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TUTORIAL OMICS Multiparametric Assays for Cardiomyocytes Dynamic Live-Cell Monitoring of Cell Beating and Cytotoxicity on a Single Platform Figure 2. Detailed views of kinetic traces and images of cells treated Cathy Olsen, Ph.D., Jayne Hesley, and Oksana Sirenko, Ph.D. Stem cell-derived human cardiomyocytes have phenotypic characteristics and electrophysiological profles similar to those of native human cardiac cells. Cardiomyocytes in culture are able to form a beating syncytium, which behaves similarly to native cardiomyocytes. Oscillation of intracellular calcium levels occurring with synchronized contractions of the cells can be monitored using a calcium-sensitive dye, and treatmentinduced changes in the pattern of oscillation can be monitored via changes in fuorescent signal over time. Recently, it has been shown that changes in intracellular calcium fux associated with cardiomyocyte contractions can be monitored on a fuorescence imaging plate reader (FLIPR® Tetra System). Now, the Spectra- Max® i3 Multi-Mode Microplate Platform with SpectraMax MiniMax™ Imaging Cytometer from Molecular Devices enables detection of cardiomyocyte beating and fuorescence imaging of cell viability in a single instrument. A cardiac beating assay was performed on the SpectraMax i3 Multi-Mode Microplate Platform using the EarlyTox™ Cardiotoxicity Kit from Molecular Devices for assessment of compound effects upon cardiomyocytes. iCell Cardiomyocytes, human induced pluripotent stem cell (iPSC)-derived cardiomyocytes from Cellular Dynamics International, were cultured in 384-well microplates until a synchronously beating layer formed. Cells were then treated with compounds from the Screen-Well Cardiotoxicity Library (Enzo Life Sciences) at 10 µM for 24 hours and assessed for compound effects on beating and viability. with DMSO (control), digoxin, haloperidol, or staurosporine: Digoxin and haloperidol decreased the number of peaks measured in beating cells but had no significant effect on viability, while staurosporine negatively affected both beating and viability. Figure 1. Top: Representative kinetic traces from cardiomyocytes treated with compounds from the ENZO Cardiotoxicity Library. Bottom: Heat map of cell viability imaging results, expressed as percentage of the well covered by live cells, for the same assay plate as above. Blue-shaded wells indicate low viability, while red-shaded wells indicate highest viability. Examples of duplicate DMSO-treated control wells are circled in green on both plate views. Digoxin-treated wells showing reduction in peak count without loss of viability are circled in yellow, and staurosporine-treated wells where both peak count and viability are significantly reduced are circled in blue. Transfection Concentration-response curves based on peak counts for cells treated with valinomycin (red), digoxin (blue), and amiodarone (green). Bottom: Concentration-response curves for viability of cells treated with valinomycin (red), digoxin (blue), amiodarone (green), and staurosporine (orange). Continued from page 34 "As more biologically relevant cell systems are developed, such as primary cells, researchers want to adopt these within biotherapeutic and drug development activities," said Karen A. Donato, Ph.D., executive vp, global business development and marketing, at MaxCyte. MaxCyte fow electroporation was originally developed for ex vivo cell transfection, which requires reliable, high-effciency transfection of primary cells (such as human embryonic or adult stem cells, human T cells, or dendritic cells) in large numbers with rigorous safety standards. Flow electroporation melds the benefts of electroporation with simple scalability, which is advantageous in the felds of small- 36 | Figure 3. Top: molecule and therapeutic protein development, explained Dr. Donato. The MaxCyte electroporation system performs transfections of 5 × 105 cells in seconds to 2 × 1011 cells in less than 30 minutes, she continued, adding that the scalable approach results in high viability and high effciency, which in turn leads to high expression of the protein of interest. "Speed, reliability, and reproducibility are all going to become increasingly important. Pharmaceutical companies are looking for technologies that can help them make better decisions up front in the development process with fewer resources, in less time. Transient transfection is becoming a signifcant part of the drug development toolkit," September 1, 2013 | GENengnews.com | Genetic Engineering & Biotechnology News concluded Dr. Donato. In Vivo Applications An exciting transfection milestone is development of RNAi transfection tools for use in research applications as well as for the delivery of new biologics as therapeutic modalities. "Small molecules, like RNAi, are less diffcult to transfect than DNA, which needs to enter the nucleus, typically, when the cell is dividing. RNA just needs to get into the cytoplasm, eliminating a step in the transfection process," said Xavier de Mollerat du Jeu, Ph.D., senior staff scientist, molecular biology, Life Technologies. "RNAi transfection, in general, works for more cells lines, primary cells and in vivo." Previously, there were no tools to deliver RNAi in vivo. Invivofectamine 2.0, a lipidbased RNA transfection system that protects the nucleic acid from degradation in the bloodstream, allows researchers to deliver RNAi to small animal models, such as mice and rats, and to directly perform functional and biologic analysis. In vivo transfection technology opens the door to rapid design and production of knockout animals facilitating genomic and proteomic research. "Transfection technology development will continue. In addition to primary, stem cells and in vivo applications, efforts also need to focus on plant and algae cells. These systems have yet to be optimized," added Dr. de Mollerat du Jeu.