Abstract
In most conventional in vitro toxicological assays, the response of a complete cell population is averaged, and therefore, single-cell responses are not detectable. Such averaging might result in misinterpretations when only individual cells within a population respond to a certain stimulus. Therefore, there is a need for non-invasive in vitro systems to verify the toxicity of nanoscale materials. In the present study, a micro-sized cell culture chamber with a silicon nitride membrane (0.16 mm2) was produced for cell cultivation and the detection of specific cell responses. The biocompatibility of the microcavity chip (MCC) was verified by studying adipogenic and neuronal differentiation. Thereafter, the suitability of the MCC to study the effects of nanoparticles on a small cell population was determined by using a green fluorescence protein-based reporter cell line. Interleukin-8 promoter (pIL8) induction, a marker of an inflammatory response, was used to monitor immune activation. The validation of the MCC-based method was performed using well-characterized gold and silver nanoparticles. The sensitivity of the new method was verified comparing the quantified pIL8 activation via MCC-based and standard techniques. The results proved the biocompatibility and the sensitivity of the microculture chamber, as well as a high optical quality due to the properties of Si3N4. The MCC-based method is suited for threshold- and time-dependent analysis of nanoparticle-induced IL8 promoter activity. This novel system can give dynamic information at the level of adherent single cells of a small cell population and presents a new non-invasive in vitro test method to assess the toxicity of nanomaterials and other compounds.PACS: 85.35.Be, 81.16.Nd, 87.18.Mp
Highlights
There is a growing interest in improved test methods to assess biological effects of nanoparticles
No Bio-MEMS exist for long-term culturing, and long-term observation of cell response features larger, more comparable cell culture area dimensions compared to the micro-sized cell culture chamber presented in this paper [32,34,35,36,37,38,39,40]
Fabrication of the miniaturized microcavity chip An MCC was fabricated by semiconductor process technology (Figure 1a)
Summary
There is a growing interest in improved test methods to assess biological effects of nanoparticles. With regard to the application of single-cell analysis as pharmaceutical in vitro screening method, the goal of this study is the evaluation and validation of a non-invasive technique to characterize cellular processes of adherent biological cells on an individual level in a small defined cell population. No Bio-MEMS exist for long-term cultivation and non-invasive quantification of specific cellular responses of adherent individual cells in a small defined cell layer cultured on miniaturized Si3N4 membranes with cell culture areas smaller than 0.2 mm. The use of a micro-sized chip-based cell culture system in combination with reporter cells presents a powerful tool for the analysis of small cell populations and will improve the evaluation of non-invasive in vitro test methods to observe sub-toxic effects on individual adherent cells in a small cell population under physiological conditions. This article introduces a miniaturized microcavity chip (MCC)-based method for the non-invasive analysis of nanoparticle-induced effects of adherent single cells in a small defined cell layer
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