The factors controlling the proliferation and differentiation of the colonic mucosa are unknown and have proved difficult to identify mainly because of a lack of in vitro methods for studying the proliferative cells of the mucosa.

We have developed a novel method of preparing a viable single-cell suspension from isolated crypts and cloning these single cells.

We have obtained clonogenic growth from this single-cell suspension with an average of 1 colony per 10⁵ cells in control cultures. Addition of conditioned medium from the LIM1863 colon carcinoma cell line increased the mean colony number to 11 ± 3 per 10⁵ cells. The cells forming the colonies are still viable after 4 weeks in culture. The epithelial nature of the cells was confirmed by ultrastructural and immunohistochemical methods with staining for keratin 8 and 18 and anti-human epithelial membrane–specific antigen and a positive result on polymerase chain reaction for keratin 19.

We have successfully cloned single cells from disaggregated colonic crypts from both human and murine colonic mucosa. We have also demonstrated the presence of an active clonogenic factor in the conditioned medium of a colon carcinoma cell line. Assays show that the clonogenic activity in the conditioned medium is not caused by the presence of any of the epidermal growth factor family of growth factors. This is the first report of a clonogenic assay for epithelial cells of normal colonic mucosa. GASTROENTEROLOGY 1999;117:858-865