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
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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.
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.
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.
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.
KeywordsEsophageal epithelial cells Cytokeratin Proliferation Sorting Tissue engineering
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.
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