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

Published online

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Williamson, IA; Arnold, JW; Samsa, LA; Gaynor, L; DiSalvo, M; Cocchiaro, JL; Carroll, I; Azcarate-Peril, MA; Rawls, JF; Allbritton, NL; Magness, ST

Published Date

  • 2018

Published In

Volume / Issue

  • 6 / 3

Start / End Page

  • 301 - 319

PubMed ID

  • 30123820

Pubmed Central ID

  • 30123820

Electronic International Standard Serial Number (EISSN)

  • 2352-345X

Digital Object Identifier (DOI)

  • 10.1016/j.jcmgh.2018.05.004

Language

  • eng

Conference Location

  • United States