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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:Changle goose in Fujian, China is a rare genetic resource and in urgent need to be protected. Understanding the characteristics of digestive physiology and spatial variation of gastrointestinal microbiota is crucial for developing nutritional intervention strategies to improve intestinal health and production performance of goose. Hence, histomorphological assay was used for observing development status of proventriculus, jejunum, and cecum in 70-day-old Changle geese, whereas digesta from 6 alimentary canal locations (crop, proventriculus, gizzard, jejunum, cecum, and rectum) were collected for 16S rRNA gene sequencing and short chain fatty acids (SCFAs) quantitative analysis. The histomorphological observation indicated that the jejunum and cecum of Changle goose were well developed. The alpha diversity analysis revealed that, except rectum, microbiota in other noncecum sections were in high diversity as cecum. The Nonmetric MultiDimensional Scaling (NMDS) analysis showed that microbial community of proventriculus, gizzard, and jejunum formed a cluster, which distinctly discrete with the microbiota of the other gastrointestinal locations. Additionally, the proportions of Proteobacteria, Bacteroidota, and Campilobacterota at the phylum level and Lactobacillus, Streptococcus, Helicobacter, and Subdoligranulum at the genus level exhibited tremendous alternations among different gastrointestinal locations. The characteristic bacterial composition in each section was further disclosed by analyzing the core and feature Amplicon Sequence Variants (ASVs) and SCFAs pattern. Importantly, 7 body-weight-associated ASVs and 2 cecum-development-related ASVs were identified via correlation analysis. In a whole, our findings provided the first insights into the specialized digestive physiology of Changle geese and distinctive regional distribution of gastrointestinal microbiota, which laid the important foundation for improving growth performance through microbiota manipulation in geese.

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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