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Cellartis Cardiomyocytes

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1. 6 References 6 References Abnormal calcium handling properties underlie familial hypertrophic cardiomyopathy pathology in patient specific induced pluripotent stem cells Lan F Lee AS Liang P Sanchez Freire V Nguyen PK Wang L Han L Yen M Wang Y Sun N Abilez OJ Hu S Ebert AD Navarrete EG Simmons CS Wheeler M Pruitt B Lewis R Yamaguchi Y Ashley EA Bers DM Robbins RC Longaker MT Wu JC Cell Stem Cell 12 1 101 13 2013 Patient specific induced pluripotent stem cells as a model for familial dilated cardiomyopathy Sun N Yazawa M Liu J Han L Sanchez Freire V Abilez OJ Navarrete EG Hu S Wang L Lee A Pavlovic A Lin S Chen R Hajjar RJ Snyder MP Dolmetsch RE Butte MJ Ashley EA Longaker MT Robbins RC Wu JC Sci Transl Med 4 130 2012 A novel method of selecting human embryonic stem cell derived cardiomyocyte clusters for assessment of potential to influence OT interval Yamazaki K Hihara T Taniguchi T Kohmura N Yoshinaga T Ito M Sawada K J Toxicol In Vitro 26 2 335 342 2011 Usefulness of field potential as a marker of embryonic stem cell derived cardiomyocytes and endpoint analysis of embryonic stem cell test Koseki N Deguchi J Yamada T Funabashi H Seki T Toxicol Sci 35 6 899 909 2010 Improvement of the embryonic stem cell test endpoint analysis by use of field potential detection Koseki N Deguchi J Yamada T Funabashi H Seki T J Toxicol Sci 35 5 619 29 2010 P
2. Cellartis Cardiomyocytes i Contents 4 2 Field potential duration analysis cece cece cece eee eee eee e eens nen nn 14 FPD analyses available in Mobius QT software eeeeerrnnnnn nnne 15 Exporting extracted waveforms eeeeesseeeee enne nnn nnn 16 4 3 Amplit de and slope analysis 1 5 ei er a Pe OR i e x a cil a 19 5 Pacing hPSC CMs 20 S T Suppressing stiaulHs artifactSsssiuberiosus voe P Erebi A d bt Da od PT E ad 20 1 Use of platinum wire reference electrodes eeenennn n nnn 20 2 Applying bi Dpolar stimulation isssasasaxens E rex eR CUP EVE C Ve iE a RP iE R Sd cr ER 20 5 2 Acqulrmg paceu dcblViby sibus sodes odit quaa ne prd Guana muse aaa Re EU MuR 21 6 References 22 ii MED64 Application Note Cellartis Cardiomyocytes 1 Introduction 1 Introduction Human pluripotent stem cell derived cardiomyocytes hPSC CMs are the subject of intense study in both the pharmaceutical industry and academic institutions The MED64 has become a popular platform for modeling human hereditary cardiomyopathies and drug screening since hPSC CMs possess the same genetic background as primary human CMs and their pharmacology is presumably very similar The MED64 only requires culture of the hPSC CMs on the MED Probe to extract basic electrophysiological parameters such as beat frequency and field potential duration FPD In addition chronotropic drug effects can be measured thanks
3. CM Culture Medium can be used 2 6 Alternative method for coating and plating Cellartis Cardiomyo cytes Here is an alternative method for coating and plating Cellartis Cardiomyocytes Workflow Day 6 or 7 Data Acquisition Thawing onto Medium Plating onto Medium culture plate exchange MED Probe exchange Fibronectin coating MED Probe MED64 Application Note Cellartis Cardiomycoytes 5 2 Plating and culturing Cellartis Cardiomyocytes Coating the MED Probe PEI coating is omitted in this method Place a dry MED Probe in a sterile 100 mm diameter culture dish Place a sterile 35 mm diameter culture dish next to the MED Probe and fill the dish with sterile water to create moist environment Place a 2 ul bead of fibronectin 50 ug mL Becton Deckinson Cat 354008 over the recording electrode area on the MED Probe Incubate the fibronectin coated MED Probe at 4 C overnight Plating Cellartis Cardiomyocytes onto the MED Probe Aspirate 1 ul of the fibronectin from the MED Probe Place a 2 ul bead of the cardiomyocyte suspension corresponding to 30 000 45 000 viable car diomyocytes over the recording electrode area on the MED Probe Note Do not let the fibronectin coated area dry completely before seeding the cells If seeding Cellartis Cardiomyocytes on several MED Probes it is recommended to take one probe at a time CAUTION The MED Probe has 4 reference electrodes as well a
4. Cardiomyocytes onto the MED Probe Aspirate the matrigel from the MED Probe Place a 7 ul bead of the cardiomyocyte suspension corresponding to 42 000 49 000 viable cardio myocytes over the recording electrode area on the MED Probe Note Do not let the matrigel coated area dry completely before seeding the cells If seeding Cellartis Cardiomyocytes on several MED Probes it is recommended to take one probe at a time CAUTION The MED Probe has 4 reference electrodes as well as 64 recording electrodes The 4 reference electrodes MUST be free from cells but covered by medium for signal recording 4 MED64 Application Note Cellartis Cardiomyocytes 2 Plating and culturing Cellartis amp Cardiomyocytes 3 Incubate the MED Probe with the seeded Cellartis Cardiomyocytes in a cell culture incubator at 37 C 5 CO for 2 hours allowing the cells to adhere 4 Gently add 2 ml of the MEA Plating Medium with 5 uM Y 27632 to the MED Probe taking care not to dislodge the cells 5 Incubate in a cell culture incubator at 37 C 5 CO2 6 After 24 48 hours change the medium to Cellartis CM Culture Medium Cat Y10063 10 FBS After this first medium exchange half volume medium changes should be performed every 2 3 days depending on appearance pH Recordings can be started 1 day after the change to Cellartis CM Culture Medium cardiomyocytes should have started to beat in standard assay medium of choice Cellartis
5. Store the MED Probe according to instructions in a beaker with DDW in a darkened area please refer to the MED Probe product manual MED64 Application Note Cellartis Cardiomycoytes 7 3 Data acquisition 3 Data acquisition This section will describe settings that are generally recommended for data acquisition from human pluripotent stem cell derived caordiomyocytes hPSC CMs 3 1 Setting up the microenvironmental conditions 35 8 36 5 C is the recommended temperature for recording from hPSC CMs Beat frequency is very sensitive to temperature Beat rate can increase dramatically as temperature rises above 37 C Changes in pH can also cause fluctuations in beat frequency Changes in pH can be caused by changes in CO concentration in the air Thus it is very important to let the hPSC CM sit in an environment where the CO concentration is stable Two methods are recommended to achieve stability of temperature and the CO concentration 1 Use of CO incubator Place the MED Connector MED C03 inside a CO2 incubator Please note that incubators can introduce noise Please refer to page 29 36 in the MED64 Handbook vol1 to avoid noise introduced by incubators Particularly recordings can be compromised by noise introduced by the rapid temperature increases that occur as they power on Wait until the desired temperature is stable before starting acquisitions it could take several hours depending on the incubator Pla
6. St 2011 Mar D7 23 41 27 08 time secs phase 0 9809 1 8287 2 6727 4 517 4 3618 3 05 6 0545 6 9018 71991 8 59054 94264 102975 11 1503 12 0029 12 8557 chg MC co O 00 ON E45 5 647 3 Beats per Minute N amp E47 3 7C 850 B51 1 B52 8 2 4 6 8 10 12 852 8 Time seconds 70 Figure 4 Instantaneous beat freguency calculated and graphed for publication A The interspike interval ISI data exported as an ASCII file contains the timepoints and ISI in milliseconds Column C In this case Microsoft Excel has been used to open the file The instantaneous beat freguency can be obtained for each timepoint dividing 1000 by the Interspike Interval in ms and multiplying the result by 60 as seen in the function dialogue box fx 2 1000 IS1 60 B Any software package such as Excel Igorpro or MATLAB can be used to produce graphs for plotting the instanta neous beat frequency changes over time obtained from the ISI ASCII file In this example Igorpro has been used Graphing chronotropic drug effects For presentation purposes it is useful to display the steady state effect of a drug on the hPSC CM preparation s spontaneous beating This can be done using the Spike train workflow following the procedure below m Download the Spike train workflow from http www med64 com resources utilities html 2 Inthe Replay Raw Data control panel select the file Trace Trace time and Channel to be
7. all liabilities Please refer to the scientific literature for further insight on these techniques as well as the MED64 and Mobius manuals for detailed instructions on use of the MED64 System MED64 Application Note Cellartis Cardiomycoytes 1 2 Plating and culturing Cellartis amp Cardiomyocytes 2 Plating and culturing Cellartis Cardiomyocytes 2 1 Materials to be prepared Biological material Either of 1 or 2 Cellartis amp Pure Cardiomyocytes from SA121 Clontech Y10061 Cellartis amp CM Culture Base Takara Clontech Y10063 Na2B407 10H20 Sigma 59640 m 7 Caa TH Matrigel Membrane Matrix GRF Matrigel 3 Corning 356231 PBS Dulbecco s with Ca2 amp Mg2 Gibco Life Technologies or various 14040 Trypsin EDTA 0 25 phenol red Gibco Life Technologies or various 25200 Fibronectin 4 Becton Deckinson 354008 MED Probe Alpha MED Scientific MED P515A Petri dish 35 mm Various Kimwipe Various Centrifuge Various 3 Not necessary for the Alternative method in the page 5 6 4 Necessary only for the Alternative method in the page 5 6 2 MED64 Application Note Cellartis Cardiomyocytes 2 Plating and culturing Cellartis amp Cardiomyocytes 2 2 Workflow Cellartis Cardiomyocytes cryopreserved human pluripotent stem cell derived cardiomyocytes from Takara Clontech are initially thawed onto standard tissue culture plates coated with fibronectin Please r
8. doa d 2 pube indic Cr 3 2 2 Preparing the MED iPrODES wi irren ex Urpxs EU o ket yx Ur cUm hi a 3 2 3 Collecting Cellartis Cardiomyocytes from the tissue culture plate 4 2 4 Plating Cellartis Cardiomyocytes onto the MED Probe ennnee 4 2 5 Alternative method for coating and plating Cellartis Cardiomyocytes 5 Coating tne MED ProD eei db DM x QUE eM duc Rebus dot Gad cua ibo om m RU 6 Plating Cellartis Cardiomyocytes onto the MED Probe enne 6 2 6 Cleaning the used MED Probes ecce eon rre nn 7 Trypsin EDTA collagenase treatment method een nnne 7 Bl dchihd HIetliod estuve at Cb n a Brabus ke byve tena Casto h kanaa m nt 7 3 Data acquisition 8 3 1 Setting up the microenvironmental conditions eese nnne 8 1 Use of the CO gt INCUD ALON sasestuni suu tu ORAE EuEE Ne RM ID Ed eva ORE ALORS sd K yk 8 2 Use of the MED Heated Connector essen 8 32y Dataa cq iSON Nee ERE 9 Available Mobius workflow templates cesse mmm 10 Recommended acquisition settings eeeeessseeeee enne nnn 10 4 Data analysis 11 4 1 Beat Treg ericy anhalyslS suiit orani runt antenna aliens cd iV LU tau TEM Ce a 11 Instantaneous beat frequency analysis eeeeseeeeeee nnne 11 Graphing chronotropic drug effects eeeeeseeeeeeeee nnnm nnnm 12 MED64 Application Note
9. exported 3 Inthe Filter Raw Data control panel select Down sampling for the Filter Type and select a sampling frequency of 1000 Hz This can be modified depending on your signals 4 In the Export Raw Data control panel input the name of your choice for the file to be generated select the type of data format and check the Enable storage check box 5 Run the workflow with the Green Red button 12 MED64 Application Note Cellartis Cardiomyocytes 4 Data analysis Workflow Layout Help b ou b working Directory Annotations Main Replay Raw Data File Display All Channels Filename 20110920 14h00m35 modat Traces 1 to 1 Trace time O to 120000 Channels Delay ms O Trace Trace time Filter Raw Data Filter type Down sampline Sampling freq Hz 1000 Export Raw Data Filename modifier mave Enable storage Format ASCI text Figure 5 Spike train workflow The data downsampled to 1 kHz for channel 7 acquired between 0 120 seconds can be exported by clicking the Green Red button 6 Theresulting ASCII or CSV file can then be imported into the software package of choice to produce a graph with the region of interest as shown in Figure 6 Baseline 1 uM Isoproterenol II I i ii cael eel nya i kut peeled eel udha el cael as k AAA EBENEN BN HELD I KERTYI 0 2 mV 5000 ms A B Figure 6 Graphing chro
10. next to it Figure 14B MED64 Application Note Cellartis Cardiomycoytes 17 4 Data analysis m U I3 ii 0 05 5 63E 11 0 1 9 61E 10 0 15 8 11E 09 0 2 4 54E 08 0 25 1 90E 07 0 3 6 42E 07 0 35 1 82E 06 0 4 4 49E 06 5 9 86E 06 0 5 1 96E 05 0 55 3 60E 05 0 6 6 14E 05 10 0 65 9 86E 05 11 0 7 0 00015 0 75 0 00022 M 16 08 00003 a c da cn 12 a T M 4 H Sheetl 4 am b M gt MH Sheetl 4 b HB ICI pg 10056 Average 0 275 Count 10 i A B Figure 13 Creating the Excel spreadsheets to graph a waveform A Make a new spreadsheet with one column numbered in 0 05 intervals from 0 05 up to the total amount of milliseconds analyzed in the workflow B Paste the data copied from the waveform chart in the Extract Spike Measures panel 8 Now you can make a graph of the field potential waveform plotting the data vs the time using any software package with graphing capabilities such as Excel Igorpro or Matlab The averaged wave forms for each dose can be superimposed to create a figure demonstrating the dose dependence of FPD prolongation Figure 14 200 uV 100 ms Figure 14 FPD prolongation with administration of a hERG blocker FPD prolongation induced by quinidine a well known hERG blocker Any data analysis software can be used to produce graphs obtained from the ASCII file generated by Mobius In this example Igorpro Wavetetrics L
11. this example FPD analyses available in Mobius QT software There are 3 built in analysis methods for measuring FPD in Mobius QT 1 Time of Amplitude Max Min to Max Min Measures the timepoint at the signal s maximum minimum amplitude between the 2 cursors on the left and 2 cursors on the right Figure 9 Figure 9 FPD Analysis using the Time of Amplitude Max to Max analysis This method measures the duration between the maximal datapoint in the initial fast depolarizing spike between the first two cursors and the maximal datapoint in the slow repolarizing wave between the second two cursors MED64 Application Note Cellartis Cardiomycoytes 15 4 Data analysis 2 Time of Crossing Horizontal Cursor Computes the FPD by measuring the duration between the left vertical cursor Cursor 1 and the first intersection of the waveform with the horizontal cursor AFTER the right vertical cursor Cursor 2 Figure 11 Figure 10 FPD Analysis using the Time of Crossing Horizontal Cursor analysis The time between the first cursor and the first intersection of the waveform with the horizontal cursor AFTER the right vertical cursor is calculated in this measurement 3 Time of Slope Crossing Horizontal Cursor This analysis uses four cursors as shown in Figure 11 three vertical and one horizontal It computes the time between the left most vertical cursor Cursor 1 to the intersection of the linear fit slope line and th
12. to the non invasive nature of the MED Probe Nonetheless the MED64 can pace the hPSC CMs using any of the microelectrodes to study frequency or use dependent drug effects The goal of this application note is to describe how to set up experiments with human pluripotent stem cell derived cardiomyocytes from Takara Clontech Cellartis Cardiomyocytes acquire relevant data and extract the data for presentation or publication This material has been prepared by scientists with expertise in stem cell biology and cardiovascular pharmacology A complete protocol for plating culturing and carrying out experiments on hPSC CMs has been prepared based on the users experience 1 1 Acknowledgement Alpha MED Scientific would like to thank the MED64 users that have shared their knowledge Caroline Am en PhD Sofie Danielsson M Sc Kerstin Dahlenborg B Sc Daniella Steel PhD Peter Sartipy PhD Takara Bio Europe AB Gothenburg Sweden Yuki Yamamoto M Sc Takara Bio Inc Otsu Shiga Japan Michael Trujillo PhD Senior Application Scientist Alpha MED Scientific 1 2 Disclaimer This application note is a summary of information shared by MED64 users and isto be considered marketing material These methods have been developed tested and verified in the course of projects published in peer reviewed literature However Alpha MED Scientific does not guarantee that the information written in this document is correct and is free from
13. 50 fil 70 50 90 0 I 01 04 0 06 0 08 0 1 0 12 i 0 16 0 17658 Time ms Time minutes Channel 24 Average 3 w Traces Capture Labels Reset Probe 1 well w Sync Thresh 0 Window 125 w ms Hide phases V Autoscale Measures Figure 16 Analyzing the fast depolarizing spikes with Mobius In this analysis Slope 1090 Linear Fit has been chosen On the left graph in the Extract Spike Measures panel the cursors are placed for analyzing the region of interest in the waveform The right graph shows the slope values over time MED64 Application Note Cellartis Cardiomycoytes 19 5 Pacing hPSC CMs 5 Pacing hPSC CMs Field potential duration FPD is correlated with beat frequency Evaluation of FPD with pacing is useful for normalizing the experiments to the same frequency and also for determining frequency or use dependent drug effects 5 1 Suppressing stimulus artifacts When pacing please follow the instructions below to suppress stimulus artifacts 1 Use of platinum wire reference electrodes Insert the platinum wire into the MED Probe chamber and ground it using the alligator clip on the top unit of the MED Connector A B Figure 17 Grounding the platinum wire to the MED Connector A Grounding the platinum wire with an open chamber B Grounding the platinum wire with the Perfusion Cap 2 Applying bi polar stimulation Stimulate using 2 adjacent electrodes simultaneously to induce pacing ac
14. The most sensitive microelectrode array system for in vitro extracellular electrophysiology MED64 Application Note Cellartis Cardiomyocytes human pluripotent stem cell derived cardiomyocytes TakaRa C Clontech ALPHA MEE SCIENTIFIC Information in this document is subject to change without notice No part of this document may be reproduced or transmitted without the expressed written permission of Alpha MED Scientific Inc While every precaution has been taken in the preparation of this document the publisher and the authors assume no responsibility for errors omissions damages resulting from the use of information contained in this document or from the use of programs and source code that may accompany it In no event shall the publisher and or the author be liable for losses of profit or any other commercial damage caused or alleged to have been caused directly or indirectly by this document 2015 Alpha MED Scientific Inc All rights reserved Version 1 00 August 1 2015 Alpha MED Scientific I nc Saito Bio Incubator 209 7 7 15 Saito asagi Ibaraki Osaka 567 0085 Japan E mail support med64 com Website http www med64 com Contents 1 Introduction 1 TAL AcknowledgemebLt sva ra Vari S rapto PD O Gand dup EDU Edu Cad ind in a td madot 1 12 BICIS SIBI sta T TE E E EET E E TIT 1 2 Plating and culturing Cellartis amp Cardiomyocytes 2 2 1 Material tO De Drepal etd rss oui eara i a Ede o ERR Bad A da
15. ake Osego OR USA has been used to overlay field potential waveforms recorded during administration of incremental concentrations of quinidine 18 MED64 Application Note Cellartis Cardiomyocytes 4 Data analysis 4 3 Amplitude and slope analysis Amplitude and slope analysis is useful for studying the ionic components of the hPSC CM field potential For example the initial fast depolarizing spike is mainly due to sodium influx thus block of cardiac sodium channels can be examined by analyzing its amplitude and slope Below in Figure 15 an expanded view of the fast depolarizing spike in Figure 14 demonstrates quinidine s well known blockage of the fast activating sodium current Figure 15 Dose dependent changes in field potential amplitude and slope due to a sodium channel blocker Expanded view of hSC CM field potential s fast depolarizing spike demonstrating the dose dependent reduction in amplitude due to blockage of the fast activating sodium current These analyses can be performed using Mobius by selecting Amplitude or and Slope measurement in the Extract Spike Measure module in the OT recording analysis workflow template Please refer to page 108 in the Mobius Tutorial for instructions on performing amplitude and slope analyses Extract Spike Measures Sync Firing Event 12 100 K 1 Potential uv I 200 Slope 1090LinearF it u ms 300 330 1 l l i l 40
16. ce the drug aliquot to be used for your experiment in a heated water bath to minimize the temperature changes caused by drug applications Administer the drugs quickly as opening the incubator door allows the temperature to cool down and the CO concentration to change which can change beat rate When the incubator door is opened note and consider waiting until the stability in desired temperature and the CO2 concentration is achieved before re starting acquisitions it could take longer than drug effect 2 Use of the MED Heated Connector The MED Heated Connector MED CPO2H warms up the MED Probe chamber bottom The use of the MED Connector Cover MED CCO1 is recommended to maintain the desired temperature and CO con centration to avoid the pH changes around the MED Probe Provide gas mixtures the mix ratio depends on composition of your culture medium through the gas port on the top of the Connector Cover Bubble the gas through a beaker containing distilled water to maintain a humidified environment In order to achieve stability for the temperature and the CO2 concentration follow the instructions below 1 Turn on the ThermoClamp M 1 controller for the MED Heated Connector connected to the MED Heated Connector at least 30 minutes before starting acquisition 2 If When the set temperature is changed wait for the temperature measured by the ThermoClamp M 1 controller to stabilize at the new temperature It could take 5 30 min
17. coating materials However the MED Probes can be re used if they are handled and cleaned very carefully The following are protocols MED64 users recommend CAUTI ON ALWAYS avoid contact with the surface of the MED Probe to preserve the microelectrodes and insulation layer Trypsin EDTA collagenase treatment method 1 Pipette 0 5 MM Trypsin EDTA GIBCO Cat 25300 054 into the MED Probe chamber and incubate for 1 hour 2 Rinse the chamber with PBS 3 times 3 Dissolve collagenase type I Sigma Cat C0130 in PBS at a concentration of 20 unit ml 4 Pour the collagenase solution into the chamber and incubate for 1 hour at 37 C 5 Discard the used collagenase solution and rinse the MED Probe with double distilled water DDW at least 3 times 6 Dry the MED Probe in a clean area 7 Store the MED Probe according to instructions in a beaker with DDW in a darkened area please refer to the MED Probe product manual Bleaching method 1 Carefully pour or pipette 1 ml of Bleach Clorox into the MED Probe and leave it for about 15 30 seconds 2 Aspirate and repeat step 1 three times If the MED Probe is not clean after this apply 2 more rinses and a longer exposure usually 1 2 minutes is enough but as long as 15 minutes is acceptable CAUTI ON Avoid exposing the MED Probe to Bleach over 20 minutes 3 Rinse the chamber 5 times with double distilled water DDW 4 Allow it to dry or aspirate in a sterile hood 5
18. dow size ac Arbitrary s 10 Eu Channel 20 Aas R Save Beats per Minute Filename modifier Display Interspike Intervals Enable 1800 Compute Interspke Intervals Average Channel ISI 593 3 ms Save Irterspike Intervals 1 g i i i i i i O i i J Fil 0 01 02 03 04 05 06 0 7 0 8 09 1 11 12 13 14 1 5373 ilename modifier Time minutes Enable Channel 20 Autoscale Display Extracted Spikes Extracted Spies 200 J J 1 0 000 200 000 400 000 500 000 Time ms Figure 3 Screen shot of Mobius QT s built in workflow for beat frequency analysis Cardiac signals that surpass user determined thresholds are extracted Beat freguencies and ISI can be saved as ASCII files Beat freguencies and interspike intervals ISI are computed and graphed I nstantaneous beat freguency analysis The beat freguency and ISI data can be exported as ASCII files using the aforementioned workflow templates Instantaneous beat freguency can be calculated dividing 1000 by the Interspike Interval in milliseconds and multiplying the result by 60 as seen in the function dialogue box fx 1000 ISI 60 in the Figure 4 The calculated instantaneous beat freguency data can easily be graphed in Excel or any data analysis software to make a figure showing the timecourse of a drug effect on beating freguency MED64 Application Note Cellartis Cardiomycoytes 11 4 Data analysis 1 File Forma 20071201 E 2 Session
19. e horizontal cursor Cursor 2 and Cursorn 3 are used to select the portion of the waveform to be fitted with the slope line Figure 11 FPD Analysis using the Time of Slope Crossing Horizontal Cursor analysis This analysis method measures the duration between the first cursor and the timepoint the linear fit slope line between the Cursor 2 and 3 crosses the horizontal cursor NOTE The QT interval is defined as the period between the beginning of the ORS complex and the end of the T wave The most accurate correlate of this measurement can be obtained by measuring the time between the initial deviation from the isoelectric baseline preceding the beginning of the fast depolarizing spike and the return to baseline after the slow repolarizing wave 16 MED64 Application Note Cellartis Cardiomyocytes 4 Data analysis Exporting extracted waveforms Mobius QT can easily export waveforms as ASCII files Often pharmacological studies require 10 to 15 minutes to ensure the effect has stabilized but usually only the last 30 seconds of the recording are analyzed offline For presentation purposes comparison of the waveforms averaged for the last 30 seconds of the recording is often desired In order to perform this determine the number of spikes during these last 30 seconds then average the waveform this number of times in Mobius QT The averaged waveform can be obtained on the left chart in the Extract Spike Measures panel using the fol
20. efer to the Cellartis Cardiomyocytes User Manual From day 3 after thawing the cells can be plated onto the MED Probe Recordings can be started 2 days post plating on the MED Probe Day4 or5 Day5 or6 Data Acquisition Thawing onto Medium Matrigel coating Medium culture plate exchange MED Probe exchange PEI coating Plating onto MED Probe MED Probe 2 3 Preparing the MED Probes CAUTI ON Electrodes in the MED Probe are extremely fragile Avoid contact with electrodes in all of following procedures 1 Rinse a MED Probe with sterilized distilled water SDW at least three times Fill the MED Probe with 70 ethanol and leave it for 30 minutes 2 Aspirate the ethanol in a sterile hood 3 Fill the MED Probe with 0 1 Polyethylenimine PEI in 25 mM borate buffer pH 8 4 Leave it at room temperature for overnight make sure the electrodes are covered by the PEI 4 Aspirate the PEI and rinse the MED Probe with double distilled water DDW at least 4 times Note This PEI coating should be performed on each occasion before preparing the MED Probe not only the first time 5 Place a dry MED Probe in a sterile 100 mm diameter culture dish 6 Place a sterile 35 mm diameter culture dish next to the MED Probe and fill the dish with sterile water to create moist environment 7 Place a 7 ul bead of matrigel Corning Matrigel Membrane Matrix GFR Cat 356231 diluted in DPBS to a final concentration o
21. es Ch 10 for F1 Ch 18 for F2 are selected for stimulation 5 2 Acquiring paced activity The interval between stimuli should be shorter than the interspike interval during spontaneous activity to drive beating because a pacing stimulus will be ineffective during a spontaneous beat s absolute refractory period The beat rate at the recommended temperature settings 35 8 36 5 C is usually less than 50 BPM so pacing the hPSC CMs at 60 100BPM the normal adult human heart rate should not be a problem The Pacing recording workflow template s default settings will pace the hPSC CMs at 60BPM The stimulus parameters below are a good starting point for triggering beating If this fails to stimulate beating increase the stimulus amplitude and duration up to 200 uA x 0 6 msec Please refer to the MED64 Product manual for optimal stimulus current amplitudes and durations to avoid electrolysis Stimulus duration 0 4 0 6 msec Stimulus current amplitude 50 uA Successful pacing will result in a fast depolarizing spike immediately after the stimulus artifact as shown in the Figure 19 Trace 1 Stepl 0 200 0 100 0 000 Potential mV 0 100 I I D 6 2 6 8 6 4 6 6 Trace time s Figure 19 Paced activity in hPSC CMs Fast depolarizing spikes following the stimulus artifacts demonstrate the pacing stimulus is effective MED64 Application Note Cellartis Cardiomycoytes 21
22. f 200 ug ml over the recording electrode area on the MED Probe 8 Incubate the matrigel coated MED Probe in a cell culture incubator at 379C 5 CO2 for 3 hours MED64 Application Note Cellartis Cardiomycoytes 3 2 Plating and culturing Cellartis Cardiomyocytes Note Do not let the matrigel coated surface dry completely before seeding the cells 2 4 Collecting Cellartis Cardiomyocytes from the tissue culture plate L 10 11 Make Cellartis MEA Plating Medium by adding 20 FBS to Cellartis CM Culture Base Cat Y10063 Pre heat an appropriate amount of Cellartis MEA Plating Medium to 37 C Add Y 27632 to a final concentration of 5 uM prior to use Aspirate the medium from each well containing cardiomyocytes Rinse the wells with DPBS Add 0 25 trypsin EDTA to each well 80 ul per cm and incubate for 2 4 minutes Gently detach the cells by dispensing the dissociation solution over the surface using a 1 ml pipettor Add 1 volume 80 ul per cm of the MEA Plating Medium with 5 uM Y 27632 to each well to deactivate the trypsin Transfer the cell suspension into a suitable tube Count the cardiomyocytes Centrifuge the cells at 200 x g for 5 minutes at room temperature Aspirate the supernatant and gently resuspend the cell pellet in an appropriate volume of the MEA Plating Medium with 5 uM Y 27632 to a final concentration of 6 000 7 000 viable cardiomyocytes ul 2 5 Plating Cellartis
23. lowing procedure Refer to Figure 13 1 Run the data with the Green button and identify the number of spikes traces during the last 30 seconds 2 Check the Averages Check box 3 Input the number of spikes during the last 30 seconds in the Traces selector 47 at the Figure 12 4 Select the time for the last 30 seconds in the Replay Raw Data module in Main tab 5 Run Mobius by clicking the Green button The waveform for the averaged data is obtained in the left waveform chart Extract Spike Measures 162 1 162 Sync Firing Event 58 500 400 161 8 200 161 6 1614 Potential uv 200 7 1612 TimeOf AmplitudeMaxToMaxims 400 161 160 9 0 i i i i i i i l 50 100 150 200 250 300 350 400 450 500 Time ms 500 4 0 Channel 29 wh V Average 47 w Traces Labels Reset Probe 1well w Sync Thresh 0 w Window 125 w ms Hide phases 7 Autoscale Figure 12 Averaging waveforms All traces for the last 30 seconds 47 traces in this example are averaged and the resulting waveform is displayed in the left chart 6 Right click on the left waveform graph then select Copy data 7 Create an Excel spreadsheet file with one column numbered in 0 05 intervals from 0 05 up to the total amount of milliseconds in the Extract Spike Measures module panel so the data is matched to it Figure 14A Then paste the waveform data in the column
24. nding on purpose of your study High cut freq Low pass filter 1000 Hz These are guidelines that can be changed depending on the signal amplitude and or purpose of the study Particularly change the Input Range to 5 0 mV when 10 kHz is selected for the low pass filter Please refer to page 88 in the Mobius Tutorial for more details 10 MED64 Application Note Cellartis Cardiomyocytes 4 Data analysis 4 Data analysis The MED64 can easily record field potentials from beating hPSC CMs Mobius QT has a variety of built in analyses for studying different parameters Analyses can be exported for post processing using other software packages This section will introduce you to analysis which is generally used for signals acquired from hPSC CMs 4 1 Beat frequency analysis Mobius QT s built in Beat recording and Beat frequency analysis workflow templates can extract beat frequency and interspike interval ISI both during and post acquisition respectively Please refer to page 98 and 99 in the Mobius Tutorial for detailed instructions 6 Mobius win7 0 4 1 Rev 268 on 2013 03 03 Workflow Layout Help m gt wo D Working Directory Annotations Man Detect beating Averages Extract Lone Spikes Display Beats per Minute Disable vannet EXEN so lt 2 0 15 50 4 3 0 15 50 450 4 0 15 50 4 N Tn ar En Thresh Thresh Pre Post mv m ms s Compute Bests per Minute Binnine win
25. notropic drug effects A Rhythmic spontaneous field potentials at baseline B Steady state effect of isoproterenol 1 uM on beat frequency MED64 Application Note Cellartis Cardiomycoytes 13 4 Data analysis 4 2 Field potential duration analysis The hPSC CM field potential typically starts with a fast depolarizing spike due mainly to Na influx followed by a slow repolarizing wave secondary to K efflux Field potential duration FPD is the electro physiological correlate of the QT interval obtained via clinical EKG and the cardiomyocyte action potential duration obtained via patch clamping QT interval prolongation is a risk factor for Torsades de Pointes a life threatening arrhythmia Thus FPD measurement is a parameter that is used to predict cardiac safety QT Interval A Action Potential Duration B Field Potential Duration FPD Repolarizing wave Depolarizing spike C Figure 7 QT interval action potential duration and field potential duration A Classic EKG trace illustrating the QT interval B Ventricular hPSC CM action potential recorded via whole cell patch clamping C Field potentials recorded from an hPSC CM embryoid body with the MED64 Note the similarity with the EKG in panel A 14 MED64 Application Note Cellartis Cardiomyocytes 4 Data analysis Mobius QT recording QT analysis workflow templates are available for FPD analysis Refer to page 84 85 96 97 in the Mobius Tuto
26. rial in detail Mobius can automatically extract the FPD using several methods including measurement of the time between the fast depolarizing spike either positive or negative and the slow repolarizing wave Please refer to pages 102 to 105 in the Mobius Tutorial for detailed instructions on performing FPD analyses 8 Mobius Win7 0 4 1 QT analysis moflo Rev 268 on 2013 03 03 o e Kil cz C2 ie Samal Workflow Layout Help i gt O od Working Directory Annotations Mam Detect beating Measurement Averages Filter Spike Data Filter Spike Date Save Measures Data Filter type Filter type Filename modifier Bessel lowpass 9 pole None Enable Cutoff freq Hz 1000 1 Extract Spike Measures Sync Firing Event 33 Potential uv 200 400 500 1 1 1 i 0 7 i i 1 L 0 100 200 300 400 500 0 0 05 9 1 0 15 02 025 03 0 32728 Time ms Time minutes Channel 29 Average 3 Traces Capture Labels Reset Probe wel Syne Thresh 0 Window 125 ms Hide phases Autoscale Measures Edit Measures Display Results Table Recording Date 2010 10 04 14 39 59 09 TimeOfAszp tudeMaxT xim vent Phase Time c 1 Baseline O 2 150 200 j2 52 163 850 la 1 3 165 100 s 740 158 950 344 lt Figure 8 QT recording analysis workflow template Signals that surpass user determined thresholds are extracted and their FPDs are measured using the Time of Amplitude Max to Max in
27. rogressive maturation in contracting cardiomyocytes derived from human embryonic stem cells Oual itative effects on electrophysiological responses to drugs Otsuji TG Minami I Kurose Y Yamauchi K Tada M Nakatsuji N Stem Cell Res 4 3 201 13 2010 22 MED64 Application Note Cellartis Cardiomyocytes Applicaiton Note Cellartis Cardiomycoytes August 1 2015 Alpha MED Scientific I nc Manufactured by Alpha MED Scientific I nc Saito Bio Incubator 209 7 7 15 Saito asagi 2015 Alpha MED Scientific Inc Ibaraki Osaka 567 0085 Japan SCIENTIFIC Phone 81 72 648 7973 FAX 81 72 648 7974 http www med64 com support med64 com
28. s 64 recording electrodes The 4 reference electrodes MUST be free from cells but covered by medium for signal recording Incubate the MED Probe with the seeded Cellartis Cardiomyocytes ina cell culture incubator at 37 C 5 CO2 for 5 hours allowing the cells to adhere Gently add 1 ml of the MEA Plating Medium with 5 uM Y 27632 to the MED Probe taking care not to dislodge the cells Incubate in a cell culture incubator at 37 C 5 CO2 After 48 hours change the medium to 2 ml Cellartis CM Culture Medium Cat Y10063 10 FBS After this first medium exchange half volume medium changes should be performed every 2 3 days depending on appearance pH Recordings can be started 1 day after the change to Cellartis CM Culture Medium cardiomyocytes Should have started to beat in standard assay medium of choice Cellartis CM Culture Medium can be used 6 MED64 Application Note Cellartis Cardiomyocytes 2 Plating and culturing Cellartis amp Cardiomyocytes 2 7 Cleaning the used MED Probes The MED Probe s electrical characteristics are best during the first use High quality signals can be recorded and effective stimulation is possible with the MED64 System s MED Probes thanks to the electrodes characteristics the lowest impedance in a commercially available microelectrode array The electrodes impedance will increase with repeated use of the MED Probes due to damage in handling and or residual cellular debris and
29. tivity more effectively Make sure the electrodes stimuli have opposite polarities to suppress the stimulus artifact For recording paced activities the Pacing recording workflow template is available Open the workflow template and check the F2 stimulator Program the stimulus waveform with identical stimulus current amplitude Amplitude and duration Length but reversed polarity for F1 and F2 Figure 18 20 MED64 Application Note Cellartis Cardiomyocytes Stimulation Step 1 Step 2 HStep 3 HStep 4 Step 5 HStep GH HStep 74 Step 8 4 Enable step Legend MB Fi Mir 7 stim Fix gt Ch 00 60 Type Stimulation Step 1 Step 2 Step 3 Step 4 Step 5 Step GH v Enable step 7 Stim F2 Ch 18 v 604 b u Length ms Amplitude uA Legend M Fi 5 Pacing hPSC CMs HStep 7 HStep S M F2 Length ms Amplitude uA Const Pulse Const 5 00 0 00 1 E 0 60 50 00 0 994 40 0 00 4 Repeat pattem to trace end V Repeat pattem to trace end Single rep 1000 00 ms 1 pal TI All re am ms 0 2 4 6 8 uA 1 Reps Auto Inc Auto Inc dm les la A B Figure 18 Stimulus parameters for bi polar stimulation with 2 adjacent electrodes A Stimulus protocol used for F1 output channel B F2 stimulus protocol is an identical waveform with reversed polarity Adjacent electrod
30. using the MED64 System Required MED64 System Components 1 MED Probe 2 MED Connector MED C03 MED Heated Connector MED CPO2H 1 3 MED64 Head Amplifier MED A64HE1 4 MED64 Main Amplifier MED A64MD1 5 Acquisition PC 6 Mobius software Mobius QT Package or Mobius QT and EP package 1 MED Heated Connector requires the ThermoClamp M 1 controller Mount the MED Probe on to the MED Connector MED Heated Connector CAUTI ON Clean the terminals on the outer portion of the MED Probe with a Kimwipe soaked in ethanol before mounting the MED Probe Salt sediments can damage the contact pins on the MED Heated Connector MED64 Application Note Cellartis Cardiomycoytes 9 3 Data acquisition Available Mobius workflow template Data can be acquired immediately using the available Mobius workflow templates enumerated below please refer to page 83 Chapter 4 Mobius QT on the Mobius Tutorial e Beat recording For recording and real time analysis of beat frequencies and interspike inter vals ISI e QT recording For recording and real time analysis of field potentials obtained from spontane ous beating e Pacing recording For recording of paced responses and real time FPD analysis Recommended acquisition settings The following parameters are recommended to perform data acquisition for FPD analysis Input Range Maximum input signal level 2 9 mV Low cut freq High pass filter 0 1 Hz or 1Hz Change it depe
31. utes or more 3 Do NOT place the MED Heated Connector in an environment where temperature changes frequently for example in the proximity of an air conditioner 8 MED64 Application Note Cellartis Cardiomyocytes 3 Data acquisition 4 For drug application e Warm up the drugs to the same temperature or a few degrees higher than the bath media prior to administration e Use the lid for drug application Open and close it quickly e Once the lid is opened wait for 5 minutes or more until the temperature and CO concentra tion stabilize A B Figure 2 Equipment for microenvironmental control A Complete experimental set up with the MED Heated Connec tor ThermoClamp 1 controller and MED Connector Cover B Bubble the gas through a beaker containing distilled water to maintain a humidified environment 3 2 Data acquisition The MED Probe has 64 recording electrodes as well as 4 reference electrodes The differences between the field potential acquired at the recording electrodes and the potential at the reference electrodes are measured by the MED64 System Acquired signals are sent to the MED64 Head Amplifier through the MED Connector MED Heated Connector The raw signals are amplified by x10 with the Head Amplifier and then amplified further and digitized with the MED64 Main Amplifier We highly recommend reading the Product manual for each component as well as the MED64 Handbook and Mobius Tutorial before

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