Esophagus tissue engineering: in vitro generation of esophageal epithelial cell sheets and viability on scaffold Amulya K. Saxena ⁎ , Herwig Ainoedhofer, Michael E. Höllwarth Department of Pediatric and Adolescent Surgery, Medical University of Graz, A-8036 Graz, Austria Received 4 January 2009; accepted 15 January 2009 Key words: Esophagus; Tissue engineering; Squamous epithelium; Collagen; Culture Abstract Purpose: Management of long gap esophageal atresia poses challenges. The surgical techniques for esophageal replacement are associated with complications and high morbidity. The aim of this study was to develop protocols to obtain single layer sheets of esophageal epithelial cells (EECs) and to investigate their survival on collagen scaffolds. Methods: Esophageal epithelial cells were sourced from adult Sprague-Dawley rats. Briefly, the esophagus was treated with dispase to separate the epithelial layer and further trypsined to obtained EEC. The esophageal epithelial cells were cultured in vitro and seeded on to new generation of 3- dimensional collagen scaffolds. Results: Esophageal epithelial cells organized after 48 hours in culture and formed clusters after 72 to 96 hours. Organization of the EEC was completed after 7 days in culture and characteristic sheets of EEC with the histologic morphology of mature esophageal epithelium were obtained after 14 days of culture. Immunohistochemistry demonstrated pure EEC culture using cytokeratin (CK-14) markers. The esophageal epithelial cells transferred on to collagen polymers demonstrated excellent viability after 8 weeks of in vitro culture. Conclusion: Successful protocols for EEC isolation and proliferation have been established. The engineering of sheets of EEC and the viability of EEC on collagen scaffolds for 8 weeks in vitro, which are prerequisites for esophagus tissue engineering, was demonstrated. © 2009 Elsevier Inc. All rights reserved. Esophageal atresia is relatively a common congenital malformation occurring in 1:3000 to 5000 births [1]. Although the surgical repair to bridge the gap by primary anastomosis is successful in most cases, reconstruction of long gap esophageal atresia still poses challenges in surgical management. In patients with long gap atresia, delayed repair (after tacking the end of the esophagus to the prevertebral fascia or the Foker technique), myotomy, and esophageal replacement with strategies that include gastric, jejunum, or colon transposition are the possible options [2-4]. Unfortunately, these strategies are associated with a high rate of short and long-term complications that include leakage, stricture, elongation, and malnutrition because of shortening of the gastrointestinal tract [5-7]. Presented at the 40th Annual CAPS Meeting, August 21-24, 2008, Toronto, Ontario, Canada. ⁎ Corresponding author. Tel.: +43 316 3853762; fax: +43 316 3852775. E-mail address: amulya.saxena@meduni-graz.at (A.K. Saxena). www.elsevier.com/locate/jpedsurg 0022-3468/$ – see front matter © 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jpedsurg.2009.01.019 Journal of Pediatric Surgery (2009) 44, 896–901