Real-time imaging analysis of proliferation/differentiation of colonic epithelial cells in primary culture

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We have developed a novel culture method for colonic epithelial cells that allows long-term maintenance and efficient expansion of stem cells in vitro. Colonic crypts from normal adult mice were isolated and three-dimensionally cultured in serum-free medium supplemented with a combination of growth factors. The crypt cells formed a round cystic organoid consisting of epithelial monolayer of multi-lineage cells, continuously grew with increasing size of organoid structure, and could be propagated for months without losing their properties. Importantly, Lgr5+colonic stem cells were constantly maintained for a long time period in our culture. In order to test transplantability of these cultured cells, GFP+colonic cells were re-introduced into the lumen of mouse colon superficially damaged by DSS treatment. As observed 4 weeks post-transplantation, the infused organoids readily integrated into the epithelium to form self-renewing GFP+epithelial patches that were histologically normal. Similar results were obtained with colon organoids that were derived from a single Lgr5+colon stem cell after extensive in vitro expansion.The techniques that we have developed would be of significant help to build a basis to develop a novel colon stem cell therapy based on in vitro expansion of a single adult colonic stem cell.