Microarrays for the scalable production of metabolically relevant tumour spheroids: a tool for modulating chemosensitivity traits.

We report the use of thin film poly(dimethylsiloxane) (PDMS) prints for the arrayed mass production of highly uniform 3-D human HT29 colon carcinoma spheroids. The spheroids have an organotypic density and, as determined by 3-axis imaging, were genuinely spherical. Critically, the array density impacts growth kinetics and can be tuned to produce spheroids ranging in diameter from 200 to 550 µm. The diffusive limit of competition for media occurred with a pitch of ≥1250 µm and was used for the optimal array-based culture of large, viable spheroids. During sustained culture mass transfer gradients surrounding and within the spheroids are established, and lead to growth cessation, altered expression patterns and the formation of a central secondary necrosis. These features reflect the microenvironment of avascularised tumours, making the array format well suited for the production of model tumours with defined sizes and thus defined spatio-temporal pathophysiological gradients. Experimental windows, before and after the onset of hypoxia, were identified and used with an enzyme activity-based viability assay to measure the chemosensitivity towards irinotecan. Compared to monolayer cultures, a marked reduction in the drug efficacy towards the different spheroid culture states was observed and attributed to cell cycle arrest, the 3-D character, scale and/or hypoxia factors. In summary, spheroid culture using the array format has great potential to support drug discovery and development, as well as tumour biology research.

Liquid analysis dielectric capillary barrier discharge.

In this study a simple micro-tube-based system for analysis of metal-containing liquids is introduced and its analytical performance is evaluated. It is based on a miniaturised dielectric barrier discharge driven at atmospheric pressure. The emission lines of various elements are observed. The system is developed for quantitative measurements and the limits of detection are determined. Because of very low flow rates of just microL min(-1) the approach requires extremely low sample volumes.

The network formation assay: a spatially standardized neurite outgrowth analytical display for neurotoxicity screening.

We present a rapid, reproducible and sensitive neurotoxicity testing platform that combines the benefits of neurite outgrowth analysis with cell patterning. This approach involves patterning neuronal cells within a hexagonal array to standardize the distance between neighbouring cellular nodes, and thereby standardize the length of the neurite interconnections. This feature coupled with defined assay coordinates provides a streamlined display for rapid and sensitive analysis. We have termed this the network formation assay (NFA). To demonstrate the assay we have used a novel cell patterning technique involving thin film poly(dimethylsiloxane) (PDMS) microcontact printing. Differentiated human SH-SY5Y neuroblastoma cells colonized the array with high efficiency, reliably producing pattern occupancies above 70%. The neuronal array surface supported neurite outgrowth, resulting in the formation of an interconnected neuronal network. Exposure to acrylamide, a neurotoxic reference compound, inhibited network formation. A dose-response curve from the NFA was used to determine a 20% network inhibition (NI(20)) value of 260 microM. This concentration was approximately 10-fold lower than the value produced by a routine cell viability assay, and demonstrates that the NFA can distinguish network formation inhibitory effects from gross cytotoxic effects. Inhibition of the mitogen-activated protein kinase (MAPK) ERK1/2 and phosphoinositide-3-kinase (PI-3K) signaling pathways also produced a dose-dependent reduction in network formation at non-cytotoxic concentrations. To further refine the assay a simulation was developed to manage the impact of pattern occupancy variations on network formation probability. Together these developments and demonstrations highlight the potential of the NFA to meet the demands of high-throughput applications in neurotoxicology and neurodevelopmental biology.

Impact of fixation on in vitro cell culture lines monitored with Raman spectroscopy.

Raman spectroscopy provides chemical-rich information about the composition of analytes and is a powerful tool for biological studies. With the ability to investigate specific cellular components or image whole cells, compatible methods of sample preservation must be implemented for accurate spectra to be collected. Unfortunately, the effects of many commonly used sample preservation methods have not been explored with cultured cells. In this study, two human cell lineages of varying phenotypes were used to investigate the effects of sample preservation methods. Cells were cultured directly onto quartz substrates and either formalin-fixed, desiccated or air dried. The results indicate that the methodology applied to cell cultures for Raman analysis significantly influences the quality and reproducibility of the resulting spectral data. Formalin fixation was not found to be as universally efficient as anticipated for a commonly used fixative. This was due largely to the inconsistency in sample preservation between cell lines and loss of signal intensity. Sample air-drying was found to be largely inconsistent in terms of spectral reproducibility. Our study shows that sample desiccation displayed good spectral reproducibility and resulted in a good signal-to-noise ratio. Lipid and protein content in both activated and inactivated cells were maintained and provided a more controlled method compared with air-drying, revealing that the speed of drying is important for sample preservation.

Concomitant detection of CYP1A1 enzymatic activity and CYP1A1 protein in individual cells of a human urothelial cell line using a bilayer microfluidic device.

To understand molecular networking at the cellular level, analyses of processes and effects at the single-cell level are most appropriate. Usual biochemical or molecular biological analyses are based on integrated signals of numerous cells which differ, however, in their expression and activity profiles. Here we show that it is possible to determine different types of properties of individual cells by means of a specifically designed microfluidic device. As part of investigations to characterize the human urothelial cell line 5637 as a potential model system for studies of toxic and carcinogenic effects on urothelial cells, we use this cell line to assign cytochrome P450 activity, and expression of the enzymes involved, to individual cells. It is shown that the cell population is very heterogeneous with respect to the extent and kinetics of CYP1A1-dependent ethoxyresorufin O-deethylase (EROD). This is also true for the cells' CYP1A1 protein content. With some exceptions, the EROD activity largely coincides with the presence of CYP1A1 protein in the cells. The results obtained with the microfluidic device are promising and open up new perspectives with regard to multi-property determinations in individual cells and to studies focusing on the biochemical and molecular heterogeneity of cells.

Force microscopy analysis using chemometric tools.

In this paper we report the first application of multivariate data analysis techniques to force spectrometry measurement sets to enable the physicochemical assignment of spatially ordered multi-component systems. Principal component analysis (PCA) and hierarchical clustering techniques were used to reveal hidden chemical information within force-distance curves generated by high spatial resolution force microscopy. Two experimental samples were analyzed: (i) a two-component system of cytochrome c proteins on a mica surface, and (ii) a three-component system of avidin protein islands positioned on a gold and glass surface. PCA and hierarchical clustering techniques were used to discriminate the different components of the two-component system, whereas hierarchical clustering was found to be superior for the three-component system. Results were in good agreement with the topography and prior knowledge of the surface patterns. This research represents a formative step towards the combination of force spectrometry with chemometric tools for the high resolution physicochemical investigation of complex biochemical systems.

Analysis of bacterial strains with pyrolysis-gas chromatography/differential mobility spectrometry.

Eight vegetative bacterial strains and two spores were characterized by pyrolysis-gas chromatography with differential mobility spectrometry (py-GC/DMS) yielding topographic plots of ion intensity, retention time, and compensation voltage simultaneously for ions in positive and negative polarity. Biomarkers were found in the pyrolysate at characteristic retention times and compensation voltages and were confirmed by standard addition with GC/MS analyses providing discrimination between Gram negative and Gram positive bacterial types, but no recognition of individual strains within the Gram negative bacteria. Principal component analysis was applied using two dimensional data sets of ion intensity versus retention time at five compensation voltages including the reactant ion peaks all in positive and negative ion polarity. Clustering was observed with compensation voltage (CV) chromatograms associated with ion separation in the DMS detector and little or no clustering was observed with the reactant ion peaks or CV chromatograms where ion separation is poor. Consistent clustering of Gram positive B. odysseyi and Gram negative E. coli in both positive and negative polarities with the reactant ion peak chromatograms and key CV chromatograms suggests common but unknown common chemical compositions in the pyrolysate.

Two- and three-dimensional mapping of the iron distribution in the apoplastic fluid of plant leaf tissue by means of magnetic resonance imaging.

A three-dimensional nuclear magnetic resonance imaging (MRI) technique for non-invasive mapping of iron in the apoplastic fluid of plant compartments was developed. The new technique was applied to a leaf of red stem dogwood (Cornus stolonifera). The results are compared with MRI measurements of iron distributions in two dimensions and with total reflection X-ray fluorescence results.

Spectral variable selection for partial least squares calibration applied to authentication and quantification of extra virgin olive oils using Fourier transform Raman spectroscopy.

The limits of quantitative multivariate assays for the analysis of extra virgin olive oil samples from various Greek sites adulterated by sunflower oil have been evaluated based on their Fourier transform (FT) Raman spectra. Different strategies for wavelength selection were tested for calculating optimal partial least squares (PLS) models. Compared to the full spectrum methods previously applied, the optimum standard error of prediction (SEP) for the sunflower oil concentrations in spiked olive oil samples could be significantly reduced. One efficient approach (PMMS, pair-wise minima and maxima selection) used a special variable selection strategy based on a pair-wise consideration of significant respective minima and maxima of PLS regression vectors, calculated for broad spectral intervals and a low number of PLS factors. PMMS provided robust calibration models with a small number of variables. On the other hand, the Tabu search strategy recently published (search process guided by restrictions leading to Tabu list) achieved lower SEP values but at the cost of extensive computing time when searching for a global minimum and less robust calibration models. Robustness was tested by using packages of ten and twenty randomly selected samples within cross-validation for calculating independent prediction values. The best SEP values for a one year's harvest with a total number of 66 Cretian samples were obtained by such spectral variable optimized PLS calibration models using leave-20-out cross-validation (values between 0.5 and 0.7% by weight). For the more complex population of olive oil samples from all over Greece (total number of 92 samples), results were between 0.7 and 0.9% by weight with a cross-validation sample package size of 20. Notably, the calibration method with Tabu variable selection has been shown to be a valid chemometric approach by which a single model can be applied with a low SEP of 1.4% for olive oil samples across three different harvest years.