MEA-Application Note_Cardiac Slices from Adult Mouse Ventricle
Contents
1. Material Biological Materials 1 adult mouse gt 5 weeks old 1 adult mouse of your choice Technical Equipment MEA System with amplifier and data acquisition see chapter Suggested MEA System Stimulus generator internal or external MEAs microelectrode arrays Ice Stereo microscope Inverted microscope Necessary for aligning the electrode positions to the slice If you prefer to use an upright microscope mandatory in case of using 60EcoMEAs you need for example a camera and a stereo microscope for documenting the electrode position A picture of the slice on the electrode field can then be loaded into the MC_Rack program for aligning the data traces to the electrodes Heart Holder special device for blocking the ventricle in agar see picture below Oscillating microtome for example Leica Integraslice Campden and blades Adjustable pipettes and pipette tips 20 ul and 1000 ul Transfer pipettes cut Pasteur pipettes with wide opening approximately 0 5 cm Large sharp scissors or guillotine Surgical instruments for example a bone rongeur or scissors Narrow flat spatula Sharp forceps Curved straight forceps Small scissors Razor blade Special device Here called Heart Holder 2 3 2 4 2 4 1 Chemicals Oxygen gas O Carbogen gas 95 O 5 CO Nacl KCI CaCl KH PO NaHCO D Glucose MgSO Cyanoacrylate glue Pattex Henkel Dusseldorf Germany 100 alco2. multichannel systems MEA Application Note Cardiac Slices from Adult Mouse Ventricle Information in this document is subject to change without notice No part of this document may be reproduced or transmitted without the express written permission of Multi Channel Systems MCS GmbH While every precaution has been taken in the preparation of this document the publisher and the author assume no responsibility for errors or omissions or for 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 the author be liable for any loss of profit or any other commercial damage caused or alleged to have been caused directly or indirectly by this document 2013 Multi Channel Systems MCS GmbH All rights reserved Printed 09 09 2013 Multi Channel Systems MCS GmbH AspenhaustraBe 21 72770 Reutlingen Germany Fon 49 71 21 90 925 0 Fax 49 71 21 90 92 5 11 info multichannelsystems com www multichannelsystems com Products that are referred to in this document may be either trademarks and or registered trademarks of their respective holders and should be noted as such The publisher and the author make no claim to these trademarks 3 2 3 3 3 4 3 5 3 6 5 1 5 2 5 3 Table of Contents Introduction About this Application Note Material Biological Materials Technical Equipment Chemicals
3. 3 1 MEA Coating Depending on the type of selected MEA various coatings may be applied to the MEA surface to promote the adhesion of the slice Suggestions for coating methods can be found in the MEA manual available in the Download section of the MCS web site 3 1 1 Standard MEAs Standard MEAs 60MEA200 30iR Ti for example should be coated either with cellulose nitrate or with polyethylenimine PEI N nunaa S Wf Y Yy A A a M es _ yy NY ANN E E NS dissin Det 3 1 2 EcoMEAs EcoMEAs 60EcoMEA or 60EcOMEA Glass should be coated either with cellulose nitrate or with polyethylenimine PEI 3 2 MEA Application Note Setting Up the Vibratome Note As the design and handling of different vibratomes varies please consult the manual of your vibratome for more details Recommended Leica VT1000s Fill outer vibratome chamber with ice Fill vibratome chamber with frozen Ca free Tyrodes solution You might need to add some room temperature Ca free Tyrodes solution Vibratome overview Heart mounted on agar and base plate Oxygenation for Ca free Tyrodes solution filled inner chamber Slushed Ice filled outer chamber 10 3 3 Sacrificing the Animal and Isolating the Heart 1 Sacrifice the animal by cervical dislocation or decapitation 2 Mount animal on a preparation table for example icebox lid covered with tissue 3 Re
4. Media 2 4 1 Ca free Tyrodes solution 2 4 2 Agarose 2 4 3 Tyrodes Solution 2 4 4 Dulbeccos Modified Eagles Medium Methods MEA Coating 3 1 1 Planar Standard MEAs 3 1 2 EcoMEAs Setting Up the Vibratome Sacrificing the Animal and Isolating the Heart Preparations for Slicing Slicing Mounting the Slice onto the MEA Preparations for Recording Suggested MEA Systems System Configurations Microelectrode Arrays Amplifier Specifications Results amp Signals References OCOAN NN DODD O Ul O oO O O 11 12 14 15 17 18 18 18 19 20 21 1 1 Acute Cardiac Slice Introduction About this Application Note The intention of the MEA Application Notes is to show users how to set up real experiments with the MEA System on the basis of typical applications that are used worldwide The documents have been written by or with the support of experienced MEA users who like to share their experience with new users This application note includes a complete protocol for the dissection of mouse heart and the preparation of ventricular slices for acute experiments Acknowledgement Multi Channel Systems would like to thank all MEA users who shared their experience and knowledge with us A major part of this document is based on the instructions provided by the Natural and Medical Sciences Institute in Reutlingen We thank Dr Udo Kraushaar for his help with this document 2 1 2 2 MEA Application Note
5. Transfer slice to MEA using a modified slice transfer glass Pasteur pipette 14 3 6 Acute Cardiac Slice Mounting the Slice onto the MEA The recommended procedure described in the following instructions fixes the slice onto the coated MEA by adhesion An alternative method uses small weights of a few hundred milligrams to hold down the slice This method is especially recommended if uncoated 3D MEAs are used However this method might stress the slice and result in an altered potential distribution and propagation You can use either self made wire grids or a shim with a nylon mesh in the middle to apply the weight Some steel carriers with nylon meshes as slice hold downs are also commercially available for example from Warner Instruments www warneronline com Commonly used custom made weights are U shaped flattened pieces of platinum wire 80 120 mg glued onto a mesh a sort of nylon stocking or wedding veil for example You can also glue another platinum wire from the other side of the mesh symmetrically to the first piece of platinum This kind of grid is less damaging because you can vary the pressure on the slice by changing the thickness of the second wire The thickness of the wire that is placed onto the slice should match approximately the thickness of the slice If you use 350 um slices the wire should be around 300 um and not more than 350 um otherwise the grid will not hold the slice 15 MEA Applicati
6. slice z alix Me pE Moret vies Windoe jip aliz Ares Clee ko arnap Anm EENES kam O aj j ao j oa F Peai Deisis D Frema Digia Wama E10 jf pj o jj tome Ms bom 1 Di l a a a X i Bjo he j a pa j Eo f 1 2 Bm WU AO H a o DWO H ea Ea 2 b ba a Bu mh B E A 2 fe aa Ba E Pou j bn p co ore ome a 3 z 3 i HE il rt 4 E i C EMA LA 4 0 4 7 rc ou lee lt wo wi l _L J L l SE I Fer Fini perin E z z i MARETE Or 20 Acute Cardiac Slice References Most work was done by the groups of Jurgen Hescheler in Cologne Pillekamp Reppel et al 2005 Halbach Pillekamp et al 2006 and Ursula Ravens in Dresden Halbach M U Egert et al 2003 Estimation of action potential changes from field potential recordings in multicellular mouse cardiac myocyte cultures Cell Physiol Biochem 13 5 271 84 Halbach M F Pillekamp et al 2006 Ventricular slices of adult mouse hearts a new multicellular in vitro model for electrophysiological studies Cell Physiol Biochem 18 1 3 1 8 Pillekamp F M Reppel et al 2005 Establishment and characterization of a mouse embryonic heart slice preparation Cell Physiol Biochem 16 1 3 127 32 21
7. be used for synchronizing the recording with the stimulation or with external systems This is the standard configuration for low throughput academic research and high flexibility for a wide range of applications Microelectrode Arrays Available MEAs differ in electrode material diameter and spacing For an overview on available MEA types please see the Multi Channel Systems web site www multichannelsystems com or contact your local retailer The microfold structures formed by titanium nitride TiN result in a large surface area that allows the design of small electrodes with a low impedance and an excellent signal to noise ratio 60MEA200 30iR Ti Standard 8 x 8 layout TiN electrodes for recording and stimulation with substrate integrated reference electrode 60EcoMEA EcoMEAs have a standard 8 x 8 layout Gold electrodes and a substrate integrated reference electrode They are opaque that is why you need an upright microscope for controlling the slice 18 5 3 Acute Cardiac Slice Amplifier Specifications In MEA2100 Systems the sampling rate signal range and bandwidth can be adjusted via software control and is therefore suitable for a broad range of applications from single unit spike recordings to field potentials from whole heart preparations Though amplifiers with custom gain and bandwidth are available Multi Channel Systems recommends the following settings for this application in MEA1060 amplifiers Lower cuto
8. e of the MEA and of the perfusion fluid via the perfusion cannula PH01 Integrated current or voltage controlled stimulators can use any electrode as stimulation electrode The system includes amplifier data acquisition and stimulators in one compact device as well as floor and perfusion heating The filter band of the DAQ can be changed by software An additional box includes a unique freely programmable DSP for advanced closed loop experiments and many additional in and outputs for interface with other devices The MEA2100 System will fit equally well on upright and inverted microscopes The system can be upgraded to operate up to four 60 channel or two 120 channel MEAs independently from one computer Approximately 20 electrode layouts with several additional options are available at the moment The use of perforated MEAs is optional It only make sense when the slice covers the perforated area Cross sections through whole heart ventricles may not fulfill these requirement USB MEA60 System E 60 channel MEA recording system for inverted or upright microscopes The temperature controller TC01 TC02 regulates the temperature of the MEA and of the perfusion fluid via the perfusion cannula PHO1 A MEA1060 amplifier allows recording up to 60 channels from one MEA The three additional analog inputs can be used for feeding in data generated by other systems recording in parallel for example for patch clamp data The additional digital inputs can
9. ff frequency 1 Hz With an even lower value slow signal drifts will disturb the recordings and signal detection Upper cutoff frequency 3 kHz Sufficient even for rapid depolarization waveforms If only field potentials are of interest a reduction of the upper cutoff frequency might be considered to reduce high frequency noise Gain factor 800 1000 A note on gain Traditional amplifiers for extracellular recordings often have 5000 x or even 10000 x gain switch options A high gain however increases the high frequency noise level or requires a narrower filter band Considering a 20 uV extracellular signal we would receive 24 mV after a 1200 x amplification An AD converter with an input range set from 400mV to 400 mV will resolve the signal in increments of 0 36 uV which will definitely provide enough information given the noise level of such systems in general Therefore there is no need for higher amplifications Higher gains might result in clipping signals 19 MEA Application Note Results amp Signals Slices can be recorded using 60EcoMEAs or standard 60MEA200 30iR Ti MEAs The recorded cardiac field potential represents the extracellular reflection of the action potential Halbach Egert et al 2003 Every single MEA electrode picks up the local field potential at that position in the slice This allows to map propagation of cardiac action potentials in the slice and correlate field potential shape with the position in the
10. hol or acetone for cleaning the MEA contact pads Low melt agarose Roth Karlsruhe Germany 100 000 U kg BW Units per kg body weight heparin sodium Hoffmann La Roche Grenzach Wyhlen Germany Media Ca free Tyrodes solution NaCl 155 mM KCl 5 4mM NaH PO 0 33 mM Glucose 10 mM MgCl 1mM Hepes 10 mM BDM 2 3 butanedione monoxime 30mM Ca free Tyrodes solution pH 7 4 adjusted with NaOH Aerate the Tyrodes Solution with oxygen gas for 15 min Acute Cardiac Slice 2 4 2 2 4 3 2 4 4 MEA Application Note Agarose Dissolve low melting agarose in Ca free Tyrodes solution to obtain a 4 Agarose concentration Heat to boiling to dissolve then cool to and store at 37 C Tyrodes Solution Add 0 9 mM CaCl and aerate the Tyrodes Solution with oxygen gas for 15 min Dulbeccos Modified Eagles Medium CaCl 1 8 mM MgSO 0 8 mM KCI 5 3 mM NaHCO 44 mM NaCl 110 mM NaH PO 0 9 mM Prepare the media as listed above Aerate the DMEM with carbogen gas for 15 min Refrigerate 200 ml Ca free Tyrodes solution until it is partially frozen Crush the frozen Ca free Tyrodes solution thoroughly blend with hand blender and oxygenate on ice until use Note To speed up the freezing and crushing of Ca free Tyrodes solution you might consider to prepare Ca free Tyrodes solution ice cubes in advance and store the ice cubes at 20 C until use 30 45 min Acute Cardiac Slice 3 Methods
11. ller to 37 C for heating the MEA culture chamber and to 32 C for the buffer temperature Start carbogen aeration 15 min before mounting the slice Start the perfusion 15 min before mounting the slice at a low flow rate 0 5 exchanges min to maintain a stable oxygenation and pH Clean the MEA contacts with a soft tissue and pure alcohol or acetone Mount the MEA with the slice onto the amplifier as described in the MEA amplifier user manual Superfuse the slice with oxygenated Tyrodes solution prewarmed at 37 C The buffer volume should be exchanged 3 4 times per minute The slice is mechanically stressed by activating the perfusion and should be perfused for about half an hour before recording You can also control the parameter that you want to record and start the recording as soon as you get a stable baseline for example as soon as the spike rate has stabilized You are now ready for recording 17 5 1 5 2 MEA Application Note Suggested MEA Systems System Configurations Depending on the throughput and the analysis requirements desired in your laboratory different system configurations are recommended for the recording from cardiac slice preparations MEA2100 System E The MEA2100 System is the most advanced system MCS can offer today It is as flexible as the USB MEA60 System and can use the same MEAs in addition to unique 120 electrode arrays The temperature controller TC01 TCO2 regulates the temperatur
12. move fur at the chest 4 Open chest 5 Remove pericardium 6 Isolate the heart and place it in a Petri dish filled with cold Ca free Tyrodes solution Acute Cardiac Slice 11 MEA Application Note 3 4 Preparations for Slicing 1 Perfuse the heart retrogradely with a syringe with Ca free Tyrodes solution with BDM 2 Remove atria by cutting at the AV level 3 Mount the ventricles in the Heart Holder Make sure to place the heart upside down with the pin of the holder into the ventricle cavity 4 Fill Heart Holder with melted agarose 5 Cool Heart Holder to speed up hardening of the agarose 12 Acute Cardiac Slice NOTE Fill up the gap left by Heart Holder pole with agarose using a pipette tip This will prevent the heart from detaching from the agarose block during slicing 6 Trim agarose block NOTE Make sure the last cut at the heart cut area is directed as your intended slicing layer 7 Mount heart jelly block in pre refrigerated slicing platform with cyanoacrylate glue 8 Place slicing platform in slicer 13 MEA Application Note 3 5 Slicing 1 Remove 2 3 slices at the heart tip and discard those 2 Slice at 150 200 um thickness Note It is very important to move the blade very slowly to minimize tissue damage 3 Transfer slice to oxygenated Tyrodes solution with 0 9 mM CaCl 4 Incubate for 30 min 5
13. on Note Important Do not touch the slice directly The slice should not be folded to avoid damage to the tissue Be careful not to touch the MEA surface with the transfer pipette to avoid damage to the electrodes Place the slice and a drop of Tyrodes solution with the transfer pipette onto the EcoMEA center it roughly on the recording area Position the slice by gently pushing it with a pipette tip from the sides into place The region of interest should cover the recording area Remove the Tyrodes solution to fix the slice onto the EcoMEA Hold the slice in position with a small pipette tip along the side and remove excess buffer with a pipette Cover the slice with a few drops of DMEM immediately The buffer should be pipetted onto the slice carefully right from the top rather than from the side to avoid that the slice is floating up Avoid falling drops that can damage the tissue OR If you prefer the alternative method of applying a grid Position the slice in the desired position with approximately 200 ul Tyrodes solution left then apply the grid and add oxygenated Tyrodes solution Some MEA users prefer to nearly dry the slice on the array with filter paper wedges others find it not necessary when using grids and prefer to change the position of the slice if the MEA culture chamber is filled with Tyrodes solution Store slices on MEAs in incubator till use 16 10 Acute Cardiac Slice Preparation
14. s for Recording Note We recommend the perfusion cannula with temperature control PH01 for optimal environmental conditions A two channel temperature controller TCO2 allows to control both the MEA temperature via the heating integrated into the amplifier and the buffer temperature See the MC_Rack manual or online help for detailed application examples For connecting and programming the stimulus generator STG please see the respective user manual Please see the MEA manual for details on stimulation amplitudes and times that are supported by the MEA electrodes Though TiN electrodes are very stable an unsuitable stimulation pulse will irreversibly damage the electrodes We highly recommend the following preparations and tests before you start the experiment Test all connections Define your virtual rack specific to your application with the MC_Rack program and test it before use Define your stimulation file with the MC_Stimulus program and test it with the test model probe and with a MEA filled with recording buffer before use It is recommended to test a range of stimulus amplitudes and locations prior to starting your actual experiment If you are using an external stimulating electrode the position of this electrode should be optimized in this step as well Set up the perfusion system and test the perfusion with an old MEA Adjust the grounding and shielding to avoid noise pickup and 50 Hz hum Set the temperature contro
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