Dataset Information

A High-Throughput Organoid Microinjection Platform to Study Gastrointestinal Microbiota and Luminal Physiology.

ABSTRACT: 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.

SUBMITTER: Williamson IA

PROVIDER: S-EPMC6092482 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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A High-Throughput Organoid Microinjection Platform to Study Gastrointestinal Microbiota and Luminal Physiology.

Williamson Ian A IA Arnold Jason W JW Samsa Leigh Ann LA Gaynor Liam L DiSalvo Matthew M Cocchiaro Jordan L JL Carroll Ian I Azcarate-Peril M Andrea MA Rawls John F JF Allbritton Nancy L NL Magness Scott T ST

Cellular and molecular gastroenterology and hepatology 20180522 3

<h4>Background & aims</h4>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 ...[more]

PMID: 30123820

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Project description:BackgroundAlmonds contain lipid, fiber, and polyphenols and possess physicochemical properties that affect nutrient bioaccessibility, which are hypothesized to affect gut physiology and microbiota.ObjectivesTo investigate the impact of whole almonds and ground almonds (almond flour) on fecal bifidobacteria (primary outcome), gut microbiota composition, and gut transit time.MethodsHealthy adults (n = 87) participated in a parallel, 3-arm randomized controlled trial. Participants received whole almonds (56 g/d), ground almonds (56 g/d), or an isocaloric control in place of habitual snacks for 4 wk. Gut microbiota composition and diversity (16S rRNA gene sequencing), SCFAs (GC), volatile organic compounds (GC-MS), gut transit time (wireless motility capsule), stool output and gut symptoms (7-d diary) were measured at baseline and endpoint. The impact of almond form on particle size distribution (PSD) and predicted lipid release was measured (n = 31).ResultsModified intention-to-treat analysis was performed on 79 participants. There were no significant differences in mean ± SD abundance of fecal bifidobacteria after consumption of whole almonds (8.7% ± 7.7%), ground almonds (7.8% ± 6.9%), or control (13.0% ± 10.2%; q = 0.613). Consumption of almonds (whole and ground pooled) resulted in higher mean ± SD butyrate (24.1 ± 15.0 μmol/g) than control (18.2 ± 9.1 μmol/g; P = 0.046). There was no effect of almonds on gut microbiota at the phylum level or diversity, gut transit time, stool consistency, or gut symptoms. Almond form (whole compared with ground) had no effect on study outcomes. Ground almonds resulted in significantly smaller PSD and higher mean ± SD predicted lipid release (10.4% ± 1.8%) than whole almonds (9.3% ± 2.0%; P = 0.017).ConclusionsAlmond consumption has limited impact on microbiota composition but increases butyrate in adults, suggesting positive alterations to microbiota functionality. Almonds can be incorporated into the diet to increase fiber consumption without gut symptoms.This trial was registered at clinicaltrials.gov as NCT03581812.

Dataset Information

A High-Throughput Organoid Microinjection Platform to Study Gastrointestinal Microbiota and Luminal Physiology.

Publications

A High-Throughput Organoid Microinjection Platform to Study Gastrointestinal Microbiota and Luminal Physiology.

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