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Improved visualization and quantitative analysis of drug effects using micropatterned cells.

ABSTRACT: To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of every individual cell provides a tremendous opportunity for solving this critical bottleneck (1-2). To facilitate access and use of the micropatterning technology, CYTOO has developed a range of ready to use micropatterns, available in coverslip and microwell formats. In this video article, we provide detailed protocols of all the procedures from cell seeding on CYTOOchip micropatterns, drug treatment, fixation and staining to automated acquisition, automated image processing and final data analysis. With this example, we illustrate how micropatterns can facilitate cell-based assays. Alterations of the cell cytoskeleton are difficult to quantify in cells cultured on homogenous substrates, but culturing cells on micropatterns results in a reproducible organization of the actin meshwork due to systematic positioning of the cell adhesion contacts in every cell. Such normalization of the intracellular architecture allows quantification of even small effects on the actin cytoskeleton as demonstrated in these set of protocols using blebbistatin, an inhibitor of the actin-myosin interaction.

SUBMITTER: Degot S

PROVIDER: S-EPMC3159674 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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Improved visualization and quantitative analysis of drug effects using micropatterned cells.

Degot Sébastien S Auzan Muriel M Chapuis Violaine V Béghin Anne A Chadeyras Amélie A Nelep Constantin C Calvo-Muñoz Maria Luisa ML Young Joanne J Chatelain François F Fuchs Alexandra A

Journal of visualized experiments : JoVE 20101202 46

To date, most HCA (High Content Analysis) studies are carried out with adherent cell lines grown on a homogenous substrate in tissue-culture treated micro-plates. Under these conditions, cells spread and divide in all directions resulting in an inherent variability in cell shape, morphology and behavior. The high cell-to-cell variance of the overall population impedes the success of HCA, especially for drug development. The ability of micropatterns to normalize the shape and internal polarity of ...[more]

PMID: 21189468

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Project description:PurposeCartilage canal vessels are critical to the normal function of epiphyseal (growth) cartilage and damage to these vessels is demonstrated or suspected in several important developmental orthopaedic diseases. High-resolution, three-dimensional (3-D) visualization of cartilage canals has recently been demonstrated using susceptibility weighted imaging (SWI). In the present study, a quantitative susceptibility mapping (QSM) approach is evaluated for 3-D visualization of the cartilage canals. It is hypothesized that QSM post-processing improves visualization of the cartilage canals by resolving artifacts present in the standard SWI post-processing while retaining sensitivity to the cartilage canals.MethodsEx vivo distal femoral specimens from 3- and 8-week-old piglets and a 1-month-old human cadaver were scanned at 9.4 T with a 3-D gradient recalled echo sequence suitable for SWI and QSM post-processing. The human specimen and the stifle joint of a live, 3-week-old piglet also were scanned at 7.0 T. Datasets were processed using the standard SWI method and truncated k-space division QSM approach. To compare the post-processing methods, minimum/maximum intensity projections and 3-D reconstructions of the processed datasets were generated and evaluated.ResultsCartilage canals were successfully visualized using both SWI and QSM approaches. The artifactual splitting of the cartilage canals that occurs due to the dipolar phase, which was present in the SWI post-processed data, was eliminated by the QSM approach. Thus, orientation-independent visualization and better localization of the cartilage canals was achieved with the QSM approach. Combination of GRE with a mask based on QSM data further improved visualization.ConclusionsImproved and artifact-free 3-D visualization of the cartilage canals was demonstrated by QSM processing of the data, especially by utilizing susceptibility data as an enhancing mask. Utilizing tissue-inherent contrast, this method allows noninvasive assessment of the vasculature in the epiphyseal cartilage in the developing skeleton and potentially increases the opportunity to diagnose disease of this tissue in the preclinical stages, when treatment likely will have increased efficacy.

Project description:5-Aminolevulinic acid (5-ALA) induced fluorescence to augment surgical resection for high grade glioma has become a standard of care. Protoporphyrin IX (PpIX) visibility is however subject to the variability of the single tumor expression and to the interobserver interpretation. We therefore hypothesized that in different glioma cell lines with variable 5-ALA induced fluorescence, the signal can be pharmacologically increased. We therefore analyzed in three different GBM cell lines, with different expression of epidermal growth factor receptor (EGFR), the variability of 5-ALA induced PpIX fluorescence after the pharmacological blockade at different steps of PpIX breakdown and influencing the outbound transport of PpIX. Using flow cytometry, fluorescence microplate reader, and confocal microscopy the PpIX fluorescence was analyzed after exposure to tin protoporphyrin IX (SnPP), deferoxamine (DFO), and genistein. We furthermore constructed a microscope (Qp9-microscope) being able to measure quantitatively the concentration of PpIX. These values were compared with the extraction of PpIX in tumor biopsy taken during the GBM surgery. Although all three cell lines showed an increase to 5-ALA induced fluorescence their baseline activity was different. Treatment with either SnPP, DFO and genistein was able to increase 5-ALA induced fluorescence. Qp9-microscopy of tumor sample produced a color coded PpIX concentration map which was overlaid on the tumor image. The PpIX extraction from tumor sample analyzed using the plate reader gave lower values of the concentration, as compared to the expected values of the Qp9-microscope, however still in the same decimal range of ?g/mL. This may be due to homogenization of the values during extraction and cell disaggregation. In conclusion pharmacological augmentation in GBM cell lines of PpIX signal is possible. A quantitative PpIX map for surgery is feasible and may help refine surgical excision. Further correlations of tumor tissue samples and Qp9-microscopy is needed, prior to develop an intraoperative surgical adjunct to the already existing 5-ALA induced surgery.

Dataset Information

Improved visualization and quantitative analysis of drug effects using micropatterned cells.

Publications

Improved visualization and quantitative analysis of drug effects using micropatterned cells.

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