Human tissue samples
Written informed consent was obtained from adult patients undergoing elective or emergency IBD surgery in accordance with NHS Research Ethics Committee (REC) approvals (TIP-18/WM/0237; GI Biobank-16/YH/0247; IBD Biobank-09/H1204/30). Healthy control samples were sourced from patients undergoing elective colorectal cancer surgery (uninvolved tissue) or stoma reversal. Patient metadata are summarized in Supplementary Table 1.
Archived diagnostic formalin-fixed paraffin-embedded (FFPE) blocks for ST and validation were obtained from Oxford University Hospitals NHS Foundation Trust via the Oxford Radciffe Biobank (ORB; REC:19/SC/0173) and the Oxford Centre for Histopathology Research. Additional FFPE blocks were obtained from the Friedrich-Alexander University Erlangen-Nuremberg (ethics: 23–131 bp).
Sample collection, handling and processing
Full-thickness resection tissue samples, surplus to clinical needs, were obtained by the operating surgeon, who identified involved and uninvolved tissue macroscopically. Pathology was confirmed through clinical histopathology reports. Samples were stored in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal calf serum (FCS; Sigma-Aldrich), 100 U ml−1 penicillin, and 100 U ml−1 streptomycin (Sigma-Aldrich) and transported to the laboratory on ice.
Tissue samples were rinsed with phosphate-buffered saline (PBS, Gibco) and examined for anatomical landmarks. Blunt dissection was performed on ice to isolate full-thickness (FT), submucosa (SM) and mucosa (MUC) layers. Following dissection, samples were immediately preserved in CryoStor CS10 (Sigma-Aldrich) and stored in liquid nitrogen for subsequent digestion following the manufacturer’s protocol.
For FFPE sections, tissues were oriented in cassettes, fixed in 10% buffered formaldehyde (VWR International) for 48 h, and transferred to 70% ethanol before being processed through a graded alcohol series for paraffin embedding. FFPE blocks were stored at −20 °C for long-term use until required for histological staining, immunohistochemistry or ST experiments.
Cell isolation, staining and sorting
Full thickness tissue samples were dissociated into single-cell suspensions for scRNA-seq using optimized protocols for epithelial and lamina propria (LP) fractions.
Epithelial cell enrichment
... continue reading