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Home / Equine Research Bank / In Vitro Cell Dev Biol Anim / Isolation and culture of primary equine tracheal epithelial ...
In vitro cellular & developmental biology. Animal2008; 44(7); 179-184; doi: 10.1007/s11626-008-9099-8

Isolation and culture of primary equine tracheal epithelial cells.

Abstract: Culture of airway epithelial cells is a useful model to investigate physiology of airway epithelia and airway disease mechanisms. In vitro models of airway epithelial cells are established for various species. However, earlier published method for isolation and culture of equine tracheal epithelial cells requires significant improvements. In this report, the development of a procedure for efficient isolation, characterization, culture, and passage of primary equine tracheal epithelial cells are described. Epithelial cells were isolated from adult equine trachea by exposing and stripping the mucosal epithelium from the adjacent connective tissue and smooth muscle. The tissue was minced and dissociated enzymatically using 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) solution for 2 h at 37 degrees C. Cells were collected by sieving and centrifugation, and contaminating fibroblasts were removed by differential adhesion. This procedure resulted in a typical yield of 1 x 10(7) cytokeratin-positive epithelial cells per gram tracheal lining tissue. Viability was 95% by trypan blue exclusion and isolates contained approximately 94% cytokeratin-positive cells of epithelial origin. Cells seeded at a density of 6.9 x 10(4) cells/cm2 in serum-free airway epithelial cell growth medium formed monolayers near confluency within a week. Confluent cells were dissociated using dispase II and first passages (P1) and second passages (P2) were successfully established in serum-free medium. Collagen coating of tissue culture flask was not required for cell adhesion, and cultures could be maintained at the level of P2 over 30 d. In the present study, we could establish a high-yield protocol for isolation and culture of equine tracheal epithelial cells that can serve for in vitro/ex vivo studies on the (patho-)physiology of equine airway disease as well as pharmacological and toxicological targets relevant to airway diseases.
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Publication Date: 2008-07-02 PubMed ID: 18594938DOI: 10.1007/s11626-008-9099-8Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article describes the development of an improved method for isolating and culturing equine (horse) tracheal epithelial cells, with an aim to provide an efficient model for studying diseases affecting the airway in horses.

Procedure for Isolation and Culture of Epithelial Cells

  • The researchers derived epithelial cells from adult horse trachea. They accomplished this by exposing and stripping the mucosal epithelium, the lining of the airway, away from the connective tissue and smooth muscle.
  • The acquired tissue was then chopped and enzymatically disaggregated using 0.25% trypsin-EDTA solution, a common reagent employed in the process of harvesting cells.
  • Cells were gathered via sieving and centrifugation, a technique used to separate cellular components. Furthermore, fibroblasts, a type of cell that could contaminate the culture, were eliminated through differential adhesion.
  • The described procedure yielded about 1 x 107 cytokeratin-positive epithelial cells per gram of tracheal lining tissue.

Viability and Growth of Isolated Cells

  • Approximately 95% of the isolated cells were found viable through the trypan blue exclusion test, a straightforward method for determining cell viability.
  • Furthermore, 94% of the isolates were cytokeratin-positive cells of epithelial origin indicating the purity of the isolation process.
  • When seeded onto the growth medium at a specific density, the cells formed near-confluent monolayers within a week, suggesting they retained their ability to proliferate.
  • The use of dispase II, an enzyme, enabled researchers to dissociate and conduct further passages of the cells in serum-free medium, hence allowing long-term culturing of the cells.
  • The team reported no need for collagen coating of tissue culture flasks for cell adhesion, simplifying the culture process.
  • The researchers were able to maintain the cell cultures beyond 30 days in the second passage stage, providing a time scale over which experiments could be conducted.

Potential Applications of the Study

  • This improved protocol can be used for both in vitro (outside a living organism) and ex vivo (from a living organism) studies about the normal function and disease states of the horse’s respiratory system.
  • The developed protocol can aid in studying pharmacological and toxicological targets relevant to airway diseases, which could lead to better prevention strategies and treatments.

Cite This Article

APA
Shibeshi W, Abraham G, Kneuer C, Ellenberger C, Seeger J, Schoon HA, Ungemach FR. (2008). Isolation and culture of primary equine tracheal epithelial cells. In Vitro Cell Dev Biol Anim, 44(7), 179-184. https://doi.org/10.1007/s11626-008-9099-8

Publication

In vitro cellular & developmental biology. Animal
ISSN: 1071-2690
NlmUniqueID: 9418515
Country: Germany
Language: English
Volume: 44
Issue: 7
Pages: 179-184

Researcher Affiliations

Shibeshi, Workineh
  • Institute of Pharmacology, Pharmacy and Toxicology, University of Leipzig, An den Tierkliniken 15, 04103 Leipzig, Germany.
Abraham, Getu
    Kneuer, Carsten
      Ellenberger, Christin
        Seeger, Johannes
          Schoon, Heinz-Adolf
            Ungemach, Fritz R

              MeSH Terms

              • Animals
              • Cell Separation / methods
              • Cells, Cultured
              • Epithelial Cells / cytology
              • Horses / metabolism
              • Keratins / metabolism
              • Trachea / cytology
              • Vimentin / metabolism

              References

              This article includes 25 references
              1. Schumann BL, Cody TE, Miller ML, Leikauf GD. Isolation, characterization, and long-term culture of fetal bovine tracheal epithelial cells.. In Vitro Cell Dev Biol 1988 Mar;24(3):211-6.
                pubmed: 2450863doi: 10.1007/BF02623549google scholar: lookup
              2. Abraham G, Kottke C, Dhein S, Ungemach FR. Agonist-independent alteration in beta-adrenoceptor-G-protein-adenylate cyclase system in an equine model of recurrent airway obstruction.. Pulm Pharmacol Ther 2006;19(3):218-29.
                pubmed: 16084121doi: 10.1016/j.pupt.2005.05.007google scholar: lookup
              3. Bin W, Aksoy MO, Yang Y, Kelsen SG. IL-1beta enhances beta2-adrenergic receptor expression in human airway epithelial cells by activating PKC.. Am J Physiol Lung Cell Mol Physiol 2001 Apr;280(4):L675-9.
                pubmed: 11238007doi: 10.1152/ajplung.2001.280.4.L675google scholar: lookup
              4. Folkerts G, Nijkamp FP. Airway epithelium: more than just a barrier!. Trends Pharmacol Sci 1998 Aug;19(8):334-41.
                pubmed: 9745362doi: 10.1016/s0165-6147(98)01232-2google scholar: lookup
              5. Salathe M. Effects of beta-agonists on airway epithelial cells.. J Allergy Clin Immunol 2002 Dec;110(6 Suppl):S275-81.
                pubmed: 12464936doi: 10.1067/mai.2002.129412google scholar: lookup
              6. Abraham G, Kneuer C, Ehrhardt C, Honscha W, Ungemach FR. Expression of functional beta2-adrenergic receptors in the lung epithelial cell lines 16HBE14o(-), Calu-3 and A549.. Biochim Biophys Acta 2004 May 3;1691(2-3):169-79.
                pubmed: 15110997doi: 10.1016/j.bbamcr.2004.02.002google scholar: lookup
              7. Craviso GL. Generation of functionally competent single bovine adrenal chromaffin cells from cell aggregates using the neutral protease dispase.. J Neurosci Methods 2004 Aug 30;137(2):275-81.
                pubmed: 15262071doi: 10.1016/j.jneumeth.2004.02.031google scholar: lookup
              8. Abraham G, Kottke C, Ungemach FR. Equine recurrent airway obstruction does not alter airway muscarinic acetylcholine receptor expression and subtype distribution.. J Vet Pharmacol Ther 2007 Oct;30(5):401-9.
                pubmed: 17803731doi: 10.1111/j.1365-2885.2007.00897.xgoogle scholar: lookup
              9. Kaartinen L, Nettesheim P, Adler KB, Randell SH. Rat tracheal epithelial cell differentiation in vitro.. In Vitro Cell Dev Biol Anim 1993 Jun;29(6):481-92.
                pubmed: 27519750doi: 10.1007/BF02639383google scholar: lookup
              10. Davidson DJ, Kilanowski FM, Randell SH, Sheppard DN, Dorin JR. A primary culture model of differentiated murine tracheal epithelium.. Am J Physiol Lung Cell Mol Physiol 2000 Oct;279(4):L766-78.
                pubmed: 11000138doi: 10.1152/ajplung.2000.279.4.L766google scholar: lookup
              11. Abraham G, Kottke C, Ammer H, Dhein S, Ungemach FR. Segment-dependent expression of muscarinic acetylcholine receptors and G-protein coupling in the equine respiratory tract.. Vet Res Commun 2007 Feb;31(2):207-26.
                pubmed: 17180451doi: 10.1007/s11259-006-3396-zgoogle scholar: lookup
              12. Barnes PJ. Novel approaches and targets for treatment of chronic obstructive pulmonary disease.. Am J Respir Crit Care Med 1999 Nov;160(5 Pt 2):S72-9.
                pubmed: 10556174doi: 10.1164/ajrccm.160.supplement_1.17google scholar: lookup
              13. Radi ZA, Ackermann MR. Growth of differentiated ovine tracheal epithelial cells in vitro.. J Vet Med A Physiol Pathol Clin Med 2004 May;51(4):167-70.
                pubmed: 15265172doi: 10.1111/j.1439-0442.2004.00620.xgoogle scholar: lookup
              14. Kelsen SG, Anakwe O, Aksoy MO, Reddy PJ, Dhanasekaran N. IL-1 beta alters beta-adrenergic receptor adenylyl cyclase system function in human airway epithelial cells.. Am J Physiol 1997 Sep;273(3 Pt 1):L694-700.
                pubmed: 9316506doi: 10.1152/ajplung.1997.273.3.L694google scholar: lookup
              15. You Y, Richer EJ, Huang T, Brody SL. Growth and differentiation of mouse tracheal epithelial cells: selection of a proliferative population.. Am J Physiol Lung Cell Mol Physiol 2002 Dec;283(6):L1315-21.
                pubmed: 12388377doi: 10.1152/ajplung.00169.2002google scholar: lookup
              16. Martin WR, Brown C, Zhang YJ, Wu R. Growth and differentiation of primary tracheal epithelial cells in culture: regulation by extracellular calcium.. J Cell Physiol 1991 Apr;147(1):138-48.
                pubmed: 2037619doi: 10.1002/jcp.1041470118google scholar: lookup
              17. Abraham G, Kottke C, Dhein S, Ungemach FR. Pharmacological and biochemical characterization of the beta-adrenergic signal transduction pathway in different segments of the respiratory tract.. Biochem Pharmacol 2003 Sep 15;66(6):1067-81.
                pubmed: 12963495doi: 10.1016/s0006-2952(03)00460-xgoogle scholar: lookup
              18. Sime A, McKellar Q, Nolan A. Method for the growth of equine airway epithelial cells in culture.. Res Vet Sci 1997 Jan-Feb;62(1):30-3.
                pubmed: 9160421doi: 10.1016/s0034-5288(97)90176-4google scholar: lookup
              19. Kondo M, Finkbeiner WE, Widdicombe JH. Cultures of bovine tracheal epithelium with differentiated ultrastructure and ion transport.. In Vitro Cell Dev Biol 1993 Jan;29A(1):19-24.
                pubmed: 8444742doi: 10.1007/BF02634367google scholar: lookup
              20. Wu R, Zhao YH, Chang MM. Growth and differentiation of conducting airway epithelial cells in culture.. Eur Respir J 1997 Oct;10(10):2398-403.
                pubmed: 9387971doi: 10.1183/09031936.97.10102398google scholar: lookup
              21. Gruenert DC, Finkbeiner WE, Widdicombe JH. Culture and transformation of human airway epithelial cells.. Am J Physiol 1995 Mar;268(3 Pt 1):L347-60.
                pubmed: 7900815doi: 10.1152/ajplung.1995.268.3.L347google scholar: lookup
              22. Fuchs E. Keratins as biochemical markers of epithelial differentiation.. Trends Genet 1988 Oct;4(10):277-81.
                pubmed: 2474874doi: 10.1016/0168-9525(88)90169-2google scholar: lookup
              23. Lechner JF, Haugen A, McClendon IA, Pettis EW. Clonal growth of normal adult human bronchial epithelial cells in a serum-free medium.. In Vitro 1982 Jul;18(7):633-42.
                pubmed: 7141447doi: 10.1007/BF02796396google scholar: lookup
              24. Nijkamp FP. Beta-adrenergic receptors in the lung: an introduction.. Life Sci 1993;52(26):2073-82.
                pubmed: 8389950doi: 10.1016/0024-3205(93)90722-fgoogle scholar: lookup
              25. Niles R, Kim KC, Hyman B, Christensen T, Wasano K, Brody J. Characterization of extended primary and secondary cultures of hamster tracheal epithelial cells.. In Vitro Cell Dev Biol 1988 May;24(5):457-63.
                pubmed: 3372450doi: 10.1007/BF02628498google scholar: lookup

              Citations

              This article has been cited 9 times.
              1. Legere RM, Cohen ND, Poveda C, Bray JM, Barhoumi R, Szule JA, de la Concha-Bermejillo A, Bordin AI, Pollet J. Safe and effective aerosolization of in vitro transcribed mRNA to the respiratory tract epithelium of horses without a transfection agent.. Sci Rep 2021 Jan 11;11(1):371.
                doi: 10.1038/s41598-020-79855-1pubmed: 33432084google scholar: lookup
              2. Abs V, Bonicelli J, Kacza J, Zizzadoro C, Abraham G. Equine bronchial fibroblasts enhance proliferation and differentiation of primary equine bronchial epithelial cells co-cultured under air-liquid interface.. PLoS One 2019;14(11):e0225025.
                doi: 10.1371/journal.pone.0225025pubmed: 31721813google scholar: lookup
              3. Powell JD, Hess BM, Hutchison JR, Straub TM. Construction of an in vitro primary lung co-culture platform derived from New Zealand white rabbits.. In Vitro Cell Dev Biol Anim 2015 May;51(5):433-40.
                doi: 10.1007/s11626-014-9853-zpubmed: 25491427google scholar: lookup
              4. Jing Y, Zhang C, Fu T, Jiang C, Ma K, Zhang D, Hou S, Dai J, Wang H, Zhang X, Kou G, Guo Y. Combination of dextran sulfate and recombinant trypsin on aggregation of Chinese hamster ovary cells.. Cytotechnology 2016 Mar;68(2):241-8.
                doi: 10.1007/s10616-014-9774-4pubmed: 25087075google scholar: lookup
              5. Franke J, Abs V, Zizzadoro C, Abraham G. Comparative study of the effects of fetal bovine serum versus horse serum on growth and differentiation of primary equine bronchial fibroblasts.. BMC Vet Res 2014 May 26;10:119.
                doi: 10.1186/1746-6148-10-119pubmed: 24886635google scholar: lookup
              6. Yang G, Li S, Blackmon S, Ye J, Bradley KC, Cooley J, Smith D, Hanson L, Cardona C, Steinhauer DA, Webby R, Liao M, Wan XF. Mutation tryptophan to leucine at position 222 of haemagglutinin could facilitate H3N2 influenza A virus infection in dogs.. J Gen Virol 2013 Dec;94(Pt 12):2599-2608.
                doi: 10.1099/vir.0.054692-0pubmed: 23994833google scholar: lookup
              7. Shi HC, Lu D, Li HJ, Han S, Zeng YJ. In vitro isolation and cultivation of rabbit tracheal epithelial cells using tissue explant technique.. In Vitro Cell Dev Biol Anim 2013 Apr;49(4):245-9.
                doi: 10.1007/s11626-012-9572-2pubmed: 23494268google scholar: lookup
              8. Abraham G, Zizzadoro C, Kacza J, Ellenberger C, Abs V, Franke J, Schoon HA, Seeger J, Tesfaigzi Y, Ungemach FR. Growth and differentiation of primary and passaged equine bronchial epithelial cells under conventional and air-liquid-interface culture conditions.. BMC Vet Res 2011 Jun 7;7:26.
                doi: 10.1186/1746-6148-7-26pubmed: 21649893google scholar: lookup
              9. Schwab UE, Fulcher ML, Randell SH, Flaminio MJ, Russell DG. Equine bronchial epithelial cells differentiate into ciliated and mucus producing cells in vitro.. In Vitro Cell Dev Biol Anim 2010 Feb;46(2):102-6.
                doi: 10.1007/s11626-009-9258-6pubmed: 19915928google scholar: lookup
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