An impedance method for spatial sensing of 3D cell constructs--towards applications in tissue engineering.

We present the characterisation and validation of multiplexed 4-terminal (4T) impedance measurements as a method for sensing the spatial location of cell aggregates within large three-dimensional (3D) gelatin scaffolds. The measurements were performed using an array of four rectangular chambers, each having eight platinum needle electrodes for parallel analysis. The electrode positions for current injection and voltage measurements were optimised by means of finite element simulations to maximise the sensitivity field distribution and spatial resolution. Eight different 4T combinations were experimentally tested in terms of the spatial sensitivity. The simulated sensitivity fields were validated using objects (phantoms) with different conductivity and size placed in different positions inside the chamber. This provided the detection limit (volume sensitivity) of 16.5%, i.e. the smallest detectable volume with respect to the size of the measurement chamber. Furthermore, the possibility for quick single frequency analysis was demonstrated by finding a common frequency of 250 kHz for all the presented electrode combinations. As final proof of concept, a high density of human hepatoblastoma (HepG2) cells were encapsulated in gelatin to form artificial 3D cell constructs and detected when placed in different positions inside large gelatin scaffolds. Taken together, these results open new perspectives for impedance-based sensing technologies for non-invasive monitoring in tissue engineering applications providing spatial information of constructs within biologically relevant 3D environments.

Interdependence of initial cell density, drug concentration and exposure time revealed by real-time impedance spectroscopic cytotoxicity assay.

We investigated the combined effect of the initial cell density (12,500, 35,000, 75,000, and 100,000 cells cm(-2)) and concentration of the anti-cancer drug doxorubicin on HeLa cells by performing time-dependent cytotoxicity assays using real-time electrochemical impedance spectroscopy. A correlation between the rate of cell death and the initial cell seeding density was found at 2.5 µM doxorubicin concentration, whereas this was not observed at 5 or 100 µM. By sensing the changes in the cell-substrate interaction using impedance spectroscopy under static conditions, the onset of cytotoxicity was observed 5 h earlier than when using a standard colorimetric end-point assay (MTS) which measures changes in the mitochondrial metabolism. Furthermore, with the MTS assay no cytotoxicity was observed after 15 h of incubation with 2.5 µM doxorubicin, whereas the impedance showed at this time point cell viability that was below 25%. These results indicate that impedance detection reveals cytotoxic events undetectable when using the MTS assay, highlighting the importance of combining impedance detection with traditional drug toxicity assays towards a more in depth understanding of the effect of anti-cancer drugs on in vitro assays. Moreover, the detection of doxorubicin induced toxicity determined with impedance under static conditions proved to be 6 times faster than in perfusion culture.

Impedance-based Real-time Monitoring of Neural Stem Cell Differentiation.

We present here the first impedance-based characterization of the differentiation process of two human mesencephalic fetal neural stem lines. The two dopaminergic neural stem cell lines used in this study, Lund human mesencephalic (LUHMES) and human ventral mesencephalic (hVM1 Bcl-XL), have been developed for the study of Parkinsonian pathogenesis and its treatment using cell replacement therapy. We show that if only relying on impedance magnitude analysis, which is by far the most usual approach in, e.g., cytotoxicity evaluation and drug screening applications, one may not be able to distinguish whether the neural stem cells in a population are proliferating or differentiating. However, the presented results highlight that equivalent circuit analysis can provide detailed information on cellular behavior, e.g. simultaneous changes in cell morphology, cell-cell contacts, and cell adhesion during formation of neural projections, which are the fundamental behavioral differences between proliferating and differentiating neural stem cells. Moreover, our work also demonstrates the sensitivity of impedance-based monitoring with capability to provide information on changes in cellular behavior in relation to proliferation and differentiation. For both of the studied cell lines, in already two days (one day after induction of differentiation) equivalent circuit analysis was able to show distinction between proliferation and differentiation conditions, which is significantly earlier than by microscopic imaging. This study demonstrates the potential of impedance-based monitoring as a technique of choice in the study of stem cell behavior, laying the foundation for screening assays to characterize stem cell lines and testing the efficacy epigenetic control.

Ectopic expression of Cdk6 circumvents transforming growth factor-beta mediated growth inhibition.

Transforming growth factor-beta (TGF-beta) induced growth arrest of cells involves regulation of the activities of both D- and E-type cyclin kinase complexes thought to be mediated primarily by the regulation of p15(Ink4b) and p27(Kip1) cyclin kinase inhibitors. We show here that TGF-beta downregulates Cdk6 and that transient and stable expression of Cdk6 in Mv1Lu mink epithelial cells overrides TGF-beta mediated arrest. The main effect of the ectopic Cdk6 expression was to sequester TGF-beta induced p15(Ink4b) and to maintain more p27(Kip1) in cyclin D-complexes preventing the complete shift of p27(Kip1) to Cdk2 invoked by TGF-beta. This led to the presence of an active cyclinD-Cdk6-p27(Kip1) complex and partially active cyclin E-Cdk2 complex and resulted in the failure of TGF-beta to fully arrest Mv1Lu cell growth. Though dominant negative Cdk6, expressed similarly in the cells, sequestered both p15(Ink4b) and p27(Kip1), it lacks kinase activity and was unable to override the TGF-beta arrest. The results demonstrate that downregulation of Cdk6 kinase is required for the enforcement of the G(1)-phase arrest by TGF-beta and results in changes in association of the p15(Ink4b) and p27(Kip1) inhibitors with D- and E-type cyclin kinase complexes.

Towards good ecological status of surface waters in Europe--interpretation and harmonisation of the concept.

The Water Framework Directive (WFD) is a new legislative framework to manage, use, protect, and restore surface water and groundwater resources and coastal waters in the European Union (EU). The aim is to ensure sustainable water management and to reach good water quality by 2015. The assessment of the ecological status and setting of the practical management goals require several steps. The process has started with the characterisation of the river basins including identification of surface water bodies and types, and identification of significant anthropogenic pressures and impacts. The water bodies will be classified in five quality classes (high, good, moderate, poor, bad) based on the Ecological Quality Ratio, which is a ratio between reference conditions and measured status of the biological quality elements. The normative criteria for high, good and moderate ecological status described in the WFD need to be made operational because those will be used to set the practical quality targets for surface water management. National ecological assessment systems and classifications will be harmonised through the WFD intercalibration exercise in order to ensure an equal level of ambition in achieving good surface waters status all over Europe.

The eutrophication commandments.

Typically, rising atmospheric carbon dioxide concentrations are used to illustrate how humans have impacted the earth. However, we have also dramatically altered the amount of nitrogen (N) and phosphorus (P) cycling through the biosphere. Eventually these nutrients are carried to coastal receiving waters where they cause severe, often negative consequences including increased phytoplankton and macroalgae blooms, loss of submerged aquatic vegetation, low oxygen events, and decreased biodiversity. In many systems mitigation efforts are now underway to return these ecosystems to a less impacted state. While many uncertainties about the best way to manage eutrophic systems remain it is clear that we must take action to lessen our human nutrient footprint. Based on our current understanding of eutrophic systems we present ten eutrophication commandments or guidelines as a tool for scientists, policy makers, managers, and the public.

Accumulation of a form of p27(Kip1) not associated with Cdk-cyclin complexes in transforming growth factor-beta-arrested Mv1Lu cells.

The p27(Kip1) cyclin-dependent kinase inhibitor translocates in response to transforming growth factor-beta to a Cdk2-cyclin E complex inhibiting its catalytic activity, but the p27(Kip1) protein levels are unaffected [1]. We show here that transforming growth factor-beta induces the accumulation of a form of p27(Kip1) representing a subpopulation of total p27(Kip1) in growth-arrested Mv1Lu epithelial cells. The inducible p27(Kip1) is detectable only by a specific p27(Kip1) monoclonal antibody recognizing a native form of p27(Kip1). The increase in this subset of p27(Kip1) correlates with G(1) arrest and withdrawal of the cells from the cycle induced by transforming growth factor-beta, serum starvation, or contact inhibition. In contrast to the majority of p27(Kip1) in the cells, the transforming growth factor-beta-inducible p27(Kip1) is devoid of cyclin-dependent kinase/cyclin interactions. The results indicate that growth arresting treatments induce the accumulation of non-cyclin-dependent kinase-bound p27(Kip1), which may function as a reservoir for inhibition of Cdk2-cyclin E activities.

Serum hyaluronate level as a predictor of radiologic progression in early rheumatoid arthritis.

Increased serum levels of hyaluronate (HA) have been found in patients with rheumatoid arthritis (RA). This probably reflects increased leakage of HA from the inflamed joints into the circulation. In a prospective study of 40 patients with early RA, we evaluated the relationship of serum HA to clinical, laboratory, and radiologic parameters of disease activity. The patients were followed for 12 months; all had active disease at study entry. We confirmed the previous finding of higher serum HA concentrations in RA patients compared with healthy controls. At study entry, the patients' serum HA levels correlated positively with clinical and laboratory parameters of acute inflammation. Despite marked clinical improvement during therapy with second-line drugs, the serum HA levels increased during the followup period. At the end of 1 year, these levels correlated with the radiologic progression of joint lesions, whereas they showed a less pronounced correlation with clinical or laboratory parameters of inflammation. We conclude that, in early RA, serum HA levels may reflect ongoing joint destruction and may even predict subsequent joint damage.

UV radiation is a transcriptional inducer of p21(Cip1/Waf1) cyclin-kinase inhibitor in a p53-independent manner.

p53 target genes p21(Cip1/Waf1) cyclin-kinase inhibitor (p21 CKI), GADD45, bax, and cyclin G and genes affecting the redox state of the cells are implicated in p53 damage control responses. In order to attribute their functions and dependency of p53 in UV-damaged cells we undertook an analysis of UVC responses of fibroblasts derived from p53 knock-out mice. UVC radiation efficiently and rapidly inhibited DNA replication in both p53 -/- and +/+ cells. The arrest was persistent in p53 -/- fibroblasts and cells underwent apoptosis, whereas p53 +/+ cells recovered and reentered the cycle. Protein and mRNA analyses of p21 expression showed that it was induced up to sixfold with similar kinetics both in the presence and in the absence of p53. However, high doses of UV abrogated the p21 response in p53 -/- cells, whereas it was maintained in cells with normal p53. UVC radiation transcriptionally activated p21 expression as demonstrated by luciferase reporter assays using deletion constructs of the p21 promoter. The promoter assays further confirmed the independency of p53-binding sites in the activation and linked UV-responsive transcriptional regulation of p21 to two Sp1 consensus binding sites within -61 bp of the transcription initiation site. A weaker regulation was mediated by elements between -1300 to -500 bp relative to the transcription initiation site. The results suggest that in fibroblasts UVC radiation is a rapid and efficient inducer of p21 expression also in a p53-independent manner.