Primary cultures of mouse small intestinal epithelial cells using the dissociating enzyme type I collagenase and hyaluronidase

Abstract The epithelium is a highly dynamic system, which plays a crucial role in the homeostasis of the intestinal tract. However, studieson the physiological and pathophysiological functions of intestinal epithelial cells (IECs) have been hampered due to lack ofnormal epithelial cell models. In the present study, we established a reproducible method for primary culture of mouse IECs,which were isolated from the viable small intestinal crypts of murine fetuses (on embryonic day 19), using type I collagenaseand hyaluronidase in a short span of time (p20 min). With this method, continuously growing mouse IECs, which can besubcultured over a number of passages, were obtained. The obtained cell lines formed a tight cobblestone-like arrangement,displayed long and slender microvilli, expressed characteristic markers (cytokeratin 18 and Notch-1), and generated increasingtransepithelial electrical resistance and low paracellular permeability during in vitro culture. The cells also had enzymaticactivities of alkaline phosphatase and sucrase-isomaltase, and secreted various cytokines (IL-1b, IL-6, IL-8, and monocytechemoattractant protein-1), responding to the stimulation of Escherichia coli. These results show that the primary-culturedmouse IECs obtained by the method established here had the morphological and immunological characteristics of IECs. Thisculture system can be a beneficial in vitro model for studies on mucosal immunology and toxicology.Key words: Intestinal epithelial cells; Crypt; Isolation; Primary culture; Collagenase I; Hyaluronidase