A serum‐free medium developed for in vitro expansion of murine intestinal stem cells

Abstract Intestinal stem cells (ISCs) are located at the base of the intestinal crypts and have the ability to self‐renew as well as to differentiate into mature epithelial cells. Recently, ISCs have received much attention for the treatment of many intestinal diseases. However, many challenges face those studying ISCs because insufficient ISCs are available. Therefore, the development of a culture medium for ISC expansion is an important necessity for basic research and clinical application. In this study, we described the technique used to develop a serum‐free medium for expanding ISCs in vitro. Furthermore, five serum substitutes were selected and optimized in order to maintain the long‐term proliferation and enteroid‐forming ability of ISCs: (i) ethanolamine; (ii) ascorbic acid phosphate; (iii) transferrin; (iv) glutathione; and (v) sodium selenite. Analysis of gene expression of Lgr5, Bmi1, Msi1 and PTEN demonstrated that our serum‐free medium sustained the expression of genes involved in ISC‐related functions in the expanded ISCs. Additionally, the expression intensity of surface markers, including Lgr5, CD24 and CD44, on serum‐free expanded cells in crypts was greatly increased. Taken together, our results demonstrate that the number of ISCs can be expanded and their functionality maintained in our serum‐free medium, indicating the suitability of this serum‐free expansion medium for increasing the numbers of ICSs available for basic research and clinical applications in the future.