A Protocol for the Cryopreservation of Human Intestinal Mucosal Biopsies Compatible With Single-Cell Transcriptomics and Ex Vivo Studies

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.

Keywords: FACS-Seq; cryopreservation; endoscopy; gastrointestinal tract; gut epithelium; human primary culture; intestinal biopsy; single-cell transcriptomics.

FIGURE 1

Workflow for the cryopreservation of endoscopically obtained human intestinal mucosal biopsies, and subsequent preparation of this tissue to highly viable FACS-isolated single-cell suspensions compatible with deep single-cell RNA sequencing, and primary intestinal culture.

FIGURE 2

(A) Diagram and (B) corresponding FACS-plots demonstrating the sequential flow-cytometric gating strategy used to isolate cells. Average percentage of cells obtained from fresh (green circles, n = 5) and cryopreserved (blue squares, n = 13) tissue preparations throughout the population hierarchy: (C) Non-debris, (D) singlets, (E) Live and (F) Non-apoptotic cells, during FACS. Each symbol represents 1 human participant. Significance testing used a two-tailed student t-test; *p < 0.05.

FIGURE 3

Single cells suspensions were assessed for (A) cell viability and (B) RNA Integrity Number (RIN) from fresh (green; n = 3, 2, for pre/post respectively) and cryopreserved (blue; n = 3, 10, for pre/post respectively) tissue preparations during Pre-FACS isolation and 1-h Post-FACS isolation. Summary of Data Generation from scRNA-Sequencing of FACS-isolated Human Colonic Epithelial Cells Originating from Cryopreserved Biopsies of 10 participants for (C) cell captured, (D) reads/cell, (E) reads mapped to genome (%), and (F) total genes detected. (G) Single-Cell RNA-Seq Profiling of Human Colonic Epithelium. t-SNE plots of single-cell RNA-seq profiles of human intestinal epithelial cells, colored by cluster number and identity, listed by largest to smallest population, and annotated by cluster identity, determined by gene markers for subtypes. Significance testing used a two-tailed student t-test; *p < 0.05, ***p < 0.001.

FIGURE 4

Cryopreserved Human and Mouse FACS-isolated Cells Survive in Short-term Culture. FACS-isolated cells from fresh (green, n = 4) or cryopreserved (blue, n = 4) preparations of mouse colon are analyzed by flow cytometry at initial time of sorting and 72 h posting sorting for (A) live cells and (B) non-apoptotic cell proportions after incubation in standard culture conditions. (C) FACS-isolated cells from cryopreserved preparations of human biopsies (n = 4 participants) are analyzed by flow cytometry at initial time of sorting and 24 h posting sorting for live cells and non-apoptotic cell proportions after incubation in standard culture conditions. (D) GLP-1 and (E) PYY secretion from primary intestinal cultures originating from cryopreserved human tissue (n = 5 replicates) treated for 2 h with PBS-0.5% BSA (control) with or without test compounds: Conjugated Bile Acids (1 mM), meat hydrolysate (2%); data expressed as normalized to protein content, and as fold-change to the control. Significance testing used a two-tailed student t-test compared between all groupings; *p < 0.05, **p < 0.01, ***p < 0.001.