Barrier Properties of an N/TERT-Based Human Skin Equivalent

Abstract

Human skin equivalents (HSEs) can be considered a valuable tool to study aspects of human skin, including the skin barrier, or to perform chemical or toxicological screenings. HSEs are three-dimensional skin models that are usually established using primary keratinocytes and closely mimic human skin. The use of primary keratinocytes has several drawbacks, including a limited in vitro life span and large donor-donor variation. This makes them less favorable for in vitro toxicity screenings. Usage of an established keratinocyte cell line circumvents these drawbacks and enables the generation of easy-to-generate and reproducible HSEs, which can be used for pharmacological and/or toxicological screenings. For such screenings, a proper barrier function is required. In this study, we investigated the barrier properties of HSEs established with the keratinocyte cell line N/TERT (N-HSEs). N-HSEs showed comparable tissue morphology and expression of several epidermal proteins compared with HSEs established with primary keratinocytes. Our results clearly demonstrate that N-HSEs not only contain several stratum corneum (SC) barrier properties similar to HSEs, including the presence of the long periodicity phase and a comparable SC permeability, but also show some differences in lipid composition. Nonetheless, the similarities in barrier properties makes N/TERT cells a promising alternative for primary keratinocytes to generate HSEs.

Improved epidermal barrier formation in human skin models by chitosan modulated dermal matrices

“…Primary keratinocytes were isolated from the epidermis after treatment with 0.05% (w/v) trypsin (BD Falcon, Breda, The Netherlands) and cultured as described before [15]. Primary fibroblasts were isolated after incubation of the dermis in a 3:1 (w/w) mixture of collagenase (Gibco) and dispase II (Roche) for two hours at 37°C and cultured as described before [15, 29]. All isolated primary cells were tested and found negative for mycoplasma contamination.…”

“…Below a 1 mL cell-free collagen mixture with on top a 3 mL fibroblasts containing mixture (1.2–1.5 x 10 5 fibroblasts in each dermal equivalent) were produced onto filter inserts (Corning Transwell cell culture inserts, membrane diameter 24 mm, pore size 3 μm; Corning Life Sciences, The Netherlands) and allowed to polymerize at 37°C. After polymerization, the dermal equivalents were cultured as described elsewhere [8, 29] with fresh supplementation of 45 μM vitamin C (Sigma).…”

“…Primary keratinocytes (2.5x10 5 /model) in their first passage were seeded onto each dermal equivalent as described before [8, 29]. The FTMs were kept submerged for total of four days.…”

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 …”

“…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. [25,26] Compared to the prevalent "classical" 3D skin models, the development of novel complex next-generation skin models based on induced pluripotent stem cells (iPSCs) and microfluidic technology ("skin-on-chip") [27,28] or using 3D bioprinting techniques [29] will offer promising tools for the study of the microbiota-skin interplay. For example, the use of iPSC-derived cells such as keratinocytes, fibroblasts and melanocytes has already led to the successful generation of a complex 3D skin model.…”