Isolation, culture, and characterization of chicken intestinal epithelial cells

Abstract

Background Enterocytes exert an absorptive and protective function in the intestine, and they encounter many different challenging factors such as feed, bacteria, and parasites. An intestinal epithelial in vitro model can help to understand how enterocytes are affected by these factors and contribute to the development of strategies against pathogens. Results The present study describes a novel method to culture and maintain primary chicken enterocytes and their characterization by immunofluorescence and biomolecular approaches. Starting from 19-day-old chicken embryos it was possible to isolate viable intestinal cell aggregates that can expand and produce a self-maintaining intestinal epithelial cell population that survives until 12 days in culture. These cells resulted positive in immunofluorescence to Cytokeratin 18, Zonula occludens 1, Villin, and Occludin that are common intestinal epithelial markers, and negative to Vimentin that is expressed by endothelial cells. Cells were cultured also on Transwell® permeable supports and trans-epithelial electrical resistance, was measured. This value gradually increased reaching 64 Ω*cm2 7 days after seeding and it remained stable until day 12. Conclusions Based on these results it was confirmed that it is possible to isolate and maintain chicken intestinal epithelial cells in culture and that they can be suitable as in vitro intestinal model for further studies.

Novel chicken two-dimensional intestinal model comprising all key epithelial cell types and a mesenchymal sub-layer

“…The growth factor requirements for maintaining mammalian 3D organoids and intestinal monolayer cultures have been well defined, and consist of Epidermal Growth Factor, Noggin, and R-Spondin (ENR) [ 4 , 9 , 12 , 20 ]. The culture conditions of the chicken 2D enteroids were established using a modified ENR growth factor cocktail previously used in mouse intestinal monolayer cultures [ 12 ].…”

“…The generation of 2D intestinal cultures with a tight epithelium has not previously been reported in chicken [ 17 – 24 , 27 ]. Most recently, confluent cultures of primary chicken duodenal epithelial cells were described [ 20 ]. These cultures survive until day 12 with tight junctions and a low TEER value of 64 Ω·cm 2 , which is in line with some other intestinal cell models but does not indicate a tight barrier [ 41 ].…”

Avian Cell Culture Models to Study Immunomodulatory Properties of Bioactive Products

“…IEC have also been used to evaluate the effects of selected Lactobacillus probiotics on the production of avian beta-defensin 9 (AvBD9), which is responsible for maintaining the homeostasis of the gastrointestinal microbiota and the gut immune system [ 59 ]. Research on intestinal epithelial cells is increasingly popular due to their association with immunity [ 60 ].…”

“…Such an experimental approach model predicted gastrointestinal colonization using an in vitro LMH model [ 54 ]. Intestinal epithelial cell lines are used to study responses to various bioactive compounds, such as probiotics [ 55 , 60 ]. The new cell line is used in various experiments, such as testing the effects of Salmonella enterica .…”

Assessing Intestinal Health. In Vitro and Ex vivo Gut Barrier Models of Farm Animals: Benefits and Limitations

“…However, these cells lack proper epithelial morphology since IF characterization showed that Zonula occludens-1 (ZO1) was not located in their intercellular junctions, a typical trait of intestinal enterocytes. Recently, Ghiselli et al ( 62 ) developed a protocol that required various growth factors to culture cIECs. They showed that it is possible to maintain these cells for up to 14 days in culture.…”