Biomolecules & Therapeutics  
Establishment of Immortalized Primary Human Foreskin Keratinocytes and Their Application to Toxicity Assessment and Three Dimensional Skin Culture Construction
Moonju Choi1, Minkyung Park1, Suhyon Lee2, Jeong Woo Lee3, Min Chul Cho4, Minsoo Noh5 and Choongho Lee1,*
1College of Pharmacy, Dongguk University, Goyang 10326,
2R&D Institute, Biosolution Co., Ltd., Seoul 01811,
3Department of Urology, Dongguk University Ilsan Hospital, Dongguk University College of Medicine, Goyang 10326,
4Department of Urology, Seoul Metropolitan Government-Seoul National University (SMG-SNU) Boramae Medical Center, Seoul 07061,
5College of Pharmacy, Seoul National University, Seoul 08826, Republic of Korea
E-mail: choongholee@dongguk.edu
Tel: +82-31-961-5214, Fax: +82-31-961-5206
Received: February 28, 2017; Revised: March 7, 2017; Accepted: March 8, 2017; Published online: April 6, 2017.
© The Korean Society of Applied Pharmacology. All rights reserved.

Abstract
In spite of frequent usage of primary human foreskin keratinocytes (HFKs) in the study of skin biology, senescence-induced blockage of in vitro proliferation has been a big hurdle for their effective utilization. In order to overcome this passage limitation, we first isolated ten HFK lines from circumcision patients and successfully immortalized four of them via a retroviral transduction of high-risk human papillomavirus (HPV) E6 and E7 oncogenes. We confirmed expression of a keratinocyte marker protein, keratin 14 and two viral oncoproteins in these immortalized HFKs. We also observed their robust responsiveness to various exogenous stimuli, which was evidenced by increased mRNA expression of epithelial differentiation markers and pro-inflammatory genes in response to three reactive chemicals. In addition, their applicability to cytotoxicity assessment turned out to be comparable to that of HaCaT cells. Finally, we confirmed their differentiation capacity by construction of well-stratified three dimensional skin cultures. These newly established immortalized HFKs will be valuable tools not only for generation of in vitro skin disease models but also for prediction of potential toxicities of various cosmetic chemicals.
Keywords: Human foreskin keratinocyte, Immortalization, Toxicity assessment, Three-dimensional skin culture


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