Abstract
Objective
For esophagus tissue engineering, isolation and proliferation of esophageal epithelial cells (EEC) is a pre-requisite for scaffold seeding to create constructs. The aim of this study was to sort EEC expressing cytokeratin markers and their proliferative subpopulations; also, to investigate the viability of differentiated EEC subpopulations on collagen scaffolds.
Methods
Ovine esophageal epithelial cells (OEECs) from sheep esophagus were analyzed using flow cytometry for pan cytokeratin (PCK-26) and proliferation cell nuclear antigen (PCNA). Using fluorescent-activated cell sorting, OEEC were separated and analyzed for PCNA expression. The OEEC subpopulations were seeded on collagen scaffolds for a week in vitro culture.
Results
Proliferation cell nuclear antigen was expressed in >45% of OEEC isolated. In flow cytometry, 30% OEEC were PCK-26 positive which exhibited a high-proliferative capacity of 80%. PCK-26-negative OECC exhibited a low-proliferative capability of 13%. Scanning electron microscopy demonstrated organized attachment and uniform scaffold coverage in PCK-26-positive cells.
Conclusion
Ovine esophageal epithelial cells can be divided into PCK-26-positive and negative subpopulations. PCK-26-positive OEEC constitute one-third of the isolated cells with high-proliferative capability. Seeding of PCK-26-positive OEEC on collagen scaffolds leads to uniform distribution of cells in vitro. In esophagus, tissue engineering PCK-26-positive OEEC subpopulation is important for optimal construct generation.
Similar content being viewed by others
References
Spitz L (2006) Esophageal atresia: lessons I have learned in a 40-year experience. J Pediatr Surg 41:1635–1640
Arul GS, Parikh D (2008) Oesophageal replacement in children. Ann R Coll Surg Engl 90:7–12
Deurloo JA, Ekkelkamp S, Hartman EE et al (2005) Quality of life in adult survivors of correction of esophageal atresia. Arch Surg 140:976–980
Saxena AK (2005) Tissue engineering: present concepts and strategies. J Indian Assoc Pediatr Surg 10:10–15
Beckstead BL, Pan S, Bhrany AD et al (2005) Esophageal epithelial cell interaction with synthetic and natural scaffolds for tissue engineering. Biomaterials 26:6217–6228
Zhu Y, Leong MF, Ong WF et al (2007) Esophageal epithelium regeneration on fibronectin grafted poly(l-lactide-co-caprolactone) (PLLC) nanofiber scaffold. Biomaterials 28:861–868
Saxena AK, Ainoedhofer H, Höllwarth ME (2009) Esophagus tissue engineering: in vitro generation of esophageal epithelial sheets and viability on scaffold. J Pediatr Surg 44:896–901
Squier CA, Kremer MJ (2001) Biology of oral mucosa and esophagus. J Natl Cancer Inst Monogr 29:7–15
Frappier BL (2006) Epithelium. In: Eurell JA, Frappier BL (eds) Dellmann’s textbook of veterinary histology, 6th edn. Blackwell, Ames, pp 17–30
Steinert PM (2001) Keratins: dynamic, flexible structural proteins of epithelial cells. Curr Probl Dermatol 54:193–198
Pekny M, Lane EB (2007) Intermediate filaments and stress. Exp Cell Res 313:2244–2254
Moll R, Franke WW, Schiller DL et al (1982) The catalog of human cytokeratins: patterns of expression in normal epithelia, tumors and cultured cells. Cell 31:11–24
Saxena AK, Ainoedhofer H, Hollwarth ME (2009) Culture of ovine esophageal epithelial cells and in vitro esophagus tissue engineering. Tissue Eng Part C Methods. doi:10.1089/ten.tec.2009.0145
Lane EB, Alexander CM (1990) Use of keratin antibodies in tumor diagnosis. Semin Cancer Biol 1:165–179
Kuberka M, von Heimburg D, Schoof H et al (2002) Magnification of the pore size in biodegradable collagen sponges. Int J Artif Organs 25:67–73
Saxena AK, Kofler K, Ainoedhofer H et al (2009) Esophagus tissue engineering: hybrid approach with esophageal epithelium and unidirectional smooth muscle tissue component generation in vitro. J Gastrointest Surg 13:1037–1043
Nakase Y, Nakamura T, Kin S et al (2008) Intrathoracic esophageal replacement by in situ tissue-engineered esophagus. J Thorac Cardiovasc Surg 136:850–859
Doede T, Bondartschuk M, Joerck C et al (2009) Unsuccessful alloplastic esophageal replacement with porcine small intestinal submucosa. Artif Organs 33:328–333
Takimoto Y, Nakamura T, Yamamoto Y et al (1998) The experimental replacement of a cervical esophageal segment with an artificial prosthesis with the use of collagen matrix and a silicone stent. J Thorac Cardiovasc Surg 116:98–106
Landberg G, Tan EM, Roos G (1990) Flow cytometric multiparameter analysis of proliferating cell nuclear antigen/cyclin and Ki-67 antigen: a new view of the cell cycle. Exp Cell Res 187:111–118
Nowak JA, Fuchs E (2009) Isolation and culture of epithelial stem cells. Methods Mol Biol 482:215–232
Seery JP, Watt FM (2000) Asymmetric stem-cell divisions define the architecture of human oesophageal epithelium. Curr Biol 10:1447–1450
Leong MF, Chian KS, Mhaisalkar PS et al (2009) Effect of electrospun poly(d, l-lactide) fibrous scaffold with nanoporous surface on attachment of porcine esophageal epithelial cells and protein adsorption. J Biomed Mater Res A 89:1040–1048
Kidambi S, Udpa N, Schroeder SA et al (2007) Cell adhesion on polyelectrolyte multilayer coated polydimethylsiloxane surfaces with varying topographies. Tissue Eng 13:2105–2117
Discher DE, Mooney DJ, Zandstra PW (2009) Growth factors, matrices, and forces combine and control stem cells. Science 324:1673–1677
Acknowledgments
This research is funded by the European Union within the 6th Framework Program (EuroSTEC; LSHC-CT-2006-037409). We thank Prof. Wout Feitz (Radboud University Medical Centre, Nijmegen, The Netherlands), Mrs. Anna Kuess (Department of Pediatric Surgery, Medical University of Graz, Austria), Dr. Beate Rinner and Dr. Gerd Leitinger (Center for Medical Research, Medical University of Graz, Austria) for the valuable contributions toward this study.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Kofler, K., Ainoedhofer, H., Höllwarth, M.E. et al. 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 26, 97–104 (2010). https://doi.org/10.1007/s00383-009-2512-x
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00383-009-2512-x