linda Chiara
- Department of Food and Drug, University of Parma, Parma, Italy
| | - Simona Bertoni
- Department of Food and Drug, University of Parma, Parma, Italy.
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40
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Kim R, Sung JH. Recent Advances in Gut- and Gut-Organ-Axis-on-a-Chip Models. Adv Healthc Mater 2024; 13:e2302777. [PMID: 38243887 DOI: 10.1002/adhm.202302777] [Citation(s) in RCA: 4] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/22/2023] [Revised: 12/21/2023] [Indexed: 01/22/2024]
Abstract
The human gut extracts nutrients from the diet while forming the largest barrier against the outer environment. In addition, the gut actively maintains homeostasis through intricate interactions with the gut microbes, the immune system, the enteric nervous system, and other organs. These interactions influence digestive health and, furthermore, play crucial roles in systemic health and disease. Given its primary role in absorbing and metabolizing orally administered drugs, there is significant interest in the development of preclinical in vitro model systems that can accurately emulate the intestine in vivo. A gut-on-a-chip system holds great potential as a testing and screening platform because of its ability to emulate the physiological aspects of in vivo tissues and expandability to incorporate and combine with other organs. This review aims to identify the key physiological features of the human gut that need to be incorporated to build more accurate preclinical models and highlights the recent progress in gut-on-a-chip systems and competing technologies toward building more physiologically relevant preclinical model systems. Furthermore, various efforts to construct multi-organ systems with the gut, called gut-organ-axis-on-a-chip models, are discussed. In vitro gut models with physiological relevance can provide valuable platforms for bridging the gap between preclinical and clinical studies.
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Affiliation(s)
- Raehyun Kim
- Department of Biological and Chemical Engineering, Hongik University, Sejong, 30016, Republic of Korea
| | - Jong Hwan Sung
- Department of Chemical Engineering, Hongik University, Seoul, 04066, Republic of Korea
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41
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Lee JH, LeCher JC, Parigoris E, Shinagawa N, Sentosa J, Manfredi C, Goh SL, De R, Tao S, Zandi K, Amblard F, Sorscher EJ, Spence JR, Tirouvanziam R, Schinazi RF, Takayama S. Development of robust antiviral assays using relevant apical-out human airway organoids. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.01.02.573939. [PMID: 38260306 PMCID: PMC10802305 DOI: 10.1101/2024.01.02.573939] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/24/2024]
Abstract
While breakthroughs with organoids have emerged as next-generation in vitro tools, standardization for drug discovery remains a challenge. This work introduces human airway organoids with reversed biopolarity (AORBs), cultured and analyzed in a high-throughput, single-organoid-per-well format, enabling milestones towards standardization. AORBs exhibit a spatio-temporally stable apical-out morphology, facilitating high-yield direct intact-organoid virus infection. Single-cell RNA sequencing and immunohistochemistry confirm the physiologically relevant recapitulation of differentiated human airway epithelia. The cellular tropism of five severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains along with host response differences between Delta, Washington, and Omicron variants, as observed in transcriptomic profiles, also suggest clinical relevance. Dose-response analysis of three well-studied SARS-CoV-2 antiviral compounds (remdesivir, bemnifosbuvir, and nirmatrelvir) demonstrates that AORBs efficiently predict human efficacy, comparable to gold-standard air-liquid interface cultures, but with higher throughput (~10-fold) and fewer cells (~100-fold). This combination of throughput and relevance allows AORBs to robustly detect false negative results in efficacy, preventing irretrievable loss of promising lead compounds. While this work leverages the SARS-CoV-2 study as a proof-of-concept application, the standardization capacity of AORB holds broader implications in line with regulatory efforts to push alternatives to animal studies.
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Affiliation(s)
- Ji-Hoon Lee
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University School of Medicine, Atlanta, GA 30332, USA
- The Parker H. Petit Institute of Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332, USA
| | - Julia C. LeCher
- Center for ViroScience and Cure, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine and Children’s Healthcare of Atlanta, Atlanta, GA 30322, USA
- Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Eric Parigoris
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University School of Medicine, Atlanta, GA 30332, USA
- The Parker H. Petit Institute of Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332, USA
| | - Noriyuki Shinagawa
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University School of Medicine, Atlanta, GA 30332, USA
| | - Jason Sentosa
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University School of Medicine, Atlanta, GA 30332, USA
| | - Candela Manfredi
- Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA
- Children’s Healthcare of Atlanta, Atlanta, GA 30322, USA
| | - Shu Ling Goh
- Center for ViroScience and Cure, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine and Children’s Healthcare of Atlanta, Atlanta, GA 30322, USA
- Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Ramyani De
- Center for ViroScience and Cure, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine and Children’s Healthcare of Atlanta, Atlanta, GA 30322, USA
- Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Sijia Tao
- Center for ViroScience and Cure, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine and Children’s Healthcare of Atlanta, Atlanta, GA 30322, USA
- Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Keivan Zandi
- Center for ViroScience and Cure, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine and Children’s Healthcare of Atlanta, Atlanta, GA 30322, USA
- Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Franck Amblard
- Center for ViroScience and Cure, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine and Children’s Healthcare of Atlanta, Atlanta, GA 30322, USA
- Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA
| | - Eric J. Sorscher
- Department of Pediatrics, Emory University School of Medicine, Atlanta, GA 30322, USA
- Children’s Healthcare of Atlanta, Atlanta, GA 30322, USA
| | - Jason R. Spence
- Division of Gastroenterology, Department of Internal Medicine, Department of Cell and Developmental Biology, University of Michigan Medical School, Ann Arbor, MI 48109, USA
- Department of Biomedical Engineering, University of Michigan College of Engineering, Ann Arbor, MI 48109, USA
| | - Rabindra Tirouvanziam
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University School of Medicine, Atlanta, GA 30332, USA
- Children’s Healthcare of Atlanta, Atlanta, GA 30322, USA
- Center for Cystic Fibrosis & Airways Disease Research, Children’s Healthcare of Atlanta, Atlanta, GA 30322, USA
| | - Raymond F. Schinazi
- Center for ViroScience and Cure, Laboratory of Biochemical Pharmacology, Department of Pediatrics, Emory University School of Medicine and Children’s Healthcare of Atlanta, Atlanta, GA 30322, USA
| | - Shuichi Takayama
- Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University School of Medicine, Atlanta, GA 30332, USA
- The Parker H. Petit Institute of Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332, USA
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Yan J, Monlong J, Cougoule C, Lacroix-Lamandé S, Wiedemann A. Mapping the scientific output of organoids for animal and human modeling infectious diseases: a bibliometric assessment. Vet Res 2024; 55:81. [PMID: 38926765 PMCID: PMC11210181 DOI: 10.1186/s13567-024-01333-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/01/2023] [Accepted: 04/11/2024] [Indexed: 06/28/2024] Open
Abstract
The escalation of antibiotic resistance, pandemics, and nosocomial infections underscores the importance of research in both animal and human infectious diseases. Recent advancements in three-dimensional tissue cultures, or "organoids", have revolutionized the development of in vitro models for infectious diseases. Our study conducts a bibliometric analysis on the use of organoids in modeling infectious diseases, offering an in-depth overview of this field's current landscape. We examined scientific contributions from 2009 onward that focused on organoids in host‒pathogen interactions using the Web of Science Core Collection and OpenAlex database. Our analysis included temporal trends, reference aging, author, and institutional productivity, collaborative networks, citation metrics, keyword cluster dynamics, and disruptiveness of organoid models. VOSviewer, CiteSpace, and Python facilitated this analytical assessment. The findings reveal significant growth and advancements in organoid-based infectious disease research. Analysis of keywords and impactful publications identified three distinct developmental phases in this area that were significantly influenced by outbreaks of Zika and SARS-CoV-2 viruses. The research also highlights the synergistic efforts between academia and publishers in tackling global pandemic challenges. Through mostly consolidating research efforts, organoids are proving to be a promising tool in infectious disease research for both human and animal infectious disease. Their integration into the field necessitates methodological refinements for better physiological emulation and the establishment of extensive organoid biobanks. These improvements are crucial for fully harnessing the potential of organoids in understanding infectious diseases and advancing the development of targeted treatments and vaccines.
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Affiliation(s)
- Jin Yan
- Department of Gastroenterology, The Second Xiangya Hospital of Central South University, Changsha, China.
- Research Center of Digestive Disease, Central South University, Changsha, China.
- IRSD - Digestive Health Research Institute, University of Toulouse, INSERM, INRAE, ENVT, UPS, Toulouse, France.
| | - Jean Monlong
- IRSD - Digestive Health Research Institute, University of Toulouse, INSERM, INRAE, ENVT, UPS, Toulouse, France
| | - Céline Cougoule
- Institut de Pharmacologie Et Biologie Structurale, IPBS, Université de Toulouse, CNRS, UPS, Toulouse, France
| | | | - Agnès Wiedemann
- IRSD - Digestive Health Research Institute, University of Toulouse, INSERM, INRAE, ENVT, UPS, Toulouse, France.
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Liu Y, Wang D, Luan Y, Tao B, Li Q, Feng Q, Zhou H, Mu J, Yu J. The application of organoids in colorectal diseases. Front Pharmacol 2024; 15:1412489. [PMID: 38983913 PMCID: PMC11231380 DOI: 10.3389/fphar.2024.1412489] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/05/2024] [Accepted: 06/07/2024] [Indexed: 07/11/2024] Open
Abstract
Intestinal organoids are a three-dimensional cell culture model derived from colon or pluripotent stem cells. Intestinal organoids constructed in vitro strongly mimic the colon epithelium in cell composition, tissue architecture, and specific functions, replicating the colon epithelium in an in vitro culture environment. As an emerging biomedical technology, organoid technology has unique advantages over traditional two-dimensional culture in preserving parental gene expression and mutation, cell function, and biological characteristics. It has shown great potential in the research and treatment of colorectal diseases. Organoid technology has been widely applied in research on colorectal topics, including intestinal tumors, inflammatory bowel disease, infectious diarrhea, and intestinal injury regeneration. This review focuses on the application of organoid technology in colorectal diseases, including the basic principles and preparation methods of organoids, and explores the pathogenesis of and personalized treatment plans for various colorectal diseases to provide a valuable reference for organoid technology development and application.
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Affiliation(s)
- Yanxin Liu
- Department of Gastric and Colorectal Surgery, General Surgery Center, The First Hospital of Jilin University, Changchun, China
| | - Dongxu Wang
- Laboratory Animal Center, College of Animal Science, Jilin University, Changchun, China
| | - Yanhong Luan
- Laboratory Animal Center, College of Animal Science, Jilin University, Changchun, China
| | - Boqiang Tao
- Department of Oral and Maxillofacial Surgery, Hospital of Stomatology, Jilin University, Changchun, China
| | - Qirong Li
- Laboratory Animal Center, College of Animal Science, Jilin University, Changchun, China
| | - Qiang Feng
- Laboratory Animal Center, College of Animal Science, Jilin University, Changchun, China
| | - Hengzong Zhou
- Laboratory Animal Center, College of Animal Science, Jilin University, Changchun, China
| | - Jianfeng Mu
- Department of Gastric and Colorectal Surgery, General Surgery Center, The First Hospital of Jilin University, Changchun, China
| | - Jinhai Yu
- Department of Gastric and Colorectal Surgery, General Surgery Center, The First Hospital of Jilin University, Changchun, China
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Piraino F, Costa M, Meyer M, Cornish G, Ceroni C, Garnier V, Hoehnel-Ka S, Brandenberg N. Organoid models: the future companions of personalized drug development. Biofabrication 2024; 16:032009. [PMID: 38608454 DOI: 10.1088/1758-5090/ad3e30] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2023] [Accepted: 04/12/2024] [Indexed: 04/14/2024]
Abstract
High failure rates of the current drug development process are driving exemplary changes toward methodologies centered on human diseasein-vitromodeling. Organoids are self-organized tissue sub-units resembling their organ of origin and are widely acknowledged for their unique potential in recapitulating human physio-pathological mechanisms. They are transformative for human health by becoming the platform of choice to probe disease mechanisms and advance new therapies. Furthermore, the compounds' validation as therapeutics represents another point of the drug development pipeline where organoids may provide key understandings and help pharma organizations replace or reduce animal research. In this review, we focus on gastrointestinal organoid models, which are currently the most advanced organoid models in drug development. We focus on experimental validations of their value, and we propose avenues to enhance their use in drug discovery and development, as well as precision medicine and diagnostics.
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Affiliation(s)
| | - Mariana Costa
- Doppl SA, EPFL Innovation Park, Lausanne, Switzerland
| | - Marine Meyer
- Doppl SA, EPFL Innovation Park, Lausanne, Switzerland
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Sarthi JB, Trumbull AM, Abazari SM, van Unen V, Chan JE, Jiang Y, Gammons J, Anderson MO, Cil O, Kuo CJ, Sellers ZM. DRA involvement in linaclotide-stimulated bicarbonate secretion during loss of CFTR function. JCI Insight 2024; 9:e172364. [PMID: 38869953 PMCID: PMC11383163 DOI: 10.1172/jci.insight.172364] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/18/2023] [Accepted: 06/11/2024] [Indexed: 06/15/2024] Open
Abstract
Duodenal bicarbonate secretion is critical to epithelial protection, as well as nutrient digestion and absorption, and is impaired in cystic fibrosis (CF). We examined if linaclotide, typically used to treat constipation, may also stimulate duodenal bicarbonate secretion. Bicarbonate secretion was measured in vivo and in vitro using mouse and human duodenum (biopsies and enteroids). Ion transporter localization was identified with confocal microscopy, and de novo analysis of human duodenal single-cell RNA sequencing (scRNA-Seq) data sets was performed. Linaclotide increased bicarbonate secretion in mouse and human duodenum in the absence of cystic fibrosis transmembrane conductance regulator (CFTR) expression (Cftr-knockout mice) or function (CFTRinh-172). Na+/H+ exchanger 3 inhibition contributed to a portion of this response. Linaclotide-stimulated bicarbonate secretion was eliminated by down-regulated in adenoma (DRA, SLC26A3) inhibition during loss of CFTR activity. ScRNA-Seq identified that 70% of villus cells expressed SLC26A3, but not CFTR, mRNA. Loss of CFTR activity and linaclotide increased apical brush border expression of DRA in non-CF and CF differentiated enteroids. These data provide further insights into the action of linaclotide and how DRA may compensate for loss of CFTR in regulating luminal pH. Linaclotide may be a useful therapy for CF individuals with impaired bicarbonate secretion.
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Affiliation(s)
- Jessica B Sarthi
- Department of Pediatrics, Division of Gastroenterology, Hepatology, and Nutrition; and
| | - Annie M Trumbull
- Department of Pediatrics, Division of Gastroenterology, Hepatology, and Nutrition; and
| | - Shayda M Abazari
- Department of Pediatrics, Division of Gastroenterology, Hepatology, and Nutrition; and
| | - Vincent van Unen
- Department of Medicine, Division of Hematology, Stanford University, Palo Alto, California, USA
| | - Joshua E Chan
- Department of Medicine, Division of Hematology, Stanford University, Palo Alto, California, USA
| | - Yanfen Jiang
- Department of Pediatrics, Division of Gastroenterology, Hepatology, and Nutrition; and
| | - Jesse Gammons
- Department of Pediatrics, Division of Gastroenterology, Hepatology, and Nutrition; and
| | - Marc O Anderson
- Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California, USA
| | - Onur Cil
- Department of Pediatrics, University of California, San Francisco, San Francisco, California, USA
| | - Calvin J Kuo
- Department of Medicine, Division of Hematology, Stanford University, Palo Alto, California, USA
| | - Zachary M Sellers
- Department of Pediatrics, Division of Gastroenterology, Hepatology, and Nutrition; and
- Sellers Research and Clinical Development, LLC, Newark, California, USA
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46
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Eltanameli B, Piñeiro-Llanes J, Cristofoletti R. Recent advances in cell-based in vitro models for predicting drug permeability across brain, intestinal, and pulmonary barriers. Expert Opin Drug Metab Toxicol 2024; 20:439-458. [PMID: 38850058 DOI: 10.1080/17425255.2024.2366390] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2024] [Accepted: 06/06/2024] [Indexed: 06/09/2024]
Abstract
INTRODUCTION Recent years have witnessed remarkable progress in the development of cell-based in vitro models aimed at predicting drug permeability, particularly focusing on replicating the barrier properties of the blood-brain barrier (BBB), intestinal epithelium, and lung epithelium. AREA COVERED This review provides an overview of 2D in vitro platforms, including monocultures and co-culture systems, highlighting their respective advantages and limitations. Additionally, it discusses tools and techniques utilized to overcome these limitations, paving the way for more accurate predictions of drug permeability. Furthermore, this review delves into emerging technologies, particularly microphysiological systems (MPS), encompassing static platforms such as organoids and dynamic platforms like microfluidic devices. Literature searches were performed using PubMed and Google Scholar. We focus on key terms such as in vitro permeability models, MPS, organoids, intestine, BBB, and lungs. EXPERT OPINION The potential of these MPS to mimic physiological conditions more closely offers promising avenues for drug permeability assessment. However, transitioning these advanced models from bench to industry requires rigorous validation against regulatory standards. Thus, there is a pressing need to validate MPS to industry and regulatory agency standards to exploit their potential in drug permeability prediction fully. This review underscores the importance of such validation processes to facilitate the translation of these innovative technologies into routine pharmaceutical practice.
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Affiliation(s)
- Bassma Eltanameli
- Center for Pharmacometrics & Systems Pharmacology, Department of Pharmaceutics, College of Pharmacy, University of Florida, Orlando, FL, USA
- Department of Pharmaceutics, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt
| | - Janny Piñeiro-Llanes
- Center for Pharmacometrics & Systems Pharmacology, Department of Pharmaceutics, College of Pharmacy, University of Florida, Orlando, FL, USA
| | - Rodrigo Cristofoletti
- Center for Pharmacometrics & Systems Pharmacology, Department of Pharmaceutics, College of Pharmacy, University of Florida, Orlando, FL, USA
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47
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Sarthi JB, Trumbull AM, Abazari SM, van Unen V, Chan JE, Jiang Y, Gammons J, Anderson MO, Cil O, Kuo CJ, Sellers ZM. Key role of down-regulated in adenoma ( SLC26A3) chloride/bicarbonate exchanger in linaclotide-stimulated intestinal bicarbonate secretion upon loss of CFTR function. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2023.05.05.539132. [PMID: 37205513 PMCID: PMC10187319 DOI: 10.1101/2023.05.05.539132] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/21/2023]
Abstract
Duodenal bicarbonate secretion is critical to epithelial protection, nutrient digestion/absorption and is impaired in cystic fibrosis (CF). We examined if linaclotide, typically used to treat constipation, may also stimulate duodenal bicarbonate secretion. Bicarbonate secretion was measured in vivo and in vitro using mouse and human duodenum (biopsies and enteroids). Ion transporter localization was identified with confocal microscopy and de novo analysis of human duodenal single cell RNA sequencing (sc-RNAseq) datasets was performed. Linaclotide increased bicarbonate secretion in mouse and human duodenum in the absence of CFTR expression (Cftr knockout mice) or function (CFTRinh-172). NHE3 inhibition contributed to a portion of this response. Linaclotide-stimulated bicarbonate secretion was eliminated by down-regulated in adenoma (DRA, SLC26A3) inhibition during loss of CFTR activity. Sc-RNAseq identified that 70% of villus cells expressed SLC26A3, but not CFTR, mRNA. Loss of CFTR activity and linaclotide increased apical brush border expression of DRA in non-CF and CF differentiated enteroids. These data provide further insights into the action of linaclotide and how DRA may compensate for loss of CFTR in regulating luminal pH. Linaclotide may be a useful therapy for CF individuals with impaired bicarbonate secretion.
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Affiliation(s)
- Jessica B. Sarthi
- Department of Pediatrics, Division of Gastroenterology, Hepatology, and Nutrition, Stanford University, Palo Alto, CA, USA
| | - Annie M. Trumbull
- Department of Pediatrics, Division of Gastroenterology, Hepatology, and Nutrition, Stanford University, Palo Alto, CA, USA
| | - Shayda M. Abazari
- Department of Pediatrics, Division of Gastroenterology, Hepatology, and Nutrition, Stanford University, Palo Alto, CA, USA
| | - Vincent van Unen
- Department of Medicine, Division of Hematology, Stanford University, Palo Alto, CA, USA
| | - Joshua E. Chan
- Department of Medicine, Division of Hematology, Stanford University, Palo Alto, CA, USA
| | - Yanfen Jiang
- Department of Pediatrics, Division of Gastroenterology, Hepatology, and Nutrition, Stanford University, Palo Alto, CA, USA
| | - Jesse Gammons
- Department of Pediatrics, Division of Gastroenterology, Hepatology, and Nutrition, Stanford University, Palo Alto, CA, USA
| | - Marc O. Anderson
- Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, CA, USA
| | - Onur Cil
- Department of Pediatrics, University of California San Francisco, San Francisco, CA, USA
| | - Calvin J. Kuo
- Department of Medicine, Division of Hematology, Stanford University, Palo Alto, CA, USA
| | - Zachary M. Sellers
- Department of Pediatrics, Division of Gastroenterology, Hepatology, and Nutrition, Stanford University, Palo Alto, CA, USA
- Sellers Research and Clinical Development, LLC, Newark, CA, USA
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Wang H, Kim R, Wang Y, Furtado KL, Sims CE, Tamayo R, Allbritton NL. In vitro co-culture of Clostridium scindens with primary human colonic epithelium protects the epithelium against Staphylococcus aureus. Front Bioeng Biotechnol 2024; 12:1382389. [PMID: 38681959 PMCID: PMC11045926 DOI: 10.3389/fbioe.2024.1382389] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/05/2024] [Accepted: 03/28/2024] [Indexed: 05/01/2024] Open
Abstract
A complex and dynamic network of interactions exists between human gastrointestinal epithelium and intestinal microbiota. Therefore, comprehending intestinal microbe-epithelial cell interactions is critical for the understanding and treatment of intestinal diseases. Primary human colonic epithelial cells derived from a healthy human donor were co-cultured with Clostridium scindens (C. scindens), a probiotic obligate anaerobe; Staphylococcus aureus (S. aureus), a facultative anaerobe and intestinal pathogen; or both bacterial species in tandem. The co-culture hanging basket platform used for these experiments possessed walls of controlled oxygen (O2) permeability to support the formation of an O2 gradient across the intestinal epithelium using cellular O2 consumption, resulting in an anaerobic luminal and aerobic basal compartment. Both the colonic epithelial cells and C. scindens remained viable over 48 h during co-culture. In contrast, co-culture with S. aureus elicited significant damage to colonic epithelial cells within 24 h. To explore the influence of the intestinal pathogen on the epithelium in the presence of the probiotic bacteria, colonic epithelial cells were inoculated sequentially with the two bacterial species. Under these conditions, C. scindens was capable of repressing the production of S. aureus enterotoxin. Surprisingly, although C. scindens converted cholic acid to secondary bile acids in the luminal medium, the growth of S. aureus was not significantly inhibited. Nevertheless, this combination of probiotic and pathogenic bacteria was found to benefit the survival of the colonic epithelial cells compared with co-culture of the epithelial cells with S. aureus alone. This platform thus provides an easy-to-use and low-cost tool to study the interaction between intestinal bacteria and colonic cells in vitro to better understand the interplay of intestinal microbiota with human colonic epithelium.
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Affiliation(s)
- Hao Wang
- Department of Bioengineering, University of Washington, Seattle, WA, United States
| | - Raehyun Kim
- Department of Bioengineering, University of Washington, Seattle, WA, United States
- Department of Biological and Chemical Engineering, Hongik University, Sejong, Republic of Korea
| | - Yuli Wang
- Department of Bioengineering, University of Washington, Seattle, WA, United States
| | - Kathleen L. Furtado
- Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC, United States
| | - Christopher E. Sims
- Department of Bioengineering, University of Washington, Seattle, WA, United States
- Department of Medicine/Division of Rheumatology, University of Washington, Seattle, WA, United States
| | - Rita Tamayo
- Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC, United States
| | - Nancy L. Allbritton
- Department of Bioengineering, University of Washington, Seattle, WA, United States
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Dong Y, Johnson BA, Ruan L, Zeineldin M, Bi T, Liu AZ, Raychaudhuri S, Chiu I, Zhu J, Smith B, Zhao N, Searson P, Watanabe S, Donowitz M, Larman TC, Li R. Disruption of epithelium integrity by inflammation-associated fibroblasts through prostaglandin signaling. SCIENCE ADVANCES 2024; 10:eadj7666. [PMID: 38569041 PMCID: PMC10990275 DOI: 10.1126/sciadv.adj7666] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/15/2023] [Accepted: 02/27/2024] [Indexed: 04/05/2024]
Abstract
Inflammation-associated fibroblasts (IAFs) are associated with progression and drug resistance of chronic inflammatory diseases such as inflammatory bowel disease (IBD), but their direct impact on epithelial cells is unknown. Here, we developed an in vitro model whereby human colon fibroblasts are induced by specific cytokines and recapitulate key features of IAFs in vivo. When cocultured with patient-derived colon organoids (colonoids), IAFs induced rapid colonoid expansion and barrier disruption due to swelling and rupture of individual epithelial cells. Colonoids cocultured with IAFs also show increased DNA damage, mitotic errors, and proliferation arrest. These IAF-induced epithelial defects are mediated by a paracrine pathway involving prostaglandin E2 and its receptor EP4, leading to protein kinase A -dependent activation of the cystic fibrosis transmembrane conductance regulator. EP4-specific chemical inhibitors effectively prevented IAF-induced colonoid swelling and restored normal proliferation and genome stability. These findings reveal a mechanism by which IAFs could promote and perpetuate IBD and suggest a therapeutic avenue to mitigate inflammation-associated epithelial injury.
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Affiliation(s)
- Yi Dong
- Department of Cell Biology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA
| | - Blake A. Johnson
- Department of Cell Biology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA
| | - Linhao Ruan
- Department of Cell Biology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA
| | - Maged Zeineldin
- Department of Pathology, Division of GI/Liver Pathology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA
| | - Tianhao Bi
- Department of Pathology, Division of GI/Liver Pathology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA
| | - Albert Z. Liu
- Department of Cell Biology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA
| | - Sumana Raychaudhuri
- Department of Cell Biology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA
| | - Ian Chiu
- Department of Cell Biology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA
| | - Jin Zhu
- Mechanobiology Institute and Department of Biological Sciences, National University of Singapore, Singapore, Singapore
| | - Barbara Smith
- Microscope Facility, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA
| | - Nan Zhao
- Institute for Nanobiotechnology, Johns Hopkins University, Baltimore, MD 21218, USA
| | - Peter Searson
- Institute for Nanobiotechnology, Johns Hopkins University, Baltimore, MD 21218, USA
- Department of Materials Science and Engineering, Johns Hopkins University, Baltimore, MD 21218, USA
| | - Shigeki Watanabe
- Department of Cell Biology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA
| | - Mark Donowitz
- Department of Medicine, Division of Gastroenterology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA
- Department of Physiology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA
| | - Tatianna C. Larman
- Department of Pathology, Division of GI/Liver Pathology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA
| | - Rong Li
- Department of Cell Biology, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA
- Mechanobiology Institute and Department of Biological Sciences, National University of Singapore, Singapore, Singapore
- Department of Biological Sciences, National University of Singapore, Singapore, Singapore
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50
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Gandikota C, Vaddadi K, Sivasami P, Huang C, Liang Y, Pushparaj S, Deng X, Channappanava R, Metcalf JP, Liu L. The use of human iPSC-derived alveolar organoids to explore SARS-CoV-2 variant infections and host responses. J Med Virol 2024; 96:e29579. [PMID: 38572923 PMCID: PMC11603130 DOI: 10.1002/jmv.29579] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/19/2023] [Revised: 03/19/2024] [Accepted: 03/22/2024] [Indexed: 04/05/2024]
Abstract
Severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) primarily targets the respiratory system. Physiologically relevant human lung models are indispensable to investigate virus-induced host response and disease pathogenesis. In this study, we generated human induced pluripotent stem cell (iPSC)-derived alveolar organoids (AOs) using an established protocol that recapitulates the sequential steps of in vivo lung development. AOs express alveolar epithelial type II cell protein markers including pro-surfactant protein C and ATP binding cassette subfamily A member 3. Compared to primary human alveolar type II cells, AOs expressed higher mRNA levels of SARS-CoV-2 entry factors, angiotensin-converting enzyme 2 (ACE2), asialoglycoprotein receptor 1 (ASGR1) and basigin (CD147). Considering the localization of ACE2 on the apical side in AOs, we used three AO models, apical-in, sheared and apical-out for SARS-CoV-2 infection. All three models of AOs were robustly infected with the SARS-CoV-2 irrespective of ACE2 accessibility. Antibody blocking experiment revealed that ASGR1 was the main receptor for SARS-CoV2 entry from the basolateral in apical-in AOs. AOs supported the replication of SARS-CoV-2 variants WA1, Alpha, Beta, Delta, and Zeta and Omicron to a variable degree with WA1 being the highest and Omicron being the least. Transcriptomic profiling of infected AOs revealed the induction of inflammatory and interferon-related pathways with NF-κB signaling being the predominant host response. In summary, iPSC-derived AOs can serve as excellent human lung models to investigate infection of SARS-CoV-2 variants and host responses from both apical and basolateral sides.
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Affiliation(s)
- Chaitanya Gandikota
- The Lundberg-Kienlen Lung Biology and Toxicology Laboratory, Department of Physiological Sciences, Oklahoma State University, Stillwater, Oklahoma
- Oklahoma Center for Respiratory and Infectious Diseases, Oklahoma State University, Stillwater, Oklahoma
| | - Kishore Vaddadi
- The Lundberg-Kienlen Lung Biology and Toxicology Laboratory, Department of Physiological Sciences, Oklahoma State University, Stillwater, Oklahoma
- Oklahoma Center for Respiratory and Infectious Diseases, Oklahoma State University, Stillwater, Oklahoma
| | - Pulavendran Sivasami
- The Lundberg-Kienlen Lung Biology and Toxicology Laboratory, Department of Physiological Sciences, Oklahoma State University, Stillwater, Oklahoma
- Oklahoma Center for Respiratory and Infectious Diseases, Oklahoma State University, Stillwater, Oklahoma
| | - Chaoqun Huang
- The Lundberg-Kienlen Lung Biology and Toxicology Laboratory, Department of Physiological Sciences, Oklahoma State University, Stillwater, Oklahoma
- Oklahoma Center for Respiratory and Infectious Diseases, Oklahoma State University, Stillwater, Oklahoma
| | - Yurong Liang
- The Lundberg-Kienlen Lung Biology and Toxicology Laboratory, Department of Physiological Sciences, Oklahoma State University, Stillwater, Oklahoma
- Oklahoma Center for Respiratory and Infectious Diseases, Oklahoma State University, Stillwater, Oklahoma
| | - Samuel Pushparaj
- The Lundberg-Kienlen Lung Biology and Toxicology Laboratory, Department of Physiological Sciences, Oklahoma State University, Stillwater, Oklahoma
- Oklahoma Center for Respiratory and Infectious Diseases, Oklahoma State University, Stillwater, Oklahoma
| | - Xufang Deng
- Oklahoma Center for Respiratory and Infectious Diseases, Oklahoma State University, Stillwater, Oklahoma
- Department of Physiological Sciences, Oklahoma State University, Stillwater, Oklahoma
| | - Rudragouda Channappanava
- Oklahoma Center for Respiratory and Infectious Diseases, Oklahoma State University, Stillwater, Oklahoma
- Department of Veterinary Pathobiology, Oklahoma State University, Stillwater, Oklahoma
| | - Jordan P. Metcalf
- Oklahoma Center for Respiratory and Infectious Diseases, Oklahoma State University, Stillwater, Oklahoma
- Pulmonary and Critical Care Division, Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma
| | - Lin Liu
- The Lundberg-Kienlen Lung Biology and Toxicology Laboratory, Department of Physiological Sciences, Oklahoma State University, Stillwater, Oklahoma
- Oklahoma Center for Respiratory and Infectious Diseases, Oklahoma State University, Stillwater, Oklahoma
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