The oral mucosa is a barrier site which is under constant external challenges, including a diverse oral microbiome and mechanical forces from mastication (Moutsopoulos and Konkel, 2018). Despite constant triggering, this mucosal barrier maintains tissue integrity and function, and retains an impressive potential for optimal wound healing (Iglesias-Bartolome et al., 2018). However, imbalance between microbiome and host in this mucosal surface leads to prevalent human diseases such as candidiasis and periodontitis (Gaffen and Moutsopoulos, 2020; Moutsopoulos and Konkel, 2018; Williams et al., 2021). Importantly, mouse models have been instrumental in dissecting the mechanism of tissue immunity at this interface (Dutzan et al., 2018; Silva et al., 2021; Hajishengallis et al., 2015).

Techniques like flow cytometry and single-cell RNA sequencing (scRNAseq) are effective tools for understanding biological processes at a single cell level (Heumos et al., 2023; Mattanovich and Borth, 2006; Reichard and Asosingh, 2019; Sen et al., 1990). Specifically, scRNAseq has become a practical tool to delineate specific cell populations and their transcriptomic profiles in health and disease states (Villani et al., 2017; Williams et al., 2021). A critical observation in scRNAseq datasets, however, shows that tissue dissociation protocols influence cell viability, recovered cell numbers, and cell subset proportions (Denisenko et al., 2020; Nguyen et al., 2018; Uniken Venema et al., 2022). The complex anatomy of oral mucosal tissues, (Moutsopoulos and Konkel, 2018) requires extensive tissue dissociation procedures to isolate single cell suspensions and thus creates a challenge for comprehensive cell subpopulation recovery with high viability.

Major cell populations of the oral mucosa at steady state include oral epithelial cells, fibroblasts, endothelial cells, and resident immune cells (Williams et al., 2021; Wild et al., 2021., Dutzan et al., 2016). In inflammatory states like periodontitis, immune cell proportions and diversity increase while stromal cell numbers decrease. Here we present three tissue dissociation protocols which yield high viability but preferential cell population recovery of immune cells, stromal cells, or epithelial cells and can be used for both healthy and inflamed oral tissues in the mouse.