Immortalized N/TERT keratinocytes as an alternative cell source in 3D human epidermal models

Jos P H Smits,Patrick L J M Zeeuwen,Ivonne M J J Van Vlijmen-Willems,Joost Schalkwijk,Hanna Niehues,Ellen H Van Den Bogaard,Gijs Rikken,Guillaume W H J F Van De Zande

doi:10.1038/s41598-017-12041-y

Abstract

The strong societal urge to reduce the use of experimental animals, and the biological differences between rodent and human skin, have led to the development of alternative models for healthy and diseased human skin. However, the limited availability of primary keratinocytes to generate such models hampers large-scale implementation of skin models in biomedical, toxicological, and pharmaceutical research. Immortalized cell lines may overcome these issues, however, few immortalized human keratinocyte cell lines are available and most do not form a fully stratified epithelium. In this study we compared two immortalized keratinocyte cell lines (N/TERT1, N/TERT2G) to human primary keratinocytes based on epidermal differentiation, response to inflammatory mediators, and the development of normal and inflammatory human epidermal equivalents (HEEs). Stratum corneum permeability, epidermal morphology, and expression of epidermal differentiation and host defence genes and proteins in N/TERT-HEE cultures was similar to that of primary human keratinocytes. We successfully generated N/TERT-HEEs with psoriasis or atopic dermatitis features and validated these models for drug-screening purposes. We conclude that the N/TERT keratinocyte cell lines are useful substitutes for primary human keratinocytes thereby providing a biologically relevant, unlimited cell source for in vitro studies on epidermal biology, inflammatory skin disease pathogenesis and therapeutics.

Highlights

In vitro cultured 3D human skin equivalents (HSEs) and human epidermal equivalents (HEEs) are nowadays often used for studies in the fields of cell biology, tissue engineering, dermatology, and toxicology
To assess potential contamination with other cells, short tandem repeat (STR) analysis was performed by quantitative fluorescence (QF)-PCR using 21 specific targets on chromosome 13, 18, 21, and both sex chromosomes
Using immunohistochemistry we studied the expression of epidermal differentiation protein FLG, the expression of the proliferation marker Ki67, and the expression of anti-microbial peptides human beta defensin 2, and skin-derived antileukoprotease (SKALP)

Summary

Introduction

In vitro cultured 3D human skin equivalents (HSEs) and human epidermal equivalents (HEEs) ( known as organotypic skin models) are nowadays often used for studies in the fields of cell biology, tissue engineering, dermatology, and toxicology. These 3D skin models are preferred over the conventional monolayer cultures as they faithfully mimic the in vivo epidermis and skin barrier function and thereby serve as alternatives for experimental animal testing[1]. The HaCaT cell line, a spontaneously immortalized human keratinocyte cell line, is widely used in keratinocyte monolayer culture models and described for the development of organotypic skin models[2]. Wound regeneration and eczematous organotypic models with HaCaT cells have been reported[6,7,8,9,10], epidermal stratification is abnormal, aberrant epidermal differentiation protein expression is observed and a stratum corneum is lacking in these HaCaT-derived 3D skin models

Methods

Results

Conclusion