title: Real-time detection of stress in 3D tissue-engineered constructs using NF-kappaB activation in transiently transfected human dermal fibroblast cells.
abstract: The main objective of this study was to develop a nondestructive reporter system for assessing the response of human cells contained within a three-dimensional (3D) tissue-engineered construct to exogenous stress. Dermal fibroblasts were transiently transfected with a reporter construct linked to nuclear factor kappaB (NF-kappaB) activation which led to expression of a nonstable form of enhanced green fluorescent protein (d2EGFP) after stimulation. This led to a temporary production of fluorescence, which could be readily detected but was not intrinsically toxic, as cells were able to metabolize the initial cycle of d2EGFP produced. This permitted the model to be used for restimulation post recovery. To investigate the performance and predictive ability of this method for assessing cellular response to stress in 3D, we used a range of compounds known to have pro-inflammatory or oxidative properties. Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1-beta (IL-1beta) were selected for having a direct cytokine action; lipopolysaccharide (LPS) was selected for modeling bacterial-mediated inflammation; and hydrogen peroxide was selected as a crude method for delivering an oxidative stress. Transfected cells were stimulated with the above compounds in 3D and the synthesis of d2EGFP was detected as a measure of NF-kappaB activation. The resultant fluorescence was scored using a series of photomicrographs taken by epifluorescence microscopy. All agents activated NF-kappaB when cells were grown in 3D scaffolds but did not cause any significant reduction in cell viability as measured by a standard MTT-ESTA viability test. Parallel NF-kappaB activation and MTT measurements was also conducted in two-dimension (2D) and confirmed findings in 3D. The 3D model described using a fluorescent reporter gene is a highly sensitive and reliable method for detecting cellular stress and represents a key step in developing tissue engineering models with the potential for screening pharmaceutical and cosmetic compounds, as an alternative to existing in vitro and in vivo methods.
authors: Department of Engineering Materials, Kroto Research Institute, University of Sheffield, and Northern General Hospital, Sheffield, United Kingdom.
authors: Irene
authors: I
authors: Cantón
authors: Umran
authors: U
authors: Sarwar
authors: E Helen
authors: EH
authors: Kemp
authors: Anthony J
authors: AJ
authors: Ryan
authors: Sheila
authors: S
authors: MacNeil
authors: John W
authors: JW
authors: Haycock
mesh_terms: D018929:Cell Culture Techniques; D002478:Cells, Cultured; D020405:Dermis / Q000166:cytology / Q000378:metabolism*; D004353:Drug Evaluation, Preclinical; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007249:Inflammation / Q000235:genetics / Q000378:metabolism; D053583:Interleukin-1beta / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects; D023822:Tissue Engineering; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
chemical_list: D053583:Interleukin-1beta; D008070:Lipopolysaccharides; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins