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

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.

YAP1/TAZ-TEAD transcriptional networks maintain skin homeostasis by regulating cell proliferation and limiting KLF4 activity

“…To study the effects of inhibiting TEAD-dependent transcription in keratinocytes we took advantage of immortalized N/TERT2G keratinocytes that show similar epidermal differentiation in 2D culture and 3D organotypic skin models to human primary keratinocytes 24,25 . N/TERT2G keratinocytes transduced with adenoviruses expressing TEADi (ad-TEADi) showed a significant decrease in proliferation (Fig.…”

3D organ models—Revolution in pharmacological research?

“…Although the importance of these studies is undoubted, the next step must be the assessment of the actual value of organ models for in vitro drug testing including the identification of valid readout parameters, definition of their limitations, and rigorous comparison against the relevant in vivo situation. So far, only a handful of studies have performed drug testing in skin models, and of these, only 1-2 drugs were included [56][57][58][59][60], the experimental settings varied significantly between them, and the choices of readout parameters have not been sufficiently justified.…”

Skin microbiota and human 3D skin models

“…To assess the role of specific genes in the context of a 3D skin equivalent, it is also possible to use keratinocyte cell lines where stable overexpression or knockdown/inactivation of genes of interest is possible. In this context, the use of immortalised N/TERT keratinocytes seems to be a valuable approach as N/TERT keratinocytes have similar characteristics to primary keratinocytes, and successful generation of 3D skin models using N/TERT keratinocytes has been reported …”