Maintaining cell viability throughout the cell culture process is virtually a nonissue in current drug discovery efforts. But with the move toward multiplexed assays, and industry's desire for higher content without compromising throughput, it's worthwhile to think of cell viability along a spectrum and to consider cell health and responsiveness in assay development, such that cells are appropriately responsive to what a given assay intends to assess.
Informa's recent “Cell-based Assays” meeting in Berlin focused on “novel advances in assay development to improve lead discovery and increase correlation to in vivo and clinical results.” This is, of course, a key ongoing concern for drug discovery research—to ensure, as much as possible, that the investments into screening stand a good chance of eventual clinical success.
Marc Bickle, Ph.D., head of the HT technology development studio (HT-TDS) at Max Planck Institute of Molecular Cell Biology and Genetics, observed that basic culturing principles must be observed at all times.
Speaking about phenotypic profiling of primary human macrophages in automated microscopy screens during mycobacteria phagocytosis, he noted that, with macrophages, monitoring healthy morphology is crucial to successful experiments. Poor cell health can create false positives, given that mycobacteria highjack cellular metabolism for their own survival.
Dr. Bickle's laboratory thus monitors cell health on many fronts. “For viability in highcontent screens,” he said, “images provide the easiest measure for detecting DAPI- or Hoescht-stained nuclei for simple counting” to monitor for polyploidy or micronuclei (indicative of carcinogenic or teratogenic effect or other disruptions of cell cycle) or measuring aberrant nuclear morphologies.
His talk also covered mycobacteria survival measurement using high-resolution imaging on a PerkinElmer Opera System and monitoring of intracellular survival of Mycobacterium bovis, with simultaneous measurement of cellular responses of human macrophages.
With macrophages, in particular, there is the technical challenge posed by the use of postmitotic cells. Given that they do not grow, change in the numbers of cells is not an assessment option, so it is essential to quantify morphology as a cell-health indicator using multiparametric image analysis.
Also, for assays to be meaningful, cells must retain their responsiveness after preparative manipulations. Dr. Bickle described Cellectricon's Cellaxess HT® as a game changer for research in his laboratory.
Other systems his lab employed, namely electroporation in suspension or the use of shRNA, consistently caused cell death or macrophage activation, he reported. “With the Cellaxess system, the cells are perfectly happy,” requiring minimal manipulation with the automated platform that obviates both transfers to fresh plates and additional washings.
Dr. Bickle stressed the importance of extracting data correctly from assay readouts, noting that it is important either to analyze signaling pathways in conditions of cell health, or if stress conditions are being studied, to understand the parameters of the stress conditions. Further, he highlighted that common assessments of cell health, such as doubling time, are in essence physiologically removed from clinical pertinence.
“Very few cells in the body actually divide rapidly, so if you want to work with welldifferentiated cells expressing markers pertinent to a specific organ and function, you need to slow them down in the cell cycle—to take away the growth factors, for example, and provide a better matrix such as a collagen lattice or some kind of gel.”
HT-TDS is currently developing a toxicology assay that Dr. Bickle described as novel in that it assesses toxicity not via primary indicators such as cytochrome function but instead via secondary markers such as hepatocytic function. His team grows cells in a sandwich culture between two collagen layers to induce differentiation and then monitors for “caniculae formation, full polarization, and expression of normal hepatic markers.” As Dr. Bickle summarized, “If somebody is sick, he is not going to work. The same is true for cells.”
ZFN and Reporter Cell Lines
Abhishek Saharia, Ph.D., product manager for functional genomics at Sigma Life Science, presented data on the company's CompoZr® Zinc Finger Nuclease (ZFN) technology.
The technology allows researchers to bypass the protein overexpression inherent in many assays that otherwise creates “a system that doesn't truly resemble the patient,” Dr. Saharia said, given that such assays involve exogenous promoter systems.
The ZFN platform comprises engineered DNA-binding proteins that induce doublestrand breaks in DNA at user-specified sites, stimulating natural DNA-repair processes. The end result is “precisely targeted genomic edits” resulting in cell lines (including somatic) with targeted knockouts and integrations, Dr. Saharia explained.
Alternative systems may provide weaker data, he suggested, given that knockdown strategies are often partial and can lead to “incomplete effect.” Further, the ZFN platform enables the creation and assaying of robust human cell lines using any protein tag that has been endogenously incorporated into a specific locus of interest.
Dr. Saharia offered the example of use of the technology with the epidermal growth factor receptor (EGFR) protein, tagged with GFP, at the endogenous locus. “The advantage is endogenous levels of protein expression, with complete responsiveness based on endogenous promoter systems and regulatory elements.
“This puts researchers in a position to track protein localization and expression levels in a live cellular system that maintains all its natural responsive elements. Previously similar pathway responses could only be verified by endpoint experiments, either by Western blots or FACS analyses, but the ZFN system permits this visualization in a live cellular system.”
This kind of targeted disruption may prove fruitful for advancing personalized medicines, Dr. Saharia further suggested, given that it permits live visualized tracking of small molecules that disrupt specific signaling pathways such as activation of EGFR and can thus facilitate high-content screening.