Development of whole cell-based biosensor in 3D environment for nanoparticles cytotoxicity detection

The emergence of human-based models is incontestably required for the study of complex physiological pathways and validation of reliable in vitro methods as alternative for in vivo studies in experimental animals for toxicity assessment. With this objective, three dimensional environments for human cells were developed and tested using three different types of hydrogels including transglutaminase-crosslinked gelatin, collagen type I and growth-factor depleted Matrigel. Cells grown in Matrigel exhibited the greatest cell proliferation and spheroid diameter. Moreover, urea and albumin analysis indicated that the created system allows the immortalized liver cell line (HepG2) to re-establish normal hepatocyte-like properties which are not observed under the conditions of conventional cell cultures. This study presents a reproducible technology for production of complex-shaped liver multicellular spheroids as a system which improves the predictive value of cell-based assays for safety and risk assessment. The time- and dose-dependent toxicity of nanoparticles demonstrates higher cytotoxic effects when HepG2 cells grown as monolayer than embedded in hydrogels. The experimental setup provided evidence that cell environment has significant influence on cell sensitivity and that liver spheroids are useful and novel tools to examine NP dosing effect even at the level of in vitro studies. Moreover, the created cell-based biosensor responds optimally to bioactive analytes, has a fast response time, offers label-free experimentation and simple procedures. Besides, the developed NF-κB_HepG2 cells enable detect cytotoxicity of variety nanoparticles with different chemical composition, size, tendency of agglomeration or cytotoxic potential and other stimuli which activate NF-κB signaling (e.g. heavy metals), in very early stage, before cell death, in short time even after 2 h time exposure as opposed to widely used commercially available cell-based assays like MTT, XTT Alamar Blue or LDH. Successful modification of HepG2 cells resulted in generation of novel NF-κB_HepG2 sensor cells with extracellular reporter protein (human secreted alkaline phosphatase) and its application in cytotoxicity of nanomaterials detection has not been investigated before. The developed NF-κB_hepG2 cells may also provide a useful tool to study distinct molecular mechanisms of inflammation and cellular stress using low exposure concentrations of cell irritants. The presented sensor based on human cells in three dimensional milieu provides a novel application for nanoparticles screening that joins the complex in vitro model imitating living tissue with high throughput analytical methods. This system can be applied to a wide diversity of potentially hostile compounds in basic screening to provide initial warning of adverse effects and trigger subsequent analysis and remedial actions.