Objective To explore the method of small intestinal crypts isolation, organoids culture, passage, cryopreservation and recovery, and protein extraction, then study the effect of R-Spondin1 concentration on intestinal organoids maturation. Methods Isolate the small intestinal crypts, cultivate them in an in vitro 3D culture system that simulates the growth and proliferation of small intestinal stem cells in vivo, and observe the formation of intestinal organoids with an inverted microscope. The samll organoids were cut with a 1 mL syringe for passage. Set R-Spondin1 concentration gradient and compare organoids morphology at different time points. Results Successfully completed the separation culture, passage, cryopreservation, resuscitation and protein extraction of intestinal organoids and found that the intestinal crypts in the medium with a bottom concentration of less than 0. 5 μg / mL cannot grow into mature intestinal organoids. Conclusions The establishment of an in vitro culture system for small intestine organoids, especially the success of subculture, will provide a new and more physiological experimental model for long-term study of intestinal epithelium in vitro. The determination of the ratio of crypts to single cells will greatly improve the success rate of intestinal organoids culture, and the extraction of proteins will be beneficial to the study of proteomics.