Isolation and Culture of Intestinal Epithelial Cells

A disaggregation technique is described, based on collagenase and dispase, which allows the isolation of crypts from mouse colon. The crypts may be purified by selective sedimentation and plated out in 24-well plates. Application of the technique to small intestine, neonatal tissue, polyps, and tumors is also described. A separate isolation and sedimentation protocol is provided for primary culture of human colonic crypts which may be plated on to collagen or 3T3 feeder layers, but which do not subculture readily. Chelating agents give a higher yield when isolating crypts, but the viability tends to be lower. Two colony-forming assays are provided, one of which is clonogenic, and other quantitative assays include labeling index with [3H]-thymidine and estimation of cell number with crystal violet staining. Differentiation has been studied by implanting collagenase/dispase- or Acutase-generated cell suspensions in Matrigel into immune-deprived mice.