Isolation, propagation, and clonogenicity of intestinal stem cells

Intestinal stem cell research has greatly aided our understanding of the biology of intestinal self-renewal but has also shed light on the role of cancer stem cells (CSCs) in carcinogenesis, cancer growth, and dissemination. With new possibilities for CSC targeting, there is a need to have established techniques for quantifying (cancer) stem cell clonogenicity, particularly in organoid cultures. Here, we describe a detailed methodology for the isolation and expansion of mouse intestinal crypts from three different locations—the colon, proximal, and distal small intestine. In addition, we describe techniques that allow the measurement of stem cell clonogenicity and its manipulation using two approaches—organoid counting and immunohistochemistry.