Feeder layer- and animal product-free culture of neonatal foreskin keratinocytes: improved performance, usability, quali
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Feeder layer- and animal product-free culture of neonatal foreskin keratinocytes: improved performance, usability, quality and safety Peter De Corte • Gunther Verween • Gilbert Verbeken • Thomas Rose • Serge Jennes • Arlette De Coninck • Diane Roseeuw • Alain Vanderkelen Eric Kets • David Haddow • Jean-Paul Pirnay
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Received: 17 August 2010 / Accepted: 23 December 2010 / Published online: 11 March 2011 The Author(s) 2011. This article is published with open access at Springerlink.com
Abstract Since 1987, keratinocytes have been cultured at the Queen Astrid Military Hospital. These keratinocytes have been used routinely as auto and allografts on more than 1,000 patients, primarily to accelerate the healing of burns and chronic wounds. Initially the method of Rheinwald and Green was used to prepare cultured epithelial autografts, starting from skin samples from burn patients and using animalderived feeder layers and media containing animal-
P. De Corte G. Verween G. Verbeken T. Rose J.-P. Pirnay (&) Skin- and Keratinocyte Bank, Laboratory for Molecular and Cellular Technology, Burn Wound Centre, Queen Astrid Military Hospital, Bruynstraat 1, 1120 Brussels, Belgium e-mail: [email protected] S. Jennes Burn Wound Centre, Queen Astrid Military Hospital, 1120 Brussels, Belgium A. De Coninck D. Roseeuw Department of Dermatology, Universitair Ziekenhuis Brussel—Vrije Universiteit Brussel, 1090 Brussels, Belgium A. Vanderkelen E. Kets Queen Astrid Military Hospital, 1120 Brussels, Belgium
derived products. More recently we systematically optimised our production system to accommodate scientific advances and legal changes. An important step was the removal of the mouse fibroblast feeder layer from the cell culture system. Thereafter we introduced neonatal foreskin keratinocytes (NFK) as source of cultured epithelial allografts, which significantly increased the consistency and the reliability of our cell production. NFK master and working cell banks were established, which were extensively screened and characterised. An ISO 9001 certified Quality Management System (QMS) governs all aspects of testing, validation and traceability. Finally, as far as possible, animal components were systematically removed from the cell culture environment. Today, quality controlled allograft production batches are routine and, due to efficient cryopreservation, stocks are created for off-theshelf use. These optimisations have significantly increased the performance, usability, quality and safety of our allografts. This paper describes, in detail, our current cryopreserved allograft production process. Keywords Cell and tissue banking Cell and tissue engineering Allograft Keratinocytes Burns Ulcers
D. Haddow Altrika Ltd, 217 Portobello, Sheffield S1 4DP, UK
Introduction
D. Haddow University of Sheffield, Sheffield, UK
In the late 1970s, Rheinwald and Green described a method for serial cultivation of human epidermal
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keratinocytes on a feeder layer of mouse fibroblasts in a mitogen-rich medium cont
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