Establishment of Immortalized Primary Human Foreskin Keratinocytes and Their Application to Toxicity Assessment and Three Dimensional Skin Culture Construction

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.

Allele-Specific Small Interfering RNA Corrects Aberrant Cellular Phenotype in Keratitis-Ichthyosis-Deafness Syndrome Keratinocytes

“…from patient skin biopsy would have been ideal for this study, these cells have a limited lifespan that restricts us from performing multiple experiments. To bypass the restrictions, we used immortalized patient KCs because of their indefinite lifespan and capability of proliferation and differentiation, features of primary KCs (Choi et al, 2017). This was further confirmed in our in vivo skin graft experiments, where regenerated skin grafts from immortalized KID-KCs recapitulated the epidermal architecture of the skin of patients with KID syndrome.…”

Optimization of human papillomavirus-based pseudovirus techniques for efficient gene transfer

“…2 E). Primary cell lines such as HFKs only replicate for a limited number of passages, and proliferation slows as passages increase 25 . The number of passages undergone by HFKs at the time of infection influences infection efficiency.…”

In vitro assessment of cytotoxic activities of Lachesis muta muta snake venom

“…More than 95% of epidermal tissue is composed of keratinocytes. These cells have been used for the study of skin related diseases, toxicity assessments [ 18 ] and an in vitro model for cutaneous loxoscelism (dermonecrosis) caused by spider venoms [ 25 , 26 ]. Our data show that L .…”

“…muta cytotoxicity and the involvement of cell death mechanisms using normal human keratinocytes. These cells have an essential role in skin biology and this tissue is commonly used to evaluate the toxicity of various agents [ 18 ], in addition of being remarkably affected in snakebite victims. Therefore, the determination of events that underlie cellular damage and tissue destruction will help to identify pathways perturbed by crude L .…”