Novel three-dimensional live skin-like in vitro composite for bioluminescence reporter gene assay.

Autor: Tomita T; Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan., Nakajima Y; Health and Medical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu, Japan., Ohmiya Y; Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Ikeda, Japan.; Osaka Institute of Technology (OIT), Omiya, Japan., Miyazaki K; Department of Life Science and Biotechnology, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.
Jazyk: angličtina
Zdroj: The FEBS journal [FEBS J] 2024 Oct; Vol. 291 (20), pp. 4619-4632. Date of Electronic Publication: 2024 Aug 15.
DOI: 10.1111/febs.17246
Abstrakt: We genetically manipulated HaCaT cells, a spontaneously immortalised normal keratinocyte cell line, to stably express two different coloured luciferase reporter genes, driven by interleukin 8 (IL-8) and ubiquitin-C (UBC) promoters, respectively. Subsequently, we generated a three-dimensional (3D) skin-like in vitro composite (SLIC) utilising these cells, with the objective of monitoring bioluminescence emitted from the SLIC. This SLIC was generated on non-woven silica fibre membranes in differentiation medium. Immunohistochemical analyses of skin differentiation markers in the SLIC revealed the expression of keratins 2 and 10, filaggrin, and involucrin, indicating mature skin characteristics. This engineered SLIC was employed for real-time bioluminescence monitoring, allowing the assessment of time- and dose-dependent responses to UV stress, as well as to hydrophilic and hydrophobic chemical loads. Notably, evaluation of responses to hydrophobic substances has been challenging with conventional 2D cell culture methods, suggesting the need for a new approach, which this technology could address. Our observations suggest that engineered SLIC with constitutively expressing reporters driven by selected promoters which are tailored to specific objectives, significantly facilitates assays exploring the physiological functions of skin cells based on genetic response mechanisms. It also highlights new avenues for evaluating the physiological impacts of various compounds designed for topical application to human skin.
(© 2024 The Author(s). The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)
Databáze: MEDLINE
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