Ex vivo culture, proliferation and expansion of intestinal epithelium

A method for generating a long term culture of mammalian intestinal cells, wherein the culture this thus generated provides for multilineage differentiation that maintains (a) three-dimensional structure of intestinal epithelium and (b) differentiation markers characteristic of intestinal absorptive enterocytes, goblet cells and enteroendocrine cells, in which intestinal cells grow, differentiate and are viable for more than 3 months and up to 350 days, said culture comprising a primary intestinal tissue explant cultured in the presence of exogenous R-spondin;

• the method comprising:
• initiating a culture by mixing fragments of a primary intestinal tissue with a gel solution;
• pouring the gel solution mixed with fragments of the primary intestinal tissue over a layer of gel formed in a transwell container with a lower semi-permeable support;
• placing the transwell in an outer dish comprising culture medium comprising exogenous R-spondin, wherein the level of the culture medium is maintained such that the gel containing the tissue is not submerged in the culture medium;
• and
• maintaining the long term culture for more than 30 days and up to 350 days;
• wherein cells in the resulting long term culture exhibit normal Wnt-signaling and are responsive to proteins that stimulate or inhibit intestinal growth, wherein the R-spondin is present at a concentration of from 500 ng/ml to 1 mg/ml.