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Microfluidic Device to Manipulate 3D Human Epithelial Cell-Derived Intestinal Organoids.

ABSTRACT: In this study, we propose a microfluidic organoid-trapping device used to immobilize human intestinal organoids and apply fluidic stimuli to them. The proposed device has a microchannel with a trapping region with wall gaps between the channel walls and the bottom surface, and a constriction to clog the organoids in the channel. Since the introduced culture medium escapes from the gap, organoids can be cultured without excessive deformation by hydrostatic pressure. Owing to the characteristics of the organoid-trapping device, we succeeded in trapping human intestinal organoids in the channel. Furthermore, to demonstrate the applicability of the device for culturing intestinal organoids, we induced organoid fusion to form large organoids by aligning the organoids in the channel and applying fluidic shear stress to the organoids to regulate their surface structures. Therefore, we believe that organoid-trapping devices will be useful for investigating organoids aligned or loaded with fluidic stimulation.

SUBMITTER: Matsumoto M

PROVIDER: S-EPMC9785580 | biostudies-literature | 2022 Nov

REPOSITORIES: biostudies-literature

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Microfluidic Device to Manipulate 3D Human Epithelial Cell-Derived Intestinal Organoids.

Matsumoto Miki M Morimoto Yuya Y Sato Toshiro T Takeuchi Shoji S

Micromachines 20221126 12

In this study, we propose a microfluidic organoid-trapping device used to immobilize human intestinal organoids and apply fluidic stimuli to them. The proposed device has a microchannel with a trapping region with wall gaps between the channel walls and the bottom surface, and a constriction to clog the organoids in the channel. Since the introduced culture medium escapes from the gap, organoids can be cultured without excessive deformation by hydrostatic pressure. Owing to the characteristics o ...[more]

PMID: 36557386

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Project description:UnlabelledDirected differentiation of stem cell lines into intestine-like tissue called induced human intestinal organoids (iHIOs) is now possible (J. R. Spence, C. N. Mayhew, S. A. Rankin, M. F. Kuhar, J. E. Vallance, K. Tolle, E. E. Hoskins, V. V. Kalinichenko, S. I. Wells, A. M. Zorn, N. F. Shroyer, and J. M. Wells, Nature 470:105-109, 2011). We tested iHIOs as a new model to cultivate and study fecal viruses. Protocols for infection of iHIOs with a laboratory strain of rotavirus, simian SA11, were developed. Proof-of-principle analyses showed that iHIOs support replication of a gastrointestinal virus, rotavirus, on the basis of detection of nonstructural viral proteins (nonstructural protein 4 [NSP4] and NSP2) by immunofluorescence, increased levels of viral RNA by quantitative reverse transcription-PCR (qRT-PCR), and production of infectious progeny virus. iHIOs were also shown to support replication of 12/13 clinical rotavirus isolates directly from stool samples. An unexpected finding was the detection of rotavirus infection not only in the epithelial cells but also in the mesenchymal cell population of the iHIOs. This work demonstrates that iHIOs offer a promising new model to study rotaviruses and other gastrointestinal viruses.ImportanceGastrointestinal viral infections are a major cause of illness and death in children and adults. The ability to fully understand how viruses interact with human intestinal cells in order to cause disease has been hampered by insufficient methods for growing many gastrointestinal viruses in the laboratory. Induced human intestinal organoids (iHIOs) are a promising new model for generating intestine-like tissue. This is the first report of a study using iHIOs to cultivate any microorganism, in this case, an enteric virus. The evidence that both laboratory and clinical rotavirus isolates can replicate in iHIOs suggests that this model would be useful not only for studies of rotaviruses but also potentially of other infectious agents. Furthermore, detection of rotavirus proteins in unexpected cell types highlights the promise of this system to reveal new questions about pathogenesis that have not been previously recognized or investigated in other intestinal cell culture models.

Project description:Human intestinal epithelial organoids (enteroids and colonoids) are tissue cultures used for understanding the physiology of the human intestinal epithelium. Here, we explored the effect on the transcriptome of common variations in culture methods, including extracellular matrix substrate, format, tissue segment, differentiation status, and patient heterogeneity. RNA-sequencing datasets from 276 experiments performed on 37 human enteroid and colonoid lines from 29 patients were aggregated from several groups in the Texas Medical Center. DESeq2 and gene set enrichment analysis (GSEA) were used to identify differentially expressed genes and enriched pathways. PERMANOVA, Pearson's correlation, and dendrogram analysis of the data originally indicated three tiers of influence of culture methods on transcriptomic variation: substrate (collagen vs. Matrigel) and format (3-D, transwell, and monolayer) had the largest effect; segment of origin (duodenum, jejunum, ileum, colon) and differentiation status had a moderate effect; and patient heterogeneity and specific experimental manipulations (e.g., pathogen infection) had the smallest effect. GSEA identified hundreds of pathways that varied between culture methods, such as IL1 cytokine signaling enriched in transwell versus monolayer cultures and E2F target genes enriched in collagen versus Matrigel cultures. The transcriptional influence of the format was furthermore validated in a synchronized experiment performed with various format-substrate combinations. Surprisingly, large differences in organoid transcriptome were driven by variations in culture methods such as format, whereas experimental manipulations such as infection had modest effects. These results show that common variations in culture conditions can have large effects on intestinal organoids and should be accounted for when designing experiments and comparing results between laboratories. Our data constitute the largest RNA-seq dataset interrogating human intestinal epithelial organoids.

Dataset Information

Microfluidic Device to Manipulate 3D Human Epithelial Cell-Derived Intestinal Organoids.

Publications

Microfluidic Device to Manipulate 3D Human Epithelial Cell-Derived Intestinal Organoids.

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