Characterization of cultured epithelial cells using a novel technique not requiring enzymatic digestion for subculturing
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ORIGINAL PAPER
Characterization of cultured epithelial cells using a novel technique not requiring enzymatic digestion for subculturing Antonio Peramo • Stephen E. Feinberg Cynthia L. Marcelo
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Received: 28 August 2012 / Accepted: 6 October 2012 / Published online: 13 November 2012 Springer Science+Business Media Dordrecht 2012
Abstract Our laboratory had developed a methodology to expand epithelial cells in culture by growing keratinocyte monolayers, under large volumes of medium that produces large numbers of keratinocytes that leave the monolayer and move into suspension. The cells have been defined as epithelial Pop Up Keratinocytes or ePUKs cells and appear to be highly suitable for clinical applications. In this publication we extend the characterization of the cells with a detailed analysis of the capabilities of the monolayer of a single culture flask to produce, over time, ePUK cells. The cells were characterized using standard epithelial markers for proliferation and differentiation. Analysis of morphology of the monolayer formed and total number of cells produced is presented for a variety of human epithelial cell strains. These keratinocytes provide an additional controlled human cell system for investigation of the mechanisms regulating epithelia cell growth and differentiation and since they Electronic supplementary material The online version of this article (doi:10.1007/s10561-012-9343-z) contains supplementary material, which is available to authorized users. A. Peramo (&) C. L. Marcelo Department of Surgery, University of Michigan, Ann Arbor, MI 48109, USA e-mail: [email protected] Present Address: A. Peramo S. E. Feinberg Department of Oral and Maxillofacial Surgery, University of Michigan, Ann Arbor, MI 48109, USA
are produced in large numbers, they are highly suitable for use in epithelial cell banking. Keywords Keratinocyte ePUK Epithelia cell Cell culture Oral mucosa
Introduction Keratinocytes derived from epidermis, oral mucosa, and urothelium are used in construction of cell based tissue engineering and regenerative medicine applications. Several methods (Oliveira and Hodges 2005; Bavister et al. 2005; Mignone et al. 2010; Lei and Andreadis 2008; Hodgkinson et al. 2010) are being developed to obtain cells with functional plasticity to construct artificial tissue for transplantation, to ‘correct’ specific systemic diseases and as a source for cell-mediated wound healing therapies. It would be advantageous to develop a methodology to grow adult somatic cells with maximum plasticity, from human tissue, to circumvent many of the well-known and currently debated ethical and scientific problems associated with the use of embryonic derived stem cells or induced pluripotent stem cells (Lister et al. 2011). Previously, we have shown that human epithelia keratinocytes in primary culture can be induced by tissue culture manipulation to produce, without the use of enzymes for passaging, large numbers of small cells in a combined suspension/monolayer culture. The
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