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.
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.


CONCLUSIONS
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.
Kofler
Kristina
K
Department of Pediatric and Adolescent Surgery, Medical University of Graz, Auenbruggerplatz 34, 8036, Graz, Austria.
Ainoedhofer
Herwig
H
Höllwarth
Michael E
ME
Saxena
Amulya K
AK
D000818:Animals; D002470:Cell Survival; D004195:Disease Models, Animal; D004847:Epithelial Cells / Q000648:ultrastructure*; D004935:Esophageal Diseases / Q000473:pathology / Q000628:therapy*; D004947:Esophagus / Q000648:ultrastructure*; D005434:Flow Cytometry / Q000379:methods*; D007150:Immunohistochemistry; D007413:Intestinal Mucosa / Q000648:ultrastructure; D007633:Keratins / Q000276:immunology*; D008855:Microscopy, Electron, Scanning; D012756:Sheep; D023822:Tissue Engineering / Q000379:methods*; D054457:Tissue Scaffolds
D007633:Keratins