Cellular and Molecular Biotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan.
Health and Medical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu, Japan.
FEBS J. 2024 Oct;291(20):4619-4632. doi: 10.1111/febs.17246. Epub 2024 Aug 15.
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.
我们通过基因操作，使 HaCaT 细胞（一种自发永生化的正常角质形成细胞系）稳定表达两种不同颜色的荧光素酶报告基因，分别由白细胞介素 8（IL-8）和泛素 C（UBC）启动子驱动。随后，我们利用这些细胞生成了一种三维（3D）皮肤样体外复合（SLIC），旨在监测 SLIC 发出的生物发光。该 SLIC 是在分化培养基中在无纺硅纤维膜上生成的。SLIC 中皮肤分化标志物的免疫组织化学分析显示角蛋白 2 和 10、丝聚合蛋白和兜甲蛋白的表达，表明具有成熟皮肤特征。该工程 SLIC 可用于实时生物发光监测，可评估对 UV 应激、亲水性和疏水性化学负荷的时间和剂量依赖性反应。值得注意的是，用传统的 2D 细胞培养方法评估对疏水性物质的反应具有挑战性，这表明需要一种新的方法，而这项技术可以解决这个问题。我们的观察表明，具有由特定启动子驱动的组成型表达报告基因的工程 SLIC，可显著促进基于遗传反应机制探索皮肤细胞生理功能的测定。它还为评估各种设计用于局部应用于人体皮肤的化合物的生理影响提供了新的途径。
