ISOLATION OF INTESTINAL EPITHELIAL-CELLS FOR THE STUDY OF DIFFERENTIAL GENE-EXPRESSION ALONG THE CRYPT-VILLUS AXIS

Methods for the differential isolation of intestinal epithelial cells from crypt and villus compartments of small intestine have been used in the study of intestinal functions. However, the use of different methods has resulted in discrepant conclusions as to the localization of expressed genes. Therefore, we undertook a careful comparison of two methods of intestinal epithelial cell isolation, the distended intestinal sac method and the everted intestinal sac method. The isolated cell fractions (distended sac fractions 1-10, everted sac fractions 1-5) were evaluated for the expression of two mRNAs whose localization along the crypt-villus axis had been previously elucidated by in situ hybridization: cytochrome P-450IIB1 (expressed in villus cells) and cryptdin (expressed in crypt cells). Northern blots of total or poly(A)+ RNA from each cell population showed that the first fractions from both methods contained P-450IIB1 mRNA, suggesting that both methods allowed the isolation of cells originating from the villus. Cryptdin mRNA was detected only in cell fractions 5-10 using the distended sac method and was not detected in any fractions from the everted sac method. [H-3]-thymidine incorporation demonstrated that dividing (crypt) cells were successfully removed by the distended sac method, but remained with the everted sac intestinal remnant. Finally, light and electron microscopy of the isolated cells as well as the intestinal remnants confirmed that while undifferentiated crypt cells were present in distended sac cell fractions 9 and 10, they remained with the everted sac remnant. Thus the distended sac protocol was useful for the isolation of cells from tip and crypt compartments. Furthermore, RNA extracted from cells isolated using the distended sac method is suitable for the construction of cDNA libraries that will be useful for cloning of genes that are differentially expressed along the crypt-villus axis.