Establishment of human intestinal epithelial cell-derived organoid culture system

Abstract

Objective To establish the human intestinal epithelial cells-derived organoid culture system ex-vivo. Methods The chelating buffer containing 2 mmol/L EDTA was used to isolate the crypts from human intestinal mucosa. The isolated crypts were embedded in Matrigel and then covered by the culture medium enriched with growth factors. The growth of organoids was observed under the microscope. The expression of the stem cell and mature epithelial cell markers was analyzed by immunohistochemistry or in situ hybridization. Cell proliferation was labeled with EdU staining. Cell apoptosis was detected by TUNEL assay. Results After 4-5 days, crypts embedded in Matrigel grew into organoids. On day 8, organoids were digested, re-embedded in Matrigel, and cell fractions grew into organoids again. Lgr5+ cells were detected by in situ hybridization. On day 5 after the removement of Wnt3a, R-Spodin, SB202190, Nicotinamide, organoids were differentiated to mature cells which were labeled as Fabp1+ cells, Muc2+ cells, Chga+ cells and Lyz+ cells by immunofluorescence. Proliferating cells were detected in organoids while few apoptotic cells were detected. Conclusions The human intestinal epithelial organoid culture system can be successfully established. The culture system is a reliable ex-vivo model to study the pathophysiology of digestive tract diseases. Key words: Inflammatory bowel disease; Organoid; Intestinal mucosa