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Slide 2 Electric Field Stimulation (EFS) of iPSC-derived cardiomyocytes using the Cellaxess Elektra Discovery Platform with high frequency sampling Note: This is an interactive slideshow with voiceover. Make sure your loudspeakers are turned on! Slide 3 Paul Karila, PhD Susanne Lardell, MSc www.cellectricon.com Electric Field Stimulation (EFS) of iPSC-derived cardiomyocytes using the Cellaxess Elektra Discovery Platform with high frequency sampling Slide 4 Contents Cellaxess Elektra discovery platform, core technology overview Case Study, electric field stimulation of iPSC-derived cardiomyocytes using the Cellaxess Elektra discovery platform with high frequency sampling The viewer of this presentation will: Get a comprehensive understanding of the principles of operation of the Cellaxess Elektra Platform Understand how the Cellaxess platform can be applied to enable novel, excitable cellular assays. Learn workflows and principles for key assay development and protocol steps. 3 Slide 5 Technology overview 4 Slide 6 Main unit Fully integrated robotic system Liquid handling capability. 96-tip electrode array for parallel recordings Imager Extremely sensitive imaging-based reader Fast acquisition rates, up to 50 Hz High resolution, single cell detection capability Consumables HCA compatible microplates (96-, 384-well) Electroporation buffer & accelerator solution Tips and electrode modules Cellaxess Elektra - Platform Overview 5 An automated system for in-situ electric field manipulation of cell cultures Slide 7 Cellaxess Elektra in-situ electrostimulation Capillary electrode tip arrays 96-tip array, EP 96-tip array, EFS Optimized Microplates 96-well plates 384-well plates Fully automated procedure Stand alone operation LAS integrated operation 6 Slide 8 1. Cells are cultured in 96 well or 384 well plates 2. Cells are loaded with reporter assay (typically indicator dye) 3. Compounds are incubated with cells prior to EFS 4. Physiologically relevant EFS protocols are employed using the CXE platform The cell culture is monitored, and fluorescence data is captured to provide dynamic read-out. 5. Off-line image processing through custom SW analysis suite enables generation of pharmacology data Cellaxess Elektra - Typical assay concept 7 Slide 9 Example Electric field stimulation 8 pre-pulse post-pulse Slide 10 Cellaxess Elektra Plate Reader Sample pictures GFP transfected N2A cells in 384 well plate, 5HZ Aq 4x Microscopy image Digitally zoomed, reader 9 Slide 11 Cellaxess ROI-based analysis workflow description 5. Responses to stimuli are quantified (e.g. amplitude or half-width, the parameters that give best pharmacology) 6. Pharmacological effects determined (cell data matched to meta data such as well ID, and compound ID and concentration) for the different treatments 4. From the ROIs, traces over time are generated from the image stack 1. The entire MTP is imaged dynamically, and the image stack is exported to HCA analysis software 2. The images are segmented for allocation of data to individual wells 3. ROI are created on basis of certain response criteria, and applied to the entire image stack 10 Slide 12 Case Study: 11 Electric Field Stimulation of iPSC-derived cardiomyocytes using the Cellaxess Elektra Discovery Platform with high frequency sampling Slide 13 Background The objective was to explore the feasibility of using the Cellaxess Elektra platform to identify molecules which have cardiac rhythm liabilities through EFS-induced pacing of iPSC-derived cardiomyocytes Specifially, we wanted to explore the high speed sampling capability of the Cellaxess Elektra in combination with pacing of the iCMs at physiologically relevant frequencies To reach this goal, a POC study was designed 12 Slide 14 Assay Development Plan Week/ Dec 10th Dec 17th Jan 14th Jan 21st Jan 28th Feb 4th Feb 11th Feb 18th Feb 25th March 4th March 11th Aim Evaluate different calcium assay kits Determine Calcium Indicator and assay workflow Optimise cell density Determine EFS protocol and readout rate Assay stability reserveAssay stability reserveCompile data for report PhI report finalization and review Decide on parameters to analyse and data workflow Explore analysis/pro gramming options Establish analysis/re adouts/sta tistics Validate heating system Hardware & software scripting and modificatio ns 13 Slide 15 Data analysis Export ROI data Igor (Wavemetrics) Scripts for normalization, extracting parameters Graphing CX Elektra pre-read Assay workflow Calcium indicator @ 37C, 7% CO 2 iCell CMs cultured @ 37C, 7% CO 2 14 Compound incubation CX Elektra post-read Slide 16 Cell culture optimization, investigated parameters Optimization performed to identify parameters with respect to response rate, signal, reproducibility and cell health Different calcium indicators investigated FLIPR Calcium 5 Assay Kit chosen Different cell densities were investigated (40k, 30k, 20k, 15k, 10k, 5k, 3k, 1k and 100 viable cells/well) Optimal time in culture was investigated: 48h, 72h, 1 week and 2 weeks Highly consistent cell cultures were generated with 40 000, 30 000, 20 000 and 15 000 cells/well All wells responded to 1Hz and 2Hz pacing Spontaneous activity in all wells with little variation (in amplitude and frequency) between wells 15 Slide 17 Hardware optimization, detection Protocols were optimized for voltage and pulse duration to obtain reproducible pacing and to keep cultures viable throughout experiment Reader scripts were developed to optimize the recording of Ca 2+ transients The platform is capable of up to 70 Hz aquisition rate (example below) but frame rates were optimised to different pacing protocols to be able to analyse relevant parameters without compromising signal (usually between 20 and 35 FPS) 16 Tau = 0.754 0.03 (n=2) HWD = 0.95 0.01 (n=2) Average intensity Time (s) Slide 18 Pacing vs Spontaneously beating cells -Example data 17 EFS (35 V, 2 Hz) EFS (35 V, 1 Hz) Time (s) Spontaneous activity Pacing @ 1 Hz Pacing @ 2 Hz Fluorescence intensity Slide 19 Data analysis 18 Maximum rate change (RFU/s) Largest distance between two samples/sampling interval Decay fitted to single exp between max and min signal Peak width at half peak height ROIs from the camera readout are exported and calculations performed using Igor software. Calculations frequently performed: Frequency, Amplitude, Tau, HWD and TTP can be calculated from the Ca2+ transients. Ability to follow pace can be detected (irregularity-index, occurrence of arrhythmic beats). IC/EC 50 calculations can be derived from any of above parameters. Heat maps, Z Half width duration, HWD (s) Tau ( ) Slide 20 Pharmacology; Z calculations - Isoproterenol vs control Experimental details: Isoproterenol 3M vs control (0.1 % DMSO). Each HWD value is an average of 3 peaks/well. Spontaneous activity and EFS: 0.66 Hz, 35V, 2ms pulse duration, 35 fps, binning 8x8. HWD (paced cells) Frequency (spontaneous activity) z= 0.588 z= 0.617 Both parameters provide an accurate measure of assay performance 19 Slide 21 Pharmacology; Use dependence of Flecainide detected by pacing IC50 was lower for paced cells (1Hz) compared to spontaneously beating cultures (0.4-0.6 Hz) n=2 20 IC 50 (M) Spontaneous activity (amplitude) IC 50 (M) @ 1Hz pacing (amplitude) Experiment 12.740.40 Experiment 21.110.64 Experiment details:15 000 cells/well, cultured for 2 weeks, Calcium 5 (diluted 1:2) Spontaneous activity and 1 Hz pacing,5fps. Data were fitted to Hills equation, each data point is an average of 10 peaks. Slide 22 Pharmacology - Pacing enables pharmacology where spontaneous activity is blocked/non-existing Experimental details: Digoxin 3 M (serial dilution 1:3, 6 conc.) vs control (0.1 % DMSO). Spontaneous activity and EFS: 0.3 Hz, 35V, 2ms pulse duration, 35 fps, binning 8x8. Each value is an average of 3 peaks/well represented with stdev Digoxin (Na + /K + ATPase inhibitor); HWD (spont act) HWD (paced cells) EC50 (M) SD HWD_Paced0.280.03 HWD_Spont0.550.84 Pacing enables visualization of drug effects also where spontaneous activity is blocked (better pharmacology) Time (s) 21 Slide 23 Pacing enables pharmacology also where spontaneous activity is blocked Tau = 0.77 Spontaneous activity Pacing (0.3Hz, 35V, 2ms duration) Digoxin, 3M 22 Time (s) Fluorescence intensity Slide 24 Conclusions In a robust iPSC-derived cardiomyocyte assay, we have demonstrated feasibility of EFS pacing with the Cellaxess Elektra in 96-well format : o A unique combination of high speed, high resolution imaging o Precise pacing of cells in the culture within the physiological frequency range Detects use dependent drugs and the ability to follow pace Enables visualization of drug effects also where spontaneous activity is blocked Enables native pharmacology from mature myocyte cultures o Pharmacology agrees well with literature data (Flecainide, Digoxin, Forskolin, Isoproterenol, Milrinone, Nifedipine, Thapsigargin, Cisapride tested) 23 For further information, please contact us! susanne.lardell@cellectricon.com Slide 25 For further information, please contact us! susanne.lardell@cellectricon.com Thank you for your attention!