Abstract
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° 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° 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.
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Acknowledgments
The authors would like to thank Prof. Beyenbach and Prof. Quaroni for providing the monoclonal antibody TS23 specific to the glycosylated form of Notch-1 and Dr. Herring and Prof. Beaulieu for providing the MIM 1/39 antibody, an intestinal crypt cell marker. Furthermore, the authors would like to acknowledge Ursula Luginbühl for sucrase activity measurement, and many thanks are addressed to Eliane Mueller for advice in immunofluorescence staining.
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Editor: J. Denry Sato
Julia L. Golaz and Nathalie Vonlaufen contributed equally to this work and are joint first authors. Supported in part by the Vetsuisse research foundation, the Foundation Research 3R (project No. 85/03), and the Swiss National Science Foundation (3100A0-112532).
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Golaz, J.L., Vonlaufen, N., Hemphill, A. et al. Establishment and characterization of a primary canine duodenal epithelial cell culture. In Vitro Cell.Dev.Biol.-Animal 43, 176–185 (2007). https://doi.org/10.1007/s11626-007-9034-4
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DOI: https://doi.org/10.1007/s11626-007-9034-4