Isolation, culture, and characterization of chicken intestinal epithelial cells

BMC Molecular and Cell Biology - 2021
Federico Ghiselli1, Barbara Rossi2, Martina Felici1, Maria Parigi3, Giovanni Tosi3, L. Fiorentini3, Paola Massi3, Andrea Piva1,2, Ester Grilli4,1
1DIMEVET, University of Bologna, Bologna, Italy
2Vetagro S.p.A., Reggio Emilia, Italy
3Istituto Zooprofilattico Sperimentale Della Lombardia e Dell’Emilia Romagna, Sede Territoriale di Forlì, Forlì, Italy
4Vetagro Inc., Chicago, USA

Tóm tắt

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.

Từ khóa


Tài liệu tham khảo

Groschwitz KR, Hogan SP. Intestinal barrier function: molecular regulation and disease pathogenesis. J Allergy Clin Immunol. 2009;124:3–22.

Roda G, Sartini A, Zambon E, Calafiore A, Marocchi M, Caponi A, et al. Intestinal epithelial cells in inflammatory bowel diseases. World J Gastroenterol WJG. 2010;16:4264–71.

Kaur G, Dufour JM. Cell lines: valuable tools or useless artifacts. Spermatogenesis. 2012;2:1–5.

Bussière FI, Niepceron A, Sausset A, Esnault E, Silvestre A, Walker RA, et al. Establishment of an in vitro chicken epithelial cell line model to investigate Eimeria tenella gamete development. Parasit Vectors. 2018;11:44.

Immerseel FV, Buck JD, Smet ID, Pasmans F, Haesebrouck F, Ducatelle R. Interactions of butyric acid– and acetic acid–treated Salmonella with chicken primary Cecal epithelial cells in vitro. Avian Dis. 2004;48:384–91.

Dimier-Poisson IH, Bout DT, Quéré P. Chicken primary enterocytes: inhibition of Eimeria tenella replication after activation with crude interferon-γ supernatants. Avian Dis. 2004;48:617–24.

Byrne CM, Clyne M, Bourke B. Campylobacter jejuni adhere to and invade chicken intestinal epithelial cells in vitro. Microbiology. 2007;153:561–9.

Borrmann E, Berndt A, Hänel I, Köhler H. Campylobacter-induced interleukin-8 responses in human intestinal epithelial cells and primary intestinal chick cells. Vet Microbiol. 2007;124:115–24.

Guo S, Li C, Liu D, Guo Y. Inflammatory responses to a Clostridium perfringens type a strain and α -toxin in primary intestinal epithelial cells of chicken embryos. Avian Pathol. 2015;44:81–91.

Yuan C, He Q, Li J, Azzam MM, Lu J, Zou X. Evaluation of embryonic age and the effects of different proteases on the isolation and primary culture of chicken intestinal epithelial cells in vitro: primary culture of chicken IEC In Vitro. Anim Sci J. 2015;86:588–94.

Kaiser A, Willer T, Steinberg P, Rautenschlein S. Establishment of an In Vitro intestinal epithelial cell culture model of avian origin. Avian Dis. 2017;61:229–36.

Bar Shira E, Friedman A. Innate immune functions of avian intestinal epithelial cells: Response to bacterial stimuli and localization of responding cells in the developing avian digestive tract. PLOS ONE. 13(7):e0200393.

Rath NC, Liyanage R, Gupta A, Packialakshmi B, Lay JO. A method to culture chicken enterocytes and their characterization. Poult Sci 2018;0:1–8.

Niu N, Wang L. In vitro human cell line models to predict clinical response to anticancer drugs. Pharmacogenomics. 2015;16:273–85.

Xu H, Jiao Y, Qin S, Zhao W, Chu Q, Wu K. Organoid technology in disease modelling, drug development, personalized treatment and regeneration medicine. Exp Hematol Oncol. 2018;7:30.

Fair KL, Colquhoun J, Hannan NRF. Intestinal organoids for modelling intestinal development and disease. Philos Trans R Soc B Biol Sci. 2018;373:20170217.

Pierzchalska M, Grabacka M, Michalik M, Zyla K, Pierzchalski P. Prostaglandin E2 supports growth of chicken embryo intestinal organoids in Matrigel matrix. BioTechniques. 2012;52:307–15.

Powell RH, Behnke MS. WRN conditioned media is sufficient for in vitro propagation of intestinal organoids from large farm and small companion animals. Biol Open. 2017;6:698–705.

Li J, Li J, Zhang SY, Li RX, Lin X, Mi YL, et al. Culture and characterization of chicken small intestinal crypts. Poult Sci. 2018;97:1536–43.

Panek M, Grabacka M, Pierzchalska M. The formation of intestinal organoids in a hanging drop culture. Cytotechnology. 2018;70:1085–95.

Takahashi T. Organoids for drug discovery and personalized medicine. Annu Rev Pharmacol Toxicol. 2019;59:447–62.

Yu J, Peng S, Luo D, March JC. In vitro 3D human small intestinal villous model for drug permeability determination. Biotechnol Bioeng. 2012;109:2173–8.

Bermudez-Brito M, Plaza-Díaz J, Fontana L, Muñoz-Quezada S, Gil A. In vitro cell and tissue models for studying host–microbe interactions: a review. Br J Nutr. 2013;109:S27–34.

Ren HJ, Zhang CL, Liu RD, Li N, Li XG, Xue HK, et al. Primary cultures of mouse small intestinal epithelial cells using the dissociating enzyme type I collagenase and hyaluronidase. Braz J Med Biol Res. 2017;50(5):e5831.

Bai S, Zhang K, Ding X, Wang J, Zeng Q, Peng H, et al. Uptake of manganese from the manganese-lysine complex in primary chicken intestinal epithelial cells. Anim Open Access J MDPI. 2019;9:559.

Assimakopoulos SF, Papageorgiou I, Charonis A. Enterocytes’ tight junctions: from molecules to diseases. World J Gastrointest Pathophysiol. 2011;2:123–37.

Thorne CA, Chen IW, Sanman LE, Cobb MH, Wu LF, Altschuler SJ. Enteroid Monolayers Reveal an Autonomous WNT and BMP Circuit Controlling Intestinal Epithelial Growth and Organization. Dev Cell. 2018;44:624–33 e4.

Hao Y-H, Lafita-Navarro MC, Zacharias L, Borenstein-Auerbach N, Kim M, Barnes S, et al. Induction of LEF1 by MYC activates the WNT pathway and maintains cell proliferation. Cell Commun Signal CCS. 2019;17:129.

Jho E, Zhang T, Domon C, Joo C-K, Freund J-N, Costantini F. Wnt/β-catenin/Tcf signaling induces the transcription of Axin2, a negative regulator of the signaling pathway. Mol Cell Biol. 2002;22:1172–83.

Yamashita Y, Nakagomi K, Takeda T, Hasegawa S, Mitsui Y. Effect of heparin on pulmonary fibroblasts and vascular cells. Thorax. 1992;47:634–9.

Hickman J. Transepithelial/endothelial electrical resistance (TEER) theory and applications for microfluidic body-on-a-chip devices. J Rare Dis Res Treat. 2016;1:46–52.

Steensma A, Noteborn HPJM, Kuiper HA. Comparison of Caco-2, IEC-18 and HCEC cell lines as a model for intestinal absorption of genistein, daidzein and their glycosides. Environ Toxicol Pharmacol. 2004;16:131–9.

Srinivasan B, Kolli AR, Esch MB, Abaci HE, Shuler ML, Hickman JJ. TEER Measurement Techniques for In Vitro Barrier Model Systems. J Lab Autom. 2015;20:107–26.

Thông tin
Thông tin xuất bản

BMC Molecular and Cell Biology

Thông tin tác giả