Establishment and characterization of a primary canine duodenal epithelial cell culture.
Many mechanisms involved in the pathogenesis of chronic enteropathies or host-pathogen interactions in canine intestine have not been elucidated so far. Next to the clinical and in vivo research tools, an in vitro model of canine intestinal cell culture would be very helpful for studies at the cellular level. Therefore, the purpose of this study was to establish and characterize a primary canine duodenal epithelial cell culture. Neonatal duodenum was disrupted with trypsin-ethylenediaminetetraacetic acid (EDTA) and the mucosa scraped off and digested with collagenase and dispase. After centrifugation on a 2% sorbitol gradient, the cells were incubated at 37 degrees C in OptiMEM supplemented with Primocin, epidermal growth factor, insulin, hydrocortisone, and 10% fetal calf serum (FCS). After 24 h, the FCS concentration was reduced to 2.5%, and the temperature decreased to 33 degrees C. With this method, the cultures were growing to confluent monolayers within 5-6 d and remained viable for an average of 2 wk. Their epithelial nature was confirmed by electron microscopy and immunofluorescence staining using antibodies directed against specific cytokeratins, desmosomes, and tight junctions. The intestinal cells proliferated, as evidenced by immunolabeling with a Ki-67 antibody, and cryptal cell subpopulations could be identified. Furthermore, alkaline phosphatase and sucrase activity were detected.
Golaz
Julia L
JL
Department of Clinical Veterinary Medicine, Division of Small Animal Internal Medicine, Vetsuisse Faculty of the University of Bern, Länggassstrasse 122, 3001 Bern, Switzerland.
Vonlaufen
Nathalie
N
Hemphill
Andrew
A
Burgener
Iwan A
IA
D000818:Animals; D000831:Animals, Newborn; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques / Q000379:methods*; D002460:Cell Line; D002478:Cells, Cultured; D004285:Dogs; D004386:Duodenum / Q000166:cytology*; D004847:Epithelial Cells / Q000166:cytology* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D007382:Intermediate Filaments / Q000378:metabolism
D015415:Biomarkers