Culture and characterization of mouse intestinal organoids

Abstract

Background & Aim Organoids formed by self-organizing stem cells resemble their native counterparts in cellular content, multicellular architecture and functional features. As such, they are emerging as powerful tools in basic and translational research. Intestinal organoids possess a particularly high level of multicellular organization and a wide range of potential applications. However, these cultured intestinal tissue pieces from the intestinal epithelial could be maintained in culture for several days at maximum, and thus were not self-renewing. Lgr5+ cells isolated from intestine, colon, or stomach can form organoids in long-term culture. An essential component of these cultures is R-spondin, the ligand of Lgr5. Thus, when cultured under the appropriate 3D conditions, single Lgr5-expressing intestinal stem cells (ISCs) undergo cycles of self-renewal, differentiation and morphogenesis, and self-organize into crypt-villus domains that house cycling ISCs and differentiated intestinal cells. In this study, we performed isolation and characterization of ISCs from BALB/c-nude mouse. Methods, Results & Conclusion Crypts were isolated from the mouse small intestine by minor modification of the method described by Khalil. Freshly isolated mouse crypts from dissociated mouse ISCs colonies were embedded in Matrigel, cast into 40 mL droplets at the bottom of well in a 48 well plate. Following polymerization, the gels were overlaid with ISC expansion medium containing B27, N2, epidermal growth factor, noggin, and R-spondin 1. As a result, mouse ISCs had enteroids and spheroid morphologies. We also confirmed by quantitative real-time RT-PCR that expression of ISCs related genes (Lgr5, mucin-1, mucin-2, defensin-5, chromogranin A, and villin) was maintained at 8 passage or more. In this study, we observed that expression of specific ISC markers and consecutive self-renewing in the mouse intestinal organoids.