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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!.

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  1. Electric Field Stimulation (EFS) of iPSC-derived cardiomyocytes using the Cellaxess® Elektra Discovery Platform with high frequency sampling Note: This is an interactive slideshow withvoiceover. Make sure yourloudspeakersareturned on!

  2. Electric Field Stimulation (EFS) of iPSC-derived cardiomyocytes using the Cellaxess® Elektra Discovery Platform with high frequency sampling Paul Karila, PhD Susanne Lardell, MSc www.cellectricon.com

  3. Contents • CellaxessElektra 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 CellaxessElektra 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

  4. Technology overview 4

  5. Cellaxess Elektra - Platform Overview An automated system for in-situelectricfield manipulation of cell cultures • 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 5

  6. Cellaxess Elektra – in-situelectrostimulation 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

  7. Cellaxess Elektra - Typical assay concept 1. Cells arecultured in 96 well or 384 wellplates 2. Cells areloadedwith reporter assay (typicallyindicatordye) 3. Compoundsareincubatedwith cells prior to EFS 4. Physiologically relevant EFS protocolsareemployedusing the CXE platform The cell culture is monitored, and fluorescence data is capturedtoprovidedynamic read-out. 5. Off-line image processingthroughcustom SW analysissuiteenables generation ofpharmacology data 7

  8. Example – Electric field stimulation pre-pulse post-pulse 8

  9. Cellaxess Elektra Plate Reader – Sample pictures GFP transfected N2A cells in 384 wellplate, 5HZ Aq Digitallyzoomed, reader 4x Microscopy image 9

  10. Cellaxess ROI-based analysis – workflow description 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 toindividualwells 3. ROI are created on basis of certain response criteria, and applied to the entire image stack 4. From the ROIs, traces over time are generated from the image stack 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 10

  11. Case Study: Electric Field Stimulation of iPSC-derived cardiomyocytes using the Cellaxess® Elektra Discovery Platform with high frequency sampling 11

  12. 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

  13. Assay Development Plan 13

  14. Assay workflow iCell CMs cultured @ 37°C, 7% CO2 Calcium indicator @ 37°C, 7% CO2 CX Elektra pre-read Compound incubation CX Elektra post-read • Data analysis • Export ROI data • Igor (Wavemetrics) • Scripts for normalization, extracting parameters • Graphing 14

  15. Cell cultureoptimization, investigatedparameters • Optimizationperformedtoidentify parameters withrespecttoresponse rate, signal, reproducibility and cell health • Different calcium indicatorsinvestigated – FLIPR Calcium 5 Assay Kit chosen • Different cell densitieswereinvestigated (40k, 30k, 20k, 15k, 10k, 5k, 3k, 1k and 100 viablecells/well) • Optimal time in culturewasinvestigated: 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

  16. Hardware optimization, detection • Protocols were optimized for voltage and pulse duration to obtain reproducible pacing and to keep cultures viable throughout experiment • Reader scripts weredevelopedtooptimize the recording of Ca2+ transients • The platformis capableofupto 70 Hz aquisition rate (examplebelow) butframe rates wereoptimisedto different pacingprotocolsto be abletoanalyse relevant parameters withoutcompromising signal (usuallybetween 20 and 35 FPS) Average intensity Tau = 0.754 ±0.03 (n=2) HWD = 0.95 ±0.01 (n=2) 16 Time (s)

  17. Pacing vs Spontaneouslybeatingcells -Example data Spontaneous activity Pacing @ 1 Hz Fluorescence intensity EFS (35 V, 1 Hz) Pacing @ 2 Hz EFS (35 V, 2 Hz) 17 Time (s)

  18. Data analysis ROIs from the camerareadoutare exported and calculationsperformedusing Igor software. Calculationsfrequentlyperformed: 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/EC50calculations can be derived from any of above parameters. Heat maps, Z’ Tau () Maximum rate change (RFU/s) Halfwidth duration , HWD (s) Decay fitted to single exp between max and minsignal Largest distance between two samples/sampling interval Peak widthat half peakheight 18

  19. Pharmacology; Z’ calculations - Isoproterenolvscontrol Frequency(spontaneousactivity) HWD (paced cells) z’= 0.617 z’= 0.588 • Both parameters provide an accuratemeasureofassay performance Experimental details: Isoproterenol 3µM vs control (0.1 % DMSO). Each HWD value is an averageof 3 peaks/well. Spontaneousactivity and EFS: 0.66 Hz, 35V, 2ms pulse duration, 35 fps, binning8x8. 19

  20. Pharmacology;UsedependenceofFlecainidedetected by pacing IC50 was lower for paced cells (1Hz) compared to spontaneously beating cultures (0.4-0.6 Hz) n=2 Experiment details:15 000 cells/well, cultured for 2 weeks, Calcium 5 (diluted 1:2) Spontaneousactivity and 1 Hz pacing ,5fps. Data werefittedtoHill’sequation, each data point is an averageof 10 peaks. 20

  21. Pharmacology- Pacing enables pharmacology where spontaneous activity is blocked/non-existing Digoxin (Na+/K+ ATPase inhibitor); HWD (spont act) HWD (paced cells) Time (s) Pacingenablesvisualizationof drug effects also where spontaneousactivityis blocked (better pharmacology) Experimental details: Digoxin 3 µM (serialdilution 1:3, 6 conc.) vs control (0.1 % DMSO). Spontaneousactivity and EFS: 0.3 Hz, 35V, 2ms pulse duration, 35 fps, binning 8x8. Eachvalue is an averageof 3 peaks/wellrepresentedwithstdev 21

  22. Pacing enables pharmacology also where spontaneous activity is blocked Digoxin, 3µM Spontaneous activity Fluorescence intensity Pacing (0.3Hz, 35V, 2ms duration) Fluorescence intensity Tau = 0.77 Time (s) 22

  23. Conclusions • In a robust iPSC-derived cardiomyocyteassay, we have demonstrated feasibility of EFS pacing with the CellaxessElektra in 96-well format : • Aunique combination of high speed, high resolution imaging • Precise pacing of cells in the culture within the physiological frequency range • Detects use dependent drugs and the ability to follow pace • Enablesvisualizationofdrugeffectsalsowherespontaneousactivity is blocked • Enablesnativepharmacology from maturemyocytecultures • Pharmacologyagreeswellwithliteraturedata (Flecainide, Digoxin, Forskolin, Isoproterenol, Milrinone, Nifedipine, Thapsigargin, Cisapride tested) For further information, pleasecontactus! susanne.lardell@cellectricon.com 23

  24. Thankyou for your attention! For further information, pleasecontactus! susanne.lardell@cellectricon.com

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