GEN Exclusives

More »

Feature Articles

More »
Aug 1, 2011 (Vol. 31, No. 14)

Cell Viability & Responsiveness in Assays

Keeping Cells “Happy” and Healthy Helps Support Complex Multiplexed Assays

  • Scaling HCS to HTS

    Fabio Gasparri, Ph.D., principal research scientist at Nerviano Medical Sciences, stressed the importance of tending to basics. Nerviano conducts HCS assays to evaluate mechanisms of action of lead compounds affecting regulation of cell cycle or apoptosis.

    Cytostatic or cytotoxic effects are determined through specific marker assessments. For confounding compound classes, such as those inducing polynuclear formation or that increase cell volume, which in turn can affect monoparametric readouts such as ATP content, his lab applies alternative methods such as cell count to better determine the real effects of lead compounds.

    Currently, the lab employs time-lapse microscopy among its tools. “The time variable is crucial for better evaluation and good characterization of lead compounds. Focusing only on endpoint assays has some limitations; for instance, they may not clearly distinguish between cytostatic and cytotoxic effects.”

    Dr. Gasparri is looking toward adoption of technologies that will further strengthen the robustness of Nerviano's R&D efforts. He said image-based technologies, like TAP Biosystems' cell- IQ® platform or impedence-based technology such as Roche's xCELLigence system for monitoring real-time cell proliferation in plates, “could be useful to specifically monitor cytostatic or cytotoxic effects and improve early characterization studies.”

    Dr. Gasparri's lab currently conducts highcontent mechanism-based assays using the Thermo Fisher Scientific Cellomics ArrayScan HCS reader.

    “In particular, we monitor the regulation of the cell cycle in response to targets we've identified at Nerviano related to cell-cycle control and to signal transduction. We employ assays based on immunofluorescence or reporter cell lines stably transfected with GFP proteins. Recently, we've been performing primary HC screening assays and are integrating these to analyze multiparametric data arising across different HC primary screens.”

    Ultimately, this is all intended to increase the predictive contribution of earlier in vitro data gathering toward in vivo outcomes. After all, he insisted, when discussing in vivo work, “we are talking about models, so even as we try to increase the relevance of our models, they remain models.”

  • Stem Cell-Based Assays

    Davide Danovi, M.D., Ph.D., research associate in Steven Pollard’s laboratory at the University College of London’s Cancer Institute, described approaches to carry out live image-based chemical screens using glioma stem cells.

    Pollard’s laboratory has developed a method to isolate cells from normal brain tissue and also from brain tumor (glioblastoma) samples. The cell culture protocol essentially involves defined medium with growth factors and the use of laminin, which avoids cell suspension (and thus the formation of neurospheres).

    “Neurospheres can be used for different applications, but we argue that if you want to screen for drugs to cure glioma, you need to test drugs on the relevant cell population, which is the stem cell-like population,” Dr. Danovi explained.

    The lab then applies image-based assays to quantitate morphological changes and assess proliferative behaviors, “in order to be able to isolate drug leads that specifically affect a particular subtype of glioma, targeting glioma stem cells versus normal neural stem cells.”

    Modules from various sources are used, including CyBio’s Cybi®-Selma liquid-handling device, an IncuCyte image-based platform from Essen BioScience, and Cell Profiler freeware from the Broad Institute.

    In-house bioinformatics efforts led by Amos Folarin allow “proper image analysis, so we can track cells, morphological changes, and mitoses, for example. Our approach uses proliferation as a readout to score compounds that are cytotoxic or cytostatic.” Among cytostatic compounds, those that induce terminal differentiation of glioma stem cells are considered for potential glioblastoma therapies.


Add a comment

  • You must be signed in to perform this action.
    Click here to Login or Register for free.
    You will be taken back to your selected item after Login/Registration.

Related content

Jobs

GEN Jobs powered by HireLifeScience.com connects you directly to employers in pharma, biotech, and the life sciences. View 40 to 50 fresh job postings daily or search for employment opportunities including those in R&D, clinical research, QA/QC, biomanufacturing, and regulatory affairs.
 Searching...
More »

GEN Poll

More » Poll Results »

Mindclones and Immortality

Is the idea of making a software copy of a human mind something in the realm of reality or science fiction?