To establish human intestinal organoid culture in vitro and effective gene delivery system mediating by lentivirus into organoid, it will be essential to develop colorectal cancer research and gene therapy.

(1) The biological specimen of ileum or colon by surgical resection is taken 3 to 5 cm and separated mucous membrane from muscular layer. It is digested and separated by ethylene diamine tetraacetic acid Chelation Buffer for collecting intestinal crypts. The intestinal crypts is resuspended with in Matrigel. The sample is seeded on pre-warmed96-well plate, after solidification, which is added with culture medium including growth factors. The organoid status can be observed in everyday, the medium be exchanged in each 2 or 3 days, and the organoid structure be passaged in each 6 or 7 days. (2) Packaging high-titer lentivirus carrying with reporter gene, green fluorescent protein (GFP), infect into intestinal organoid, and the expression quantity of fluorescent is observed in organoid.

We could culture mini-gut organoid with "budding" spherical structures using sporadic intestinal crypts ex vivo, and the livability of both groups of crypt/organoid are greater than 70%. Intestinal organoid can express a protein marker about absorptive cell which is located in normal intestinal epithelium. Reporter gene mediating by lentivirus vector can be stably expressed in organoids.

This new intestinal organoid culture model not only is used to research gene functions in maintaining intestinal homeostasis.

Intestinal organoid; Crypt base columnar cells; Lentivirus; Gene-editing; Gene delivery system