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.”