Background

Inflammatory bowel disease (IBD) is a chronic inflammatory condition of the gastrointestinal (GI) tract that encompasses two distinct pathologies: ulcerative colitis and Crohn’s disease. The global prevalence of IBD has been increasing since 2000 and now affects approximately 1.4 million individuals in America and 2.3 million in Europe (Ng et al., 2017). Current medical treatment for IBD involves the use of immunomodulatory agents to empirically dampen inflammation; however, up to one-third of patients fail initial treatment, and half of the initial responders will eventually relapse.

The interplay between the epithelial and stromal compartments supports homeostasis of the GI tract. Epithelial cells form a dynamic cellular layer with high regeneration potential that separates the luminal content from the underlying stromal tissue while supporting nutrient and water absorption. Simultaneously, the resident stromal cells of the lamina propria provide structural support, immunity against pathogens, and tolerance to the commensal microbiota. Breakdown of this equilibrium by dysregulation of host–microbiome interactions, defects in intestinal epithelial barrier permeability, and loss of immune tolerance can lead to the development of IBD (Maloy and Powrie, 2011). Therefore, it is important to consider the complex cellular heterogeneity when designing new experimental approaches to better understand the molecular mechanisms underlying disease.

The recent development of single-cell technologies has provided a finer picture of complex biology and deciphered heterogeneity present in tissue, enabling the identification of new cell populations, cellular interactions, and signaling pathways associated with pathology. However, these powerful approaches require the isolation of high-quality single cells from tissues to provide a representative and reliable tissue-associated cellular landscape. While the field of single-cell technology is moving fast, scientists have had to adapt tissue dissociation protocols depending on the cell types and contexts of interest. In the context of intestinal biology, the complex cellular heterogeneity of the gut mucosa and sensitivity to detachment-mediated cell death has hindered protocols for tissue dissociation that aim to represent the cellular repertoire of the original tissue. The main limitation is the fragility of the epithelial cells that cannot support most enzymes used to dissociate tissues. This becomes even more challenging when working with tissues under inflammatory conditions where cells have been exposed to a harmful and often cytotoxic environment. Some studies have already developed protocols to obtain epithelial and stromal cells from the same tissues (Smillie et al., 2019; Elmentaite et al., 2020); however, there is variability across different studies and laboratories. Our goal was to establish a detailed and easy-to-follow protocol to isolate simultaneously epithelial and stromal cells from mouse and human colon samples under inflammatory conditions. For the mouse protocol, we used the common dextran sodium sulfate (DSS) model of intestinal damage; for the human, we optimized the protocol for processing cryo-preserved colon biopsies from patients with Crohn’s disease. We validated the protocols using downstream flow cytometry and single-cell RNA sequencing analyses and demonstrated that both recover the main cell populations found in the gut mucosa.