Obtaining purified human intestinal epithelia for single-cell analysis and organoid culture

Alexander D B Ross; Francesca Perrone; Rasa Elmentaite; Sarah A Teichmann; Matthias Zilbauer

doi:10.1016/j.xpro.2021.100597

Obtaining purified human intestinal epithelia for single-cell analysis and organoid culture

STAR Protoc. 2021 Jun 12;2(2):100597. doi: 10.1016/j.xpro.2021.100597. eCollection 2021 Jun 18.

Authors

Alexander D B Ross^{1

2

3}, Francesca Perrone¹, Rasa Elmentaite⁴, Sarah A Teichmann^{4

5

6}, Matthias Zilbauer^{1

2

7}

Affiliations

¹ Department of Paediatrics, University of Cambridge, Cambridge CB2 0QQ, UK.
² Wellcome Trust, MRC Cambridge Stem Cell Institute, University of Cambridge, Cambridge CB2 0SZ, UK.
³ Department of Surgery, Universisty of Cambridge, Cambridge CB2 0QQ, UK.
⁴ Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton CB10 1SA, UK.
⁵ Theory of Condensed Matter, Cavendish Laboratory, Department of Physics, University of Cambridge, Cambridge CB3 0HE, UK.
⁶ European Molecular Biology Laboratory, European Bioinformatics Institute (EBI), Wellcome Genome Campus, Hinxton CB10 1SA, UK.
⁷ Department of Paediatric Gastroenterology, Hepatology and Nutrition, Cambridge University Hospitals Trust, Cambridge CB2 0QQ, UK.

Abstract

Here, we describe protocols for the preparation and dissociation of human fetal and pediatric intestinal tissue to a high-viability epithelial single-cell suspension. This epithelium-enriched single-cell suspension can then be used to generate single-cell RNA sequencing data as well as to create human intestinal organoids from both the fetal and pediatric intestine. Finally, this protocol details the dissociation of the intestinal organoids for use in single-cell analysis or passaging of organoids. For complete details on the use and execution of this protocol, please refer to Elmentaite et al. (2020).

Keywords: Cell isolation; Clinical Protocol; Organoids; Single Cell; Stem Cells.

MeSH terms

Biopsy
Child
Humans
Intestinal Mucosa / cytology*
Organoids / cytology*
Single-Cell Analysis / methods*

Grants and funding

MR/T001917/1/MRC_/Medical Research Council/United Kingdom