Volume 117, Issue 4, Pages 858-865 (October 1999) Clonogenic growth of epithelial cells from normal colonic mucosa from both mice and humans  Robert H. Whitehead, Kirsten Demmler, Steven P. Rockman, Nadine K. Watson  Gastroenterology  Volume 117, Issue 4, Pages 858-865 (October 1999) DOI: 10.1016/S0016-5085(99)70344-6 Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 1 A single suspension of cells prepared from mouse colonic crypts using pancreatin. The cell suspension comprises mainly single cells with occasional doublets (original magnification 100×). Gastroenterology 1999 117, 858-865DOI: (10.1016/S0016-5085(99)70344-6) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 2 Two colonies derived from single cells from disaggregated mouse colonic crypts after 21 days of culture in agarose (original magnification 100×). Gastroenterology 1999 117, 858-865DOI: (10.1016/S0016-5085(99)70344-6) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 3 Mouse colon colony assay. Titration of 2 batches of LIM1863 conditioned medium using a single-cell suspension isolated from murine colonic crypts. Both colonies (>40 cells) and clusters (15-40 cells) were counted after 23 days. Each assay was performed in triplicate. Gastroenterology 1999 117, 858-865DOI: (10.1016/S0016-5085(99)70344-6) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 4 Immunofluorescence staining of cytocentrifuge preparations of cells from colonies picked from an agar cloning assay of single human colonic crypt cells after 4 weeks in culture. (A) Fluorescein isothiocyanate conjugate control; (B) keratin 18; (C) human epithelial-specific antigen. Gastroenterology 1999 117, 858-865DOI: (10.1016/S0016-5085(99)70344-6) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 5 PCR reaction using primers specific for human keratin 19. Lane 1, size markers; lane 2, H2O control; lane 3, colonic mucosal tissue; lane 4, primary mucosal culture; lane 5, crypts isolated from human colonic mucosa; lane 6, colonies of human colonic cells picked from agarose; lane 7, H2O control. Gastroenterology 1999 117, 858-865DOI: (10.1016/S0016-5085(99)70344-6) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 (A) Electron micrograph of a section through 4 cells of a colony of mouse colonic cells picked from agar after 28 days in culture. The viability of the cells is confirmed by the intact nuclei and cytoplasmic organelles. Lipid droplets can be seen in the cytoplasm of some cells. Tight junctions are present at the apical junctions between cells (arrows). No microvilli or mucin inclusions were found (original magnification 3600×). (B) Apical junctional complex (arrow) between 2 adjacent cells in a colony derived from mouse colonic epithelial cells. Each cell was joined to neighboring cells by a similar junction in the apical region (original magnification 12,600×). Gastroenterology 1999 117, 858-865DOI: (10.1016/S0016-5085(99)70344-6) Copyright © 1999 American Gastroenterological Association Terms and Conditions

Fig. 6 (A) Electron micrograph of a section through 4 cells of a colony of mouse colonic cells picked from agar after 28 days in culture. The viability of the cells is confirmed by the intact nuclei and cytoplasmic organelles. Lipid droplets can be seen in the cytoplasm of some cells. Tight junctions are present at the apical junctions between cells (arrows). No microvilli or mucin inclusions were found (original magnification 3600×). (B) Apical junctional complex (arrow) between 2 adjacent cells in a colony derived from mouse colonic epithelial cells. Each cell was joined to neighboring cells by a similar junction in the apical region (original magnification 12,600×). Gastroenterology 1999 117, 858-865DOI: (10.1016/S0016-5085(99)70344-6) Copyright © 1999 American Gastroenterological Association Terms and Conditions