Methods for the isolation of intact epithelium from the mouse intestine

Abstract Methods are described for the isolation of intact intestinal epithelium in several novel forms. The first method described is for application to segments of bowel that must be studied without killing the animal, e.g., a resected length of bowel. In this method, the isolated segment is everted onto a glass rod and incubated at 37°C in fresh 30 mM ethylenediaminetetraacetic acid (EDTA) in calcium‐magnesium‐free Hank's balanced salt solution (CMF) for either 15 or 20 minutes. At the end of the incubation, the epithelium is isolated by vibration into a tube of cold CMF. If it is incubated for 20 minutes, the entire epithelium is isolated in the form of single crypt‐villus units. If it is incubated for 15 minutes, the epithelium is isolated in the form of small intact sheets, but not all of the epithelium is removed. The second method requires the death of the animal but yields better results. The anesthetized animal is perfused through the left ventricle with a fresh 37° solution of EDTA (0.01–30 mM) in CMF. Then the segment of gut is removed, everted onto a glass rod, and the epithelium isolated by vibration into cold CMF. The whole of the epithelium is isolated with this procedure and, with higher concentrations of EDTA, it is isolated in the form of large sheets (1–2 cm 2 ). Structurally inact units could be obtained with either procedure. There was no contamination with underlying nonepithelial elements. The best results, as determined by morphology and viability, were obtained with the perfusion method using 30 mM EDTA. With this concentration, the whole procedure, from sacrifice of the animal to isolation of the epithelium, requires less than 4 minutes. Procedures are also described for the isolation of either pure intact crypts or of pure intact vill from small intestine. Each of the methods described could be used to isolate the epithelium from any region of the intestinal tract with equal ease. Some applications of the methods are also discussed.