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, studies on the physiological and pathophysiological functions of intestinal epithelial cells (IECs) have been hampered due to lack of normal 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 collagenase and hyaluronidase in a short span of time (≤20 min). With this method, continuously growing mouse IECs, which can be subcultured 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 increasing transepithelial electrical resistance and low paracellular permeability during in vitro culture. The cells also had enzymatic activities of alkaline phosphatase and sucrase-isomaltase, and secreted various cytokines (IL-1β, IL-6, IL-8, and monocyte chemoattractant protein-1), responding to the stimulation of Escherichia coli. These results show that the primary-cultured mouse IECs obtained by the method established here had the morphological and immunological characteristics of IECs. This culture system can be a beneficial in vitro model for studies on mucosal immunology and toxicology.

Isolation, culture, and characterization of chicken intestinal epithelial cells

“…Later, Yuan et al (2015) [10] obtained cIECs starting from 14-days-old embryos using the enzyme collagenase type I to recover intestinal aggregates. These were morphologically similar to the mouse intestinal crypts isolated by Ren and colleagues [24] and when seeded on coated plastic, they were able to generate a monolayer, that survived until 9d in culture; however, a clear cell characterization was missing. Both Kaiser et al (2017) [11] and, more recently, Bai et al (2019) [25] obtained cIECs with a proper morphology but those degenerated after 7-10d in culture.…”

A novel method for isolation and culture of primary swine gastric epithelial cells

“…Collagenase type I is widely used in digest protocols and cell culture due to its protease potential for disassembling collagen brils from the connective tissue. Furthermore, collagenase type I led to the isolation of broblasts [31] and the culture of small intestinal epithelial cells [32]. Dispase II is a collagenase type IV with a mild proteolytic activity that has also been used in animal tissues for isolating labile primary cells such as porcine urothelial cells, lymphatic and embryonic endothelial cells, stem cells and Schwann cells [33][34][35][36].…”

A Novel Methodology For Isolation and Primary Culture of Swine Gastric Epithelial Cells.

“…Collagenase type I is widely used in methodologies related to tissue dissociation and cell culture due to its protease potential, capable of digesting collagen bers present in connective tissue. On the other hand, collagenase type I has been used for broblast isolation [31] and culture of small intestinal epithelial cells [32]. Dispase II is a collagenase type IV with a mild proteolytic activity that has been used for isolation of labile primary cells such as porcine urothelial cells, lymphatic and embryonic endothelial cells, stem cells and Schwan cells [33][34][35][36].…”