tion, spheroids are more suitable to study organotypic properties in vitro [38]. Moreover, we established a novel fourfold mutated tau variant (K280q) that showed a drastically increased pathology in comparison to widely used wildtype tau (WT), single mutated tau (P301L, DK280) or even threefold tau variant [14,32,39]. In contrast to previous OA-based tau hyperphosphorylation pathol- ogy models [8,17], our novel system provides a very fast and intensified pathology development. Moreover, the comparative analysis of the WT and K280q tau cells allows detailed quantitative information for tau-specific degenerative processes (Fig. 4A). A more detailed understanding of the underlying tau patholog- ical degenerative processes could be obtained combining the analysis of spheroid impedance spectroscopy and cross section area (Fig. 5). Interestingly, especially for the WT tau expressing spheroids low OA concentrations led to a relative impedance increase coexistent with a constant cross section area, whereas higher OA concentrations caused a cross section area increase accompanied by strongly decreased relative impedance. Recently, it has been shown that AD pathology induces a continuing process of axon sprouting and swelling in neurons leading to increased membrane and decreased electrolyte properties [40,41] assuming similar effects that could be detected after 24 hours incubation with 5 nM OA. Subsequently, the retraction of neuronal processes is initiated by microtubule instability leading to the breakdown of the neuronal network and cell disintegration of spheroidal structure [42] determined after 72 hours OA treatment. In contrast, accelerated cell damage is characteristic at higher OA concentrations of 25 nM. Furthermore, swelling of the neuronal cell body could be visualized via microscopic images of SH-SY5Y spheroids after 24 hours incubation. Cell swelling is characteristic for accelerated neurodegeneration leading to necrosis/apoptosis, which were found to coexist in AD patient brains [43]. Despite the cellular swelling, a continuous impedance decrease was observed. This can be correlated to extended degeneration processes that accompany loss of cell-cell-/cell-matrix-contacts and cause there- with an increased intercellular volume. Finally, progressive cell loss also induces spheroid size reduction and a strong proceeding of conductive elements that evoke further impedance decrease after 72 hours treatment with 25 nM OA. Herewith, we could show that the distinction of neurite retraction versus massive cell death and therewith the selective detection of tau-specific degeneration and regeneration is possible using our impedance-based screening system in combination with optical monitoring without the need for further molecular end point analysis. Furthermore, our results reveal evidences for processes, which are highly analogue to in vivo- like explant culture models [15,40] demonstrating the in vivo-like complex character of our 3D culture model. More strikingly, we were able for the first time to sensitively detect the attenuating effect of the potential API candidates AR- A014418, hymenialdisine, 17-AAG and methylene blue against OA-induced tauopathy in the low nanomolar concentration range. Especially the very selective GSK-3b inhibitor AR-A014418 performed very well when considering potential cytotoxic side effects that could be detected simultaneously. Taken together, we demonstrate that our novel 3D tauopathy culture model in combination with our impedance spectroscopy-based label-free monitoring system is suitable for high content compound testing. Since our system uses single spheroids that are individually traced and analyzed over time with only 3000 to 5000 cells per spheroid the bioelectronic-based read-out fulfills the demands of modern high-content screening systems regarding cell content and material efforts. Therefore, our neuronal 3D in vitro model is positioned A Novel Tauopathy 3D-Culture Model on a Biochip between simple 2D culture and complex, cost and time consuming animal models and is therefore suitable to improve the evaluation of APIs in preclinical trials. Moreover, the established 3D in vitro model can be easily modified and extended to investigate further aspects like induction of taupathological consequences (e.g. toxic b-amyloid species or shift from 3R to 4R tau isoforms). Furthermore, the established cell culture model is highly appropriate for further applications in the field of neurodegener- ative diseases (e.g. patient-specific induced pluripotent stem cells offering a platform for personalized medicine) [5]. Conclusions In conclusion, the presented results show the high suitability and advantages of our novel neuronal 3D in vitro tauopathy cell culture model. In combination with our microcavity array-based label-free monitoring system we successfully demonstrated the quantitative monitoring of tau-specific neurodegenerative processes in individ- ual 3D-cultures and linking them to distinct pathological mechanisms. More strikingly, we demonstrate the capability of our system for compound efficacy as well as side effect testing. This makes our 3D model system suitable for high content screening systems especially in the focus of API development against AD and related tauopathies and a valid alternative model to 2D cell culture and complex animal models. Materials and Methods SH-SY5Y Cell Cultivation, Spheroid Formation and Okadaic Acid-induced Tau Hyperphosphorylation SH-SY5Y cells (ATCC) were cultured in DMEM medium supplemented with 15% fetal bovine serum, 1% non-essential amino acids, 2 mM glutamax, 100 units/ml streptomycin/ penicillin and 0.4% gentamicine (Invitrogen) at 37uC in a humidified atmosphere with 5% CO2. Spheroids were generated by seeding 2 million cells per well in 6-well plates and cultivation on a self-developed gyratory shaker (72 rpm). Tau hyperphosphorylation was induced by the application of okadaic acid (Sigma-Aldrich) in a concentration range from 5– 100 nM. Reference compounds AR-A014418 (Sigma-Aldrich), 17-allylamino-17-demothoxygeldanamycin (17-AAG) (Sigma-Al- drich), hymenialdisine (Enzo LifeScience) and methylene blue (Sigma-Aldrich) were used in a concentration range from 10– 500 nM. For individual tracking, at culture day four single SH- SY5Y spheroids were transferred into a single well of a 48-well plate and experiments were performed at culture day five. Generation of SH-SY5Ycell Lines Stably Expressing EGFP- fused Tau Constructs by Lentiviral Transduction Human wildtype tau (0N4R) cDNA was amplified by PCR from isolated human neuronal cell mRNA and cloned into the pEGFP- C1 vector C-terminal to the EGFP coding sequence. For generating replication incompetent lentiviral particles the EGFP- tau wildtype coding sequence was cloned into the pLenti6v5-topo vector (Invitrogen). Single mutated (P301L) and fourfold mutated K280q (DK280, P301L, V337M, R406W) variants were obtained by site-directed mutagenesis (QuickChange Lightning, Strata- gene). Virus particles were generated in accordance to the manufactures guide (Lentiviral TOPO Expression Kit, Invitrogen) and SH-SY5 cells were transduced and selected by incorporated blasticidine (Invitrogen) resistance (10 mg/ml). PLOS ONE | www.plosone.org 8 November 2012 | Volume 7 | Issue 11 | e49150