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A Protocol for the Cryopreservation of Human Intestinal Mucosal Biopsies Compatible With Single-Cell Transcriptomics
Alison McRae1, Maria Laura Ricardo-Silgado1, Yuanhang Liu2
1Precision Medicine for Obesity Program, Department of Gastroenterology and Hepatology, Mayo Clinic, Rochester, MN, United States.
Insights
This study presents a new workflow for cryopreserving human intestinal biopsies. This method enables single-cell RNA sequencing and ex vivo studies of intestinal epithelial cells, aiding disease research.
Area of Science:
- Gastroenterology and Molecular Biology
- Single-cell analysis of human tissues
Background:
- Human intestinal epithelium heterogeneity complicates single-cell disease studies.
- Understanding specialized cell pathophysiology requires advanced single-cell techniques.
Purpose of the Study:
- To develop a workflow for cryopreserving human intestinal biopsies for single-cell analysis.
- To enable high-viability single-cell suspensions for scRNAseq and ex vivo studies.
- To facilitate research into intestinal diseases at the single-cell level.
Main Methods:
- Cryopreservation of endoscopic human intestinal mucosal biopsies.
- Preparation of single-cell suspensions for fluorescence-activated cell sorting (FACS).
- Deep single-cell RNA sequencing (scRNAseq) and primary intestinal cell culture.
Main Results:
- Validated protocol with scRNAseq of 59,653 intestinal cells from 10 participants.
- Successfully generated primary intestinal cultures from cryopreserved tissue.
- Demonstrated suitability for physiological assays, including studying enteroendocrine cell peptide secretion.
Conclusions:
- The workflow provides accessible methods for single-cell transcriptomics and ex vivo studies from cryopreserved intestinal biopsies.
- This approach can dissect intestinal ecosystem aberrations in disease development and progression.
- Enables study of rare intestinal epithelial cell populations in human diseases.
Abstract:
The heterogeneity of the human intestinal epithelium has hindered the understanding of the pathophysiology of distinct specialized cell types on a single-cell basis in disease states. Described here is a workflow for the cryopreservation of endoscopically obtained human intestinal mucosal biopsies, subsequent preparation of this tissue to yield highly viable fluorescence-activated cell sorting (FACS)isolated human intestinal epithelial cell (IEC) single-cell suspensions compatible with successful library preparation and deep single-cell RNA sequencing (scRNAseq). We validated this protocol in deep scRNAseq of 59,653 intestinal cells in 10 human participants. Furthermore, primary intestinal cultures were successfully generated from cryopreserved tissue, capable of surviving in short-term culture and suitable for physiological assays studying gut peptide secretion from rare hormone-producing enteroendocrine cells in humans. This study offers an accessible avenue for single-cell transcriptomics and ex vivo studies from cryopreserved intestinal mucosal biopsies. These techniques may be used in the future to dissect and define novel aberrations to the intestinal ecosystem that lead to the development and progression of disease states in humans, even in rare IEC populations.
