A simple method to establish short-term cultures of normal human colonic epithelial cells from endoscopic biopsy specimens. Comparison of isolation methods, assessment of viability and metabolic activity.
BACKGROUND
Abnormalities in colonic epithelial cell function have been implicated in the pathogenesis of various intestinal disorders, especially inflammatory bowel disease (IBD). The mechanisms, however, remain obscure owing to the lack of representative human colonic epithelial cell models. The aim of this study was to develop and validate a method for establishment of short-term culture of normal human colonic epithelial cells from endoscopic biopsies.


METHODS
Epithelial cells were isolated from colonoscopic biopsies by means of ethylenediaminetetraacetic acid/ethylene glycol tetraacetic acid (EDTA/EGTA) (10 or 60 min) or by enzyme treatment and cultured in collagen-coated wells. Viability was measured with a methyltetrazoleum conversion assay, confocal laser, and electron microscopy. Metabolic function was measured by means of butyrate oxidation, 14C-leucine and 3H-glucosamine incorporation; DNA synthesis by means of 3H-thymidine incorporation, and apoptosis with an enzyme-linked immunosorbent assay (ELISA) for histone-associated DNA fragments. Cell types were identified by immunocytochemistry.


RESULTS
Ten minutes of EDTA/EGTA treatment released intact crypts and was superior to both the 60-min treatment and enzymatic treatment in terms of viability and nonepithelial cell contamination, respectively. Despite activation of detachment-induced apoptosis, a median 51% of the isolated cells was viable after 24 h of culture and metabolically active as judged by 3H-thymidine, 14C-leucine, and 3H-glucosamine incorporation. Butyrate oxidation followed more complex kinetics (substrate activation) than observed previously in other models. The apparent Km values (medians) were 0.7 mM and 4.5 mM in low and high concentration ranges, respectively.


CONCLUSIONS
We report a simple method to establish culture of human colonic epithelial cells from endoscopically obtained biopsy specimens, producing sufficient viable cells to perform metabolic studies pertinent to the pathogenesis of IBD and related human disorders.
Pedersen
G
G
Dept. of Gastroenterology C, Herlev Hospital, University of Copenhagen, Herlev, Denmark.
Saermark
T
T
Giese
B
B
Hansen
A
A
Drag
B
B
Brynskov
J
J
D001706:Biopsy; D002087:Butyrates / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002470:Cell Survival; D003106:Colon / Q000166:cytology* / Q000378:metabolism; D004724:Endoscopy; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology*; D010084:Oxidation-Reduction
D002087:Butyrates