, Volume 70, Issue 1, pp 361–373 | Cite as

Development and characterization of 2-dimensional culture for buffalo intestinal cells

  • Nidhi Chaudhary
  • Himanshu Agrawal
  • Mamta Pandey
  • Suneel Onteru
  • Dheer Singh
Original Article


Small intestinal epithelial cells (IEC) play a major role in the absorption of nutrients and toxins. Due to the similarity of genome-wide single copy protein orthologues between cattle and human, establishment of ruminant’s primary small IEC culture could be a valuable tool for toxicity studies. Therefore, the current study focused on the development and characterization of buffalo IEC culture, as cattle slaughter is banned in India. The buffalo jejunum fragments were washed consecutively several times in saline, warm phosphate buffered saline (PBS), PBS with 5 mM dithiothreitol, digesting solution and 2% sorbitol in PBS. The cells were cultured on 17 µg/cm2 collagen coated plates and transwell plates with serum (2% Fetal bovine serum (FBS) and 10% FBS) and serum-free culture conditions. The cells were differentiated into typical epithelial cobblestone morphology from day 5 onwards in 50% successful cultures. The cultured IEC were characterized by gene expression of epithelial cell markers, cytokeratin and vimentin, and enterocyte markers like villin, zonula occluden (ZO1), fatty acid binding protein 2 (FABP2) and small intestinal peptidase (IP). Based on the morphology and gene expression profile, 10% FBS has been recommended for culturing primary buffalo IEC on collagen coated plates for 10 days. However, 50% of the successful cultures could not show epithelial phenotype on 10% FBS culture conditions even on collagen coated plates. Interestingly, undifferentiated IEC showed an increasing expression of FABP2, IP and ZO1 transcripts compared to differentiated intestinal cells with 10% FBS on collagen plates. Therefore, future studies are needed to understand the role of FABP2, IP and ZO1 in differentiation of buffalo IEC.


Buffalo Small intestinal epithelial cells Collagen-coated plates Transwell plates Markers 



Intestinal epithelial cells


Fetal bovine serum


Phosphate buffered saline




Zonula occludens


Fatty acid binding protein 2


Intestinal peptidase




Ribonucleic acid


Hank’s Balanced Salt Solution


Dulbecco’s Modified Eagle’s Medium


Insulin Transferrin Selenium


complementary deoxyribonucleic acid


Deoxynucleotide triphosphate


quantitative real time polymerase chain reaction


Experiments that showed typical epithelial morphology


Experiments that did not show typical epithelial morphology


Ribosomal Protein Lateral Stalk Subunit P1



The authors are thankful to the Director, ICAR-NDRI for providing infrastructure to carry out the present study. This research work was financially supported by Department of Biotechnology, Ministry of Science and Technology, India (Grant No. 102/IFD/SAN/3670/2014-15).


  1. Arpin M, Pringault E, Finidori J, Garcia A, Jeltsch JM, Vandekerckhove J, Louvard D (1988) Sequence of human villin: a large duplicated domain homologous with other actin-severing proteins and a unique small carboxy-terminal domain related to villin specificity. J Cell Biol 107:1759–1766CrossRefGoogle Scholar
  2. Athman R, Fernandez MI, Gounon P, Sansonetti P, Louvard D, Philpott D, Robine S (2005) Shigella flexneri infection is dependent on villin in the mouse intestine and in primary cultures of intestinal epithelial cells. Cell Microbiol 7:1109–1116CrossRefGoogle Scholar
  3. Autrup H, Stoner GD, Jackson F, Harris CC, Shamsuddin AKM, Barrett LA, Trump BF (1978) Explant culture of rat colon: a model system for studying metabolism of chemical carcinogens. In vitro 14:868–877CrossRefGoogle Scholar
  4. Benya RV, Schmidt LN, Sahi J, Layden TJ, Rao MC (1991) Isolation, characterization, and attachment of rabbit distal colon epithelial cells. Gastroenterology 101:692–702CrossRefGoogle Scholar
  5. Birkner S, Weber S, Dohle A, Schmahl G, Follmann W (2004) Growth and characterisation of primary bovine colon epithelial cells in vitro. Altern Lab Anim 32:555–571Google Scholar
  6. Booth C, Patel S, Bennion GR, Potten CS (1994) The isolation and culture of adult mouse colonic epithelium. Epithel Cell Biol 4:76–86Google Scholar
  7. Chandrakasan G, Hwang CB, Ryder M, Bhatnagar RS (1990) Keratin expression in cultures of adult human epidermal cells. Cell Mol Biol 37:847–852Google Scholar
  8. Chen R, Zou Y, Mao D, Sun D, Gao G, Shi J, Liu X, Zhu C, Yang M, Ye W, Hao Q, Li R, Yu L (2014) The general amino acid control pathway regulates mTOR and autophagy during serum/glutamine starvation. J Cell Biol 206:173–182CrossRefGoogle Scholar
  9. Chopra DP, Dombkowski AA, Stemmer PM, Parker GC (2010) Intestinal epithelial cells in vitro. Stem cells Dev 19:131–142CrossRefGoogle Scholar
  10. Darimont C, Gradoux N, Persohn E, Cumin F, Pover AD (2000) Effects of intestinal fatty acid-binding protein overexpression on fatty acid metabolism in Caco-2 cells. J Lipid Res 41:84–92Google Scholar
  11. Evans GS, Flint N, Somers AS, Eyden B, Potten CS (1992) The development of a method for the preparation of rat intestinal epithelial cell primary cultures. J Cell Sci 101:219–231Google Scholar
  12. Evans GS, Flint N, Potten CS (1994) Primary cultures for studies of cell regulation and physiology in intestinal epithelium. Annu Rev Physiol 56:399–417CrossRefGoogle Scholar
  13. Fairweather SJ, Broer A, O’Mara ML, Broer S (2012) Intestinal peptidases form functional complexes with the neutral amino acid transporter BoAT1. Biochem J 446:135–148CrossRefGoogle Scholar
  14. Follmann W, Weber S, Birkner S (2000) Primary cell cultures of bovine colon epithelium: isolation and cell culture of colonocytes. Toxicol In Vitro 14:435–445CrossRefGoogle Scholar
  15. Friederich E, Huet C, Arpin M, Louvard D (1989) Villin induces microvilli growth and actin redistribution in transfected fibroblasts. Cell 59:461–475CrossRefGoogle Scholar
  16. Fukamachi HI (1992) Proliferation and differentiation of fetal rat intestinal epithelial cells in primary serum-free culture. J Cell Sci 103:511–519Google Scholar
  17. Gibson PR, Van De Pol E, Maxwell LE, Gabriel A, Doe WF (1989) Isolation of colonic crypts that maintain structural and metabolic viability in vitro. Gastroenterology 96:283–291CrossRefGoogle Scholar
  18. Gomez LC, Real SM, Ojeda MS, Gimenez S, Mayorga LS, Roque M (2007) Polymorphism of the FABP2 gene: a population frequency analysis and an association study with cardiovascular risk markers in Argentina. BMC Med Genet 8:1CrossRefGoogle Scholar
  19. Goodyear AW, Kumar A, Dow S, Ryan EP (2014) Optimization of murine small intestine leukocyte isolation for global immune phenotype analysis. J Immunol Methods 405:97–108CrossRefGoogle Scholar
  20. Hague A, Paraskeva C (1996) The intestinal epithelial cell. In: Harris A (ed) Epithelial cell culture. Cambridge University Press, New York, pp 25–41Google Scholar
  21. Hata Y, Ota S, Nagata T, Uehara Y, Terano A, Sugimoto T (1993) Primary colonic epithelial cell culture of the rabbit producing prostaglandins. Prostaglandins 45:129–141CrossRefGoogle Scholar
  22. Hoey DE, Sharp L, Currie C, Lingwood CA, Gally DL, Smith DG (2003) Verotoxin 1 binding to intestinal crypt epithelial cells results in localization to lysomomes and abrogation of toxicity. Cell Microbiol 5:85–97CrossRefGoogle Scholar
  23. Hofmann RR (1989) Evolutionary steps of ecophysiological adaptation and diversification of ruminants: a comparative view of their digestive system. Oecologia 78:443–457CrossRefGoogle Scholar
  24. Holtkamp GM, Rossem MV, Devos AF, Willekens B, Peek R, Kijlstra A (1998) Polarized secretion of IL-6 and IL-8 by human retinal pigment epithelial cells. Clin Exp Immunol 112:34–43CrossRefGoogle Scholar
  25. Kaeffer B (2002) Mammalian intestinal epithelial cells in primary culture: a mini-review. In Vitro Cell Dev Biol Anim 38:123–134CrossRefGoogle Scholar
  26. Kaeffer B, Bottreau E, Velge P, Pardon P (1993) Epithelioid and fibroblastic cell lines derived from the ileum of an adult histocompatible miniature boar (d/d haplotype) and immortalized by SV40 plasmid. Eur J Cell Biol 62:152–162Google Scholar
  27. Kaushik RS, Begg AA, Wilson HL, Aich P, Abrahamsen MS, Potter A, Babiuk LA, Griebel P (2008) Establishment of fetal bovine intestinal epithelial cell cultures susceptible to bovine rotavirus infection. J Virol Methods 148:182–196CrossRefGoogle Scholar
  28. Kedinger M, Simon-Assmann PM, Lacroix B, Marxer A, Hauri HP, Haffen K (1986) Fetal gut mesenchyme induces differentiation of cultured intestinal endodermal and crypt cells. Dev Biol 113:474–483CrossRefGoogle Scholar
  29. Kedinger M, Haffen K, Simon-Assmann P (1987) Intestinal tissue and cell cultures. Differentiation 36:71–85CrossRefGoogle Scholar
  30. Kersting S, Bruewer M, Schuermann G, Klotz A, Utech M, Hansmerten M, Krieglstein CF, Senninger N, Schulzke JD, Naim HY, Zimmer KP (2004) Antigen transport and cytoskeletal characteristics of a distinct enterocyte population in inflammatory bowel disease. Am J Pathol 165:425–437CrossRefGoogle Scholar
  31. Kishida K, Aoyama M, Masaki M, Shidoji Y (2009) The Ala54Thr polymorphism in the fatty acid-binding protein 2 gene leads to higher food intake in Japanese women. Mol Psychiatry 14:466–467CrossRefGoogle Scholar
  32. Kondo Y, Rose I, Young GP, Whitehead RH (1984) Growth and differentiation of fetal rat small intestinal epithelium in tissue culture: relationship to fetal age. Exp Cell Res 153:121–134CrossRefGoogle Scholar
  33. Lechanteur A, Almeida A, Sarmento B (2017) Elucidation of the impact of cell culture conditions of Caco-2 cell monolayer on barrier integrity and intestinal permeability. Eur J Pharm Biopharm 119:137–141. doi: 10.1016/j.ejpb.2017.06.013 CrossRefGoogle Scholar
  34. Macartney KK, Baumgart DC, Carding SR, Brubaker JO, Offit PA (2000) Primary murine small intestinal epithelial cells, maintained in long-term culture, are susceptible to rotavirus infection. J Virol 74:5597–5603CrossRefGoogle Scholar
  35. Moon C, VanDussen KL, Miyoshi H, Stappenbeck TS (2014) Development of a primary mouse intestinal epithelial cell monolayer culture system to evaluate factors that modulate IgA transcytosis. Mucosal Immunol 7:818–828CrossRefGoogle Scholar
  36. Panja A (2000) A novel method for the establishment of a pure population of nontransformed human intestinal primary epithelial cell (HIPEC) lines in long term culture. Lab Investig 80:1473–1475CrossRefGoogle Scholar
  37. Perreault N, Beaulieu JF (1996) Use of the dissociating enzyme thermolysin to generate viable human normal intestinal epithelial cell cultures. Exp Cell Res 224:354–364CrossRefGoogle Scholar
  38. Perreault N, Beaulieu JF (1998) Primary cultures of fully differentiated and pure human intestinal epithelial cells. Exp Cell Res 245:34–42CrossRefGoogle Scholar
  39. Pretlow TP, Stinson AJ, Pretlow TG (1978) Cytologic appearance of cells dissociated from rat colon and their separation by isokinetic and isopyknic sedimentation in gradients of Ficoll. J Natl Cancer Inst 61:1431–1438Google Scholar
  40. Pringault E, Robine S, Louvard D (1991) Structure of the human villin gene. Proc Natl Acad Sci USA 88:10811–10815CrossRefGoogle Scholar
  41. Quaroni A, May RJ (1980) Establishment and characterization of intestinal epithelial cell cultures. Methods Cell Biol 21:403–427CrossRefGoogle Scholar
  42. Rani P, Vashisht M, Golla N, Shandilya S, Onteru SK, Singh D (2017) Milk miRNAs encapsulated in exosomes are stable to human digestion and permeable to intestinal barrier in vitro. J Funct Foods 34:431–439CrossRefGoogle Scholar
  43. Reddy PM, Sahi J, Desai G, Vidyasagar D, Rao MC (1996) Altered growth and attachment of rabbit crypt colonocytes isolated from different developmental stages. Pediatr Res 39:287–294CrossRefGoogle Scholar
  44. Rusu D, Loret S, Peulen O, Mainil J, Dandrifosse G (2005) Immunochemical, biomolecular and biochemical characterization of bovine epithelial intestinal primocultures. BMC Cell Biol 6:1CrossRefGoogle Scholar
  45. Ryeom SW, Paul D, Goodenough DA (2000) Truncation of mutants of the tight junction protein ZO-1 disrupt corneal epithelial cell morphology. Mol Biol Cell 11:1687–1696CrossRefGoogle Scholar
  46. Sacchettini JC, Hauft SM, Van Camp SL, Cistola DP, Gordon JI (1990) Developmental and structural studies of an intracellular lipid binding protein expressed in the ileal epithelium. J Biol Chem 265:19199–19207Google Scholar
  47. Sanderson IR, Ezzell RM, Kedinger M, Erlanger M, Xu ZX, Pringault E, Leon-Robine S, Louvard D, Walker WA (1996) Human fetal enterocytes in vitro: modulation of the phenotype by extracellular matrix. Proc Natl Acad Sci USA 93:7717–7722CrossRefGoogle Scholar
  48. Schlage WK, Bulles H, Friedrichs D, Kuhn M, Teredesai A (1998) Cytokeratin expression patterns in the rat respiratory tract as markers of epithelial differentiation in inhalation toxicology I. Determination of normal cytokeratin expression patterns in nose, larynx, trachea, and lung. Toxicol Pathol 26:324–343CrossRefGoogle Scholar
  49. Sun TT, Shih C, Green H (1979) Keratin cytoskeletons in epithelial cells of internal organs. Proc Natl Acad Sci USA 76:2813–2817CrossRefGoogle Scholar
  50. Tomar A, Wang Y, Kumar N, George S, Ceacareanu B, Hassid A, Chapman KE, Aryal AM, Waters CM, Khurana S (2004) Regulation of cell motility by tyrosine phosphorylated villin. Mol Biol Cell 15:4807–4817CrossRefGoogle Scholar
  51. Tomar A, George S, Kansal P, Wang Y, Khurana S (2006) Interaction of phospholipase C-Υ1 with villin regulates epithelial cell migration. J Biol Chem 281:31972–31986CrossRefGoogle Scholar
  52. Vashisht M, Rani P, Onteru SK, Singh D (2017) Curcumin encapsulated in milk exosomes resists human digestion and possesses enhanced intestinal permeability in vitro. Appl Biochem Biotechnol. doi: 10.1007/s12010-017-2478-4 Google Scholar
  53. Velge P, Kaeffer B, Bottreau E, Langendonck N (1995) The loss of contact inhibition and anchorage-dependent growth are key steps in the acquisition of Listeria monocytogenes susceptibility phenotype by non-phagocytic cells. Biol Cell 85:55–66CrossRefGoogle Scholar
  54. Vij R, Reddi S, Kapila S, Kapila R (2016) Transepithelial transport of milk derived bioactive peptide VLPVPQK. Food Chem 190:681–688CrossRefGoogle Scholar
  55. Wang Y, Srinivasan K, Siddiqui MR, George SP, Tomar A, Khurana S (2008) A novel role for villin in intestinal epithelial cell survival and homeostasis. J Biol Chem 283:9454–9464CrossRefGoogle Scholar
  56. Weng XH, Beyenbach KW, Quaroni A (2005) Cultured monolayers of the dog jejunum with the structural and functional properties resembling the normal epithelium. Am J Physiol Gastrointest Liver Physiol 288:G705–G717CrossRefGoogle Scholar
  57. Whitehead RH, Demmler K, Rockman SP, Watson NK (1999) Clonogenic growth of epithelial cells from normal colonic mucosa from both mice and humans. Gastroenterology 117:858–865CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  • Nidhi Chaudhary
    • 1
  • Himanshu Agrawal
    • 1
  • Mamta Pandey
    • 1
  • Suneel Onteru
    • 1
  • Dheer Singh
    • 1
  1. 1.Molecular Endocrinology, Functional Genomics and Systems Biology Lab, Animal Biochemistry DivisionICAR-National Dairy Research InstituteKarnalIndia

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