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 imp licated in the pathogenesis of various intestinal disorders, especially inf lammatory bowel disease (IBD). The mechanisms, however, remain obscure owin g to the lack of representative human colonic epithelial cell models. The a im 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 biopsi es by means of ethylenediaminetetraacetic acid/ethylene glycol tetraacetic acid (EDTA/EGTA) (10 or 60 min) or by enzyme treatment and cultured in coll agen-coated wells. Viability was measured with a methyltetrazoleum conversi on assay, confocal laser, and electron microscopy. Metabolic function was m easured by means of butyrate oxidation, C-14-leucine and H-3-glucosamine in corporation; DNA synthesis by means of H-3-thymidine incorporation, and apo ptosis with an enzyme linked immunosorbent assay (ELISA) for histone-associ ated DNA fragments. Cell types were identified by immunocytochemistry. Resu lts: Ten minutes of EDTA/EGTA treatment released intact crypts and was supe rior to both the 60-min treatment and enzymatic treatment in terms of viabi lity and nonepithelial cell contamination, respectively. Despite activation of detachment-induced apoptosis, a median 51% of the isolated cells was vi able after 24 h of culture and metabolically active as judged by 3H-thymidi ne, C-14-leucine, and 3H-glucosamine incorporation. Butyrate oxidation foll owed more complex kinetics (substrate activation) than observed previously in other models. The apparent K-m values (medians) were 0.7 mM and 4.5 mM i n low and high concentration ranges, respectively. Conclusion: We report a simple method to establish culture of human colonic epithelial cells from e ndoscopically obtained biopsy specimens, producing sufficient viable cells to perform metabolic studies pertinent to the pathogenesis of IBD and relat ed human disorders.