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Electric Field Stimulation (EFS) of iPSC-derived cardiomyocytes using the Cellaxess® Elektra Discovery Platform with high frequency sampling Note: This.

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Presentation on theme: "Electric Field Stimulation (EFS) of iPSC-derived cardiomyocytes using the Cellaxess® Elektra Discovery Platform with high frequency sampling Note: This."— Presentation transcript:

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 with voiceover. Make sure your loudspeakers are turned 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

3 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

4 Technology overview 4

5 Cellaxess Elektra - Platform Overview
An automated system for in-situ electric field 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-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

7 Cellaxess Elektra - Typical assay concept
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 7

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

9 Cellaxess Elektra Plate Reader – Sample pictures
GFP transfected N2A cells in 384 well plate, 5HZ Aq 4x Microscopy image Digitally zoomed, reader 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 to individual wells 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
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 reserve Compile data for report PhI report finalization and review Decide on parameters to analyse and data workflow Explore analysis/programming options Establish analysis/readouts/statistics Validate heating system Hardware & software scripting and modifications 13

14 Assay workflow Data analysis iCell CMs cultured Calcium indicator
@ 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 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 , , and 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 were developed to optimize the recording of Ca2+ 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) Average intensity Tau = ±0.03 (n=2) HWD = 0.95 ±0.01 (n=2) 16 Time (s)

17 Fluorescence intensity
Pacing vs Spontaneously beating cells -Example data Spontaneous activity 1 Hz Fluorescence intensity EFS (35 V, 1 Hz) 2 Hz EFS (35 V, 2 Hz) 17 Time (s)

18 Maximum rate change (RFU/s)
Data analysis 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/EC50 calculations can be derived from any of above parameters. Heat maps, Z’ Tau () Maximum rate change (RFU/s) Half width duration , HWD (s) Decay fitted to single exp between max and min signal Largest distance between two samples/sampling interval Peak width at half peak height 18

19 Pharmacology; Z’ calculations - Isoproterenol vs control
Frequency (spontaneous activity) HWD (paced cells) z’= 0.588 z’= 0.617 Both parameters provide an accurate measure of assay performance Experimental details: Isoproterenol 3µM 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. 19

20 Pharmacology; Use dependence of Flecainide detected by pacing
IC50 was lower for paced cells (1Hz) compared to spontaneously beating cultures ( Hz) n=2 IC50 (µM) Spontaneous activity (amplitude) IC50 1Hz pacing (amplitude) Experiment 1 2.74 0.40 Experiment 2 1.11 0.64 Experiment details: cells/well, cultured for 2 weeks, Calcium 5 (diluted 1:2) Spontaneous activity and 1 Hz pacing ,5fps. Data were fitted to Hill’s equation, each data point is an average of 10 peaks. 20

21 Digoxin (Na+/K+ ATPase inhibitor);
Pharmacology - Pacing enables pharmacology where spontaneous activity is blocked/non-existing Digoxin (Na+/K+ ATPase inhibitor); HWD (spont act) HWD (paced cells) Time (s) EC50 (µM) SD HWD_Paced 0.28 0.03 HWD_Spont 0.55 0.84 Pacing enables visualization of drug effects also where spontaneous activity is blocked (better pharmacology) Experimental details: Digoxin 3 µM (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 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 cardiomyocyte assay, we have demonstrated feasibility of EFS pacing with the Cellaxess Elektra in 96-well format : A unique 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 Enables visualization of drug effects also where spontaneous activity is blocked Enables native pharmacology from mature myocyte cultures Pharmacology agrees well with literature data (Flecainide, Digoxin, Forskolin, Isoproterenol, Milrinone, Nifedipine, Thapsigargin, Cisapride tested) For further information, please contact us! 23

24 For further information, please contact us!
Thank you for your attention! For further information, please contact us!


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