A High-Throughput Organoid Microinjection Platform to Study Gastrointestinal Microbiota and Luminal PhysiologySummary
Background & Aims: The human gut microbiota is becoming increasingly recognized as a key factor in homeostasis and disease. The lack of physiologically relevant in vitro models to investigate host–microbe interactions is considered a substantial bottleneck for microbiota research. Organoids repr...
| Main Authors: | , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2018-01-01
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| Series: | Cellular and Molecular Gastroenterology and Hepatology |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2352345X18300808 |
| _version_ | 1818153565349216256 |
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| author | Ian A. Williamson Jason W. Arnold Leigh Ann Samsa Liam Gaynor Matthew DiSalvo Jordan L. Cocchiaro Ian Carroll M. Andrea Azcarate-Peril John F. Rawls Nancy L. Allbritton Scott T. Magness |
| author_facet | Ian A. Williamson Jason W. Arnold Leigh Ann Samsa Liam Gaynor Matthew DiSalvo Jordan L. Cocchiaro Ian Carroll M. Andrea Azcarate-Peril John F. Rawls Nancy L. Allbritton Scott T. Magness |
| author_sort | Ian A. Williamson |
| collection | DOAJ |
| description | Background & Aims: The human gut microbiota is becoming increasingly recognized as a key factor in homeostasis and disease. The lack of physiologically relevant in vitro models to investigate host–microbe interactions is considered a substantial bottleneck for microbiota research. Organoids represent an attractive model system because they are derived from primary tissues and embody key properties of the native gut lumen; however, access to the organoid lumen for experimental perturbation is challenging. Here, we report the development and validation of a high-throughput organoid microinjection system for cargo delivery to the organoid lumen and high-content sampling. Methods: A microinjection platform was engineered using off-the-shelf and 3-dimensional printed components. Microinjection needles were modified for vertical trajectories and reproducible injection volumes. Computer vision (CVis) and microfabricated CellRaft Arrays (Cell Microsystems, Research Triangle Park, NC) were used to increase throughput and enable high-content sampling of mock bacterial communities. Modeling preformed using the COMSOL Multiphysics platform predicted a hypoxic luminal environment that was functionally validated by transplantation of fecal-derived microbial communities and monocultures of a nonsporulating anaerobe. Results: CVis identified and logged locations of organoids suitable for injection. Reproducible loads of 0.2 nL could be microinjected into the organoid lumen at approximately 90 organoids/h. CVis analyzed and confirmed retention of injected cargos in approximately 500 organoids over 18 hours and showed the requirement to normalize for organoid growth for accurate assessment of barrier function. CVis analyzed growth dynamics of a mock community of green fluorescent protein– or Discosoma sp. red fluorescent protein-expressing bacteria, which grew within the organoid lumen even in the presence of antibiotics to control media contamination. Complex microbiota communities from fecal samples survived and grew in the colonoid lumen without appreciable changes in complexity. Conclusions: High-throughput microinjection into organoids represents a next-generation in vitro approach to investigate gastrointestinal luminal physiology and the gastrointestinal microbiota. Keywords: Organoid, Microinjection, High-Throughput, Fecal Microbiota, Anaerobic, Barrier Function, High-Content Sampling |
| first_indexed | 2024-12-11T14:12:38Z |
| format | Article |
| id | doaj.art-3dba638657aa4b50be50371d3a5c86ca |
| institution | Directory Open Access Journal |
| issn | 2352-345X |
| language | English |
| last_indexed | 2024-12-11T14:12:38Z |
| publishDate | 2018-01-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Cellular and Molecular Gastroenterology and Hepatology |
| spelling | doaj.art-3dba638657aa4b50be50371d3a5c86ca2022-12-22T01:03:22ZengElsevierCellular and Molecular Gastroenterology and Hepatology2352-345X2018-01-0163301319A High-Throughput Organoid Microinjection Platform to Study Gastrointestinal Microbiota and Luminal PhysiologySummaryIan A. Williamson0Jason W. Arnold1Leigh Ann Samsa2Liam Gaynor3Matthew DiSalvo4Jordan L. Cocchiaro5Ian Carroll6M. Andrea Azcarate-Peril7John F. Rawls8Nancy L. Allbritton9Scott T. Magness10Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill/North Carolina State University, Chapel Hill, North CarolinaDepartment of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North CarolinaJoint Department of Biomedical Engineering, University of North Carolina at Chapel Hill/North Carolina State University, Chapel Hill, North CarolinaGraduate Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MassachusettsJoint Department of Biomedical Engineering, University of North Carolina at Chapel Hill/North Carolina State University, Chapel Hill, North CarolinaDepartment of Molecular Genetics and Microbiology Medicine, Duke University, Durham, North CarolinaDepartment of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North CarolinaDepartment of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North CarolinaDepartment of Molecular Genetics and Microbiology Medicine, Duke University, Durham, North CarolinaJoint Department of Biomedical Engineering, University of North Carolina at Chapel Hill/North Carolina State University, Chapel Hill, North Carolina; Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, North CarolinaJoint Department of Biomedical Engineering, University of North Carolina at Chapel Hill/North Carolina State University, Chapel Hill, North Carolina; Department of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; Correspondence Address correspondence to: Scott T. Magness, PhD, University of North Carolina at Chapel Hill, 111 Mason Farm Road, CB 7032, MBRB Room 4337, Chapel Hill, North Carolina 27599. fax: (919) 843-6899.Background & Aims: The human gut microbiota is becoming increasingly recognized as a key factor in homeostasis and disease. The lack of physiologically relevant in vitro models to investigate host–microbe interactions is considered a substantial bottleneck for microbiota research. Organoids represent an attractive model system because they are derived from primary tissues and embody key properties of the native gut lumen; however, access to the organoid lumen for experimental perturbation is challenging. Here, we report the development and validation of a high-throughput organoid microinjection system for cargo delivery to the organoid lumen and high-content sampling. Methods: A microinjection platform was engineered using off-the-shelf and 3-dimensional printed components. Microinjection needles were modified for vertical trajectories and reproducible injection volumes. Computer vision (CVis) and microfabricated CellRaft Arrays (Cell Microsystems, Research Triangle Park, NC) were used to increase throughput and enable high-content sampling of mock bacterial communities. Modeling preformed using the COMSOL Multiphysics platform predicted a hypoxic luminal environment that was functionally validated by transplantation of fecal-derived microbial communities and monocultures of a nonsporulating anaerobe. Results: CVis identified and logged locations of organoids suitable for injection. Reproducible loads of 0.2 nL could be microinjected into the organoid lumen at approximately 90 organoids/h. CVis analyzed and confirmed retention of injected cargos in approximately 500 organoids over 18 hours and showed the requirement to normalize for organoid growth for accurate assessment of barrier function. CVis analyzed growth dynamics of a mock community of green fluorescent protein– or Discosoma sp. red fluorescent protein-expressing bacteria, which grew within the organoid lumen even in the presence of antibiotics to control media contamination. Complex microbiota communities from fecal samples survived and grew in the colonoid lumen without appreciable changes in complexity. Conclusions: High-throughput microinjection into organoids represents a next-generation in vitro approach to investigate gastrointestinal luminal physiology and the gastrointestinal microbiota. Keywords: Organoid, Microinjection, High-Throughput, Fecal Microbiota, Anaerobic, Barrier Function, High-Content Samplinghttp://www.sciencedirect.com/science/article/pii/S2352345X18300808 |
| spellingShingle | Ian A. Williamson Jason W. Arnold Leigh Ann Samsa Liam Gaynor Matthew DiSalvo Jordan L. Cocchiaro Ian Carroll M. Andrea Azcarate-Peril John F. Rawls Nancy L. Allbritton Scott T. Magness A High-Throughput Organoid Microinjection Platform to Study Gastrointestinal Microbiota and Luminal PhysiologySummary Cellular and Molecular Gastroenterology and Hepatology |
| title | A High-Throughput Organoid Microinjection Platform to Study Gastrointestinal Microbiota and Luminal PhysiologySummary |
| title_full | A High-Throughput Organoid Microinjection Platform to Study Gastrointestinal Microbiota and Luminal PhysiologySummary |
| title_fullStr | A High-Throughput Organoid Microinjection Platform to Study Gastrointestinal Microbiota and Luminal PhysiologySummary |
| title_full_unstemmed | A High-Throughput Organoid Microinjection Platform to Study Gastrointestinal Microbiota and Luminal PhysiologySummary |
| title_short | A High-Throughput Organoid Microinjection Platform to Study Gastrointestinal Microbiota and Luminal PhysiologySummary |
| title_sort | high throughput organoid microinjection platform to study gastrointestinal microbiota and luminal physiologysummary |
| url | http://www.sciencedirect.com/science/article/pii/S2352345X18300808 |
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