, Volume 10, Issue 3, pp 144–148 | Cite as

Isolation, identification and culture of myenteric plexus cells from ovine esophagus

  • Tanja Macheiner
  • Richard Ackbar
  • Amulya K. SaxenaEmail author
Original Article



The myenteric plexus of the esophagus consists of two major cell types, neurons and enteric glial cells, and functions to coordinate peristaltic waves. The aim of this study was to develop a protocol for the isolation of myenteric plexus and dissociation and culture of myenteric plexus cells.


The myenteric plexus was isolated from the ovine esophagus through treatment with collagenase, followed by dissociation of cells with trypsin/EDTA. Myenteric plexus cells were cultured in vitro and the different cell components were identified by immunohistochemical staining.


Isolated myenteric plexi expressed enteric glial cell markers S-100 and GFAP and enteric neuronal cell marker PGP 9.5. Furthermore, c-kit positive cells were also detected, which may represent the interstitial cells of Cajal. Despite the successful isolation of a wide range of myenteric plexus cells, dissociation of cells was poor and requires further optimization.


This study reports on the isolation and dissociation of the principle cells of the myenteric plexus from ovine esophagus. As the ovine model is a clinically relevant large animal model for esophageal disease, the isolation of ovine myenteric plexus cells is of significant importance for the understanding of pathologies of the enteric nervous system and application in gastrointestinal tissue engineering.


Enteric nervous system GFAP PGP 9.5 Tissue engineering 



This research is funded by the European Union within the 6th Framework Program (EuroSTEC; LSHC-CT-2006-037409).

Conflict of interest

The authors declare that they have no conflict of interest.


  1. 1.
    Ward SM, Burns AJ, Torihashi S, Sanders KM. Mutation of the proto-oncogene c-kit blocks development of interstitial cells and electrical rhythmicity in murine intestine. J Physiol. 1994;480:91–7.PubMedGoogle Scholar
  2. 2.
    Hitchcock RJ, Pemble MJ, Bishop AE, Spitz L, Polak JM. Quantitative study of the development and maturation of human oesophageal innervation. J Anat. 1992;180:175–83.PubMedGoogle Scholar
  3. 3.
    Li JC, Mi KH, Zhou JL, Busch L, Kuhnel W. The development of colon innervation in trisomy 16 mice and Hirschsprung’s disease. World J Gastroenterol. 2001;7:16–21.PubMedGoogle Scholar
  4. 4.
    Gabella G. Ultrastructure of the nerve plexuses of the mammalian intestine: the enteric glial cells. Neuroscience. 1981;6:425–36.PubMedCrossRefGoogle Scholar
  5. 5.
    Jessen KR, Mirsky R. Glial cells in the enteric nervous system contain glial fibrillary acidic protein. Nature. 1980;286:736–7.PubMedCrossRefGoogle Scholar
  6. 6.
    Burns AJ, Herbert TM, Ward SM, Sanders KM. Interstitial cells of Cajal in the guinea-pig gastrointestinal tract as revealed by c-Kit immunohistochemistry. Cell Tissue Res. 1997;290:11–20.PubMedCrossRefGoogle Scholar
  7. 7.
    Schäfer KH, Saffrey MJ, Burnstock G, Mestres-Ventura P. A new method for the isolation of myenteric plexus from the newborn rat gastrointestinal tract. Brain Res Brain Res Protoc. 1997;1:109–13.PubMedCrossRefGoogle Scholar
  8. 8.
    Masumoto Kouji, Suita Sachiyo, Nada Osami, Taguchi Tomo, Guo Rishu. Abnormalities of enteric neurons, intestinal pacemaker cells, and smooth muscle in human intestinal atresia. J Pediatr Surg. 1999;34:1463–8.PubMedCrossRefGoogle Scholar
  9. 9.
    Saxena AK, Ainoedhofer H, Höllwarth ME. Esophagus tissue engineering: in vitro generation of esophageal epithelial cell sheets and viability on scaffold. J Pediatr Surg. 2009;44:896–901.PubMedCrossRefGoogle Scholar
  10. 10.
    Saxena AK. Tissue engineering and regenerative medicine research perspectives for pediatric surgery. Pediatr Surg Int. 2010;26:557–73.PubMedCrossRefGoogle Scholar
  11. 11.
    Saxena AK. Congenital anomalies of soft tissues: birth defects depending on tissue engineering solutions and present advances in regenerative medicine. Tissue Eng Part B Rev. 2010;16:455–66.PubMedCrossRefGoogle Scholar
  12. 12.
    De Giorgio R, Giancola F, Boschetti E, Abdo H, Lardeux B, Neunlist M. Enteric glia and neuroprotection: basic and clinical aspects. Am J Physiol Gastrointest Liver Physiol. 2012. doi: 10.1152/ajpgi.00096.2012.PubMedGoogle Scholar
  13. 13.
    Saxena AK, Kofler K, Ainödhofer H, Höllwarth ME. Esophagus tissue engineering: hybrid approach with esophageal epithelium and unidirectional smooth muscle tissue component generation in vitro. J Gastrointest Surg. 2009;13:1037–43.PubMedCrossRefGoogle Scholar
  14. 14.
    Soltysiak P, Höllwarth ME, Saxena AK. Comparison of suture techniques in the formation of collagen scaffold tubes for composite tubular organ tissue engineering. Biomed Mater Eng. 2010;20:1–11.PubMedGoogle Scholar
  15. 15.
    Saxena AK, Ainoedhofer H, Höllwarth ME. Culture of ovine epithelial cells and in vitro esophagus tissue engineering. Tissue Eng Part C Methods. 2010;16:109–14.PubMedCrossRefGoogle Scholar
  16. 16.
    Soltysiak P, Saxena AK. Micro-computed tomography for implantation site imaging during in situ oesophagus tissue engineering in a live small animal model. J Tissue Eng Regen Med. 2009;3:573–6.PubMedCrossRefGoogle Scholar
  17. 17.
    Kofler K, Ainoedhofer H, Höllwarth ME, Saxena AK. Fluorescence-activated cell sorting of PCK-26 antigen-positive cells enables selection of ovine esophageal epithelial cells with improved viability on scaffolds for esophagus tissue engineering. Pediatr Surg Int. 2010;26:97–104.PubMedCrossRefGoogle Scholar
  18. 18.
    Ackbar R, Ainoedhofer H, Gugatschka M, Saxena AK. Decellularized ovine esophageal mucosa for esophageal tissue engineering. Technol Health Care. 2012;20:215–23.PubMedGoogle Scholar
  19. 19.
    Malvasio V, Ainoedhofer H, Ackbar R, Hoellwarth ME, Saxena AK. Effects of sodium hydroxide exposure on esophageal epithelial cells in an in vitro ovine model: implications for esophagus tissue engineering. J Pediatr Surg. 2012;47:874–80.PubMedCrossRefGoogle Scholar
  20. 20.
    Saxena AK, Baumgart H, Komann C, Ainoedhofer H, Soltysiak P, Kofler K, Höllwarth ME. Esophagus tissue engineering: in situ generation of rudimentary tubular vascularized esophageal conduit using the ovine model. J Pediatr Surg. 2010;45:859–64.PubMedCrossRefGoogle Scholar
  21. 21.
    Kofler K, Leitinger G, Kristler M, Saxena AK. Smooth muscle tissue engineering for hybrid tubular organs: scanning electron microscopic investigations of cell interactions with collagen scaffolds. J Tissue Eng Regen Med. 2009;3:321–4.PubMedCrossRefGoogle Scholar
  22. 22.
    Kofler K, Ainoedhofer H, Tausendschön J, Höllwarth ME, Saxena AK. Esophageal smooth muscle cells dedifferentiate with loss of a-smooth muscle actin expression after 8 weeks of explant expansion in vitro culture: implications on esophagus tissue engineering. Eur Surg. 2011;43:168–73.CrossRefGoogle Scholar
  23. 23.
    Ackbar R, Malvasio V, Holzer P, Saxena AK. In vitro effect of bethanechol and suberyldicholine on regions of guinea pig esophagus. J Surg Res. 2012;174:56–61.PubMedCrossRefGoogle Scholar
  24. 24.
    Feng H, Wei Y, Huajun J, Chongyang F, Ming L, Weiguo Z, Decheng L. A simple and efficient method that uses low concentration fetal bovine serum to culture and purify Schwann cells. Afr J Microbiol Res. 2011;5:2367–73.Google Scholar
  25. 25.
    Rauch U, Hänsgen A, Hagl C, Holland-Cunz S, Schäfer KH. Isolation and cultivation of neuronal precursor cells from the developing human enteric nervous system as a tool for cell therapy in dysganglionosis. Int J Colorectal Dis. 2006;21:554–9.PubMedCrossRefGoogle Scholar

Copyright information

© The Japan Esophageal Society and Springer Japan 2013

Authors and Affiliations

  • Tanja Macheiner
    • 1
    • 2
  • Richard Ackbar
    • 1
  • Amulya K. Saxena
    • 1
    Email author
  1. 1.Experimental Fetal Surgery and Tissue Engineering Unit, Department of Pediatric and Adolescent SurgeryMedical University of GrazGrazAustria
  2. 2.Biobank GrazMedical University of GrazGrazAustria

Personalised recommendations