In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture

Abstract

It is useful to be able to grow enriched populations of stem cells in vitro. Growth of stem cells as tissue spheroids is a key methodology permitting sustainable culture of adult epithelial cells. Gastrointestinal stem cells can be propagated by using conditioned medium from a supportive cell line (L-WRN). This protocol describes how to prepare conditioned medium and how to culture stem cell-enriched epithelial spheroids from the mouse gastrointestine. These spheroids are also amenable to genetic modification with recombinant lentiviruses. This system enables many types of cell biological assays that have been performed with immortalized cell lines to be applied to spheroids. Isolation of epithelial cell units from mice takes up to 2 h, and stem cell-enriched gastrointestinal spheroids are obtained within 3 d. Genetically modified spheroids with lentiviruses can be obtained in 2 weeks.

Figure 1

Development of mouse gastrointestinal epithelial organoids. (a) Collagenase digestion of the mouse stomach, small intestine, cecum, and colon. Vigorous pipetting separated single epithelial units from minced tissue fragments in collagenase solution. Bars=1 mm. (b) Isolated gastrointestinal epithelial units. Bars=200 μm. (c) Primary epithelial organoids from gastrointestinal tissues. Isolated epithelial units were embedded in Matrigel and cultured with 50% L-WRN media for 3 days. 10 μM Y27632 and 10 μM SB431542 were added for the small intestine and cecum. Bars=1 mm.

Figure 2

Typical morphology of gastroiontestinal organoids cultured in 50% L-WRN conditioned media. Bars=1 mm.

Figure 3

Schematic drawing of the stem cell culture system.

Figure 4

Isolation of lentivirus-infected organoids. (a, b) Primary organoids after infection showed mosaic GFP expression. Representative fluorescence (a) and bright field images (b) are shown. Bars=200 μm. (c) Hand-selection of secondary organoids that consist of GFP-positive cells. Bar=1 mm.