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Microelectrode Array (MEA) User Manual

1. MEA User Manual Typical noise level of a new 3 D MEA Bath grounded with a silver pellet Time axis 1000 ms voltage axis 20 p V Imi Continuous Data NN ini xl 2278000sec xis 1000 p ms 2276th sweep Y Axis 20 uv gt Typical noise level of a new standard 100 10 MEA Bath grounded with the internal reference electrode 15 Time axis 1000 ms voltage axis 100 uV il Continuous Data H lof x1 3563000sec Xwxis 1000 j ms 00 10000 3563rdsweep Y Axis 100 p uY gt 1000 1000 500 W 500 500 500 500 500 y 500 0 500 ff Ft Mind aii Casini dd tea ER ER i A EN ttn al g A a ll ae A te TERS 35 EE GEREG TONG AAA E TOA lee E 32 8 1 Stimulation Stimulation Using MEA Electrodes for Stimulation You can use any MEA electrode s for stimulation Simply connect the stimulus generator outputs to the MEA amplifier Please see the user manual for the respective MEA amplifier and stimulus generator for more details As an alternative you can also use special MEAs with four pairs of large 250x50 pm stimulating electrodes MEA 200 30 stim and a special stimulation adapter or target cells with an external electrode for stimulation This and the following chapters are intended for helping you to optimize the stimulation with MEA electrodes All electrodes suffer under electrical stimulation especially under long term stimulation The wear dep
2. 3 3 MEA User Manual Electrodes Tracks and Insulation Microfold structures result in a large surface area that allows the formation of electrodes with an excellent signal to noise ratio without compromizing on the spatial resolution TiN is a very stable material that for example is also widely used for coating heavy equipment All MEAs with TiN electrodes have a long life and can be reused several times if handled with care If used for acute slices MEAs can be used for approximately one year Long time experiments with cell cultures and rigid cleaning methods shorten the MEA lifetime but you can still reuse an MEA about 30 times depending on the coating cell culture and cleaning procedure All MEAs except FlexMEAs and 3 D MEAs show excellent temperature compatibility and are stable from 0 C to 125 C that is they can be autoclaved The impedance of a flat round titanium nitride electrode ranges between 20 and 400 kilohms depending on the diameter The smaller an electrode the higher is the impedance On one hand lower impedance seems desirable but on the other hand a smaller electrode and interelectrode distance results in a higher spatial resolution Multi Channel Systems provides MEAs with electrode sizes of 10 20 or 30 um which all show an excellent performance and low noise level The average noise level of 30 um and 10 um electrodes is less than 10 UV and 15 uV peak to peak respectively Pt electrodes 3 D ME
3. MEA types differ in the materials used for the carrier and the recording area and in the geometry that is electrode size and interelectrode distances The electrode size and interelectrode distances are used for categorizing MEAs The first number refers to the interelectrode distance for example 100 um and the second number refers to the electrode size for example 10 uim which results in the standard MEA type 100 10 for example Standard versions are available with an internal reference electrode abbreviated i r and with various culture chamber interface options Culture chambers are available with and without lid Please ask for custom layouts that is MEA layouts according to your specifications In this chapter each MEA type is briefly described and noted Standard MEAs with flat round TiN electrodes in an 8 x 8 layout grid for all applications HighDenseMEAs with the highest spatial resolution and a double recording field of 5 x 6 electrodes each HexaMEAs featuring a hexagonal layout perfect for recording from retina ThinMEAs with a thickness of only 180 um ideally suited for high resolution imaging 3 D MEAs the ideal solution for acute slices because the tip shaped Pt electrodes are intended to penetrate dead cell layers or for applications where a very high electrode surface area is required Very cost efficient and robust EcoMEAs for applications with lower spatial resolution and higher throughput especi
4. keeps your culture clean and sterile preventing contaminations by airborne pathogens It also greatly reduces evaporation and thus prevents a dry out of the culture Autoclavable Comes complete with membranes Set of 10 membranes for sealed MEA culture dishes Set of 5 membranes and MEA MEM TOOL for sealed MEA culture dishes Tool for smoothing and positioning the membrane on the ALA MEA MEM culture chamber so that the membrane is neat and flat on top of the culture chamber 45 Technical Specifications M EA Microelectrode Array MEA type Temperature compatibility Dimensions W x D x H Weight Base material Contact pad and track material Electrode diameter Interelectrode distance Electrode height Electrode type Isolation type Electrode impedance Electrode layout grid Number of electrodes TiN electrodes SiN isolator and Ti contact pads 100 10 Ti 200 10 Ti 200 10iR Ti 200 30 Ti 200 30iR Ti 200 30 Ti stim with 8 stimulation electrodes 500 10iR Ti 500 30iR Ti HexaMEA Ti 10 20 30 0 C to 125 C 49 mm x 49 mm x 1 mm 8g Glass Titanum As specified 10 20 and 30 um available As specified 100 200 and 500 um available not applicable to Hexa 10 20 30 MEA layout Planar Titanium nitride Silicon nitride 500 nm PEVCD 30 kO to 400 kO depending on electrode type and diameter 8 x 8 100 10 200 10 200 10 200 30 200 30 i r 6 x 10 500 10 i r 500
5. Sigma Aldrich Inc P7280 Fibronectin BD BioCoat Fibronectin Cellware BD Biosciences Fibronectin solution Prepare a stock solution of 25 ug ml fibronectin in distilled water or PBS and store it at 4 C 22 5 4 6 Appendix Poly D Lysine plus fibronectin solution Prepare a 0 01 96 w v poly D lysine solution and add fibronectin 1 1 resulting in a final concentration of 12 5 pg ml Procedure Pipette 10 ul of the poly D lysine plus fibronectin solution onto the recording field Pipette about 50 ul of sterile distilled water near the rim of the culture chamber Incubate for 1 h in an incubator set to 35 C 65 relative humidity 9 O 5 96 CO or 37 C 100 humidity 5 CO To avoid a dry out of the liquid place the MEA in a big Petri dish with lid on Rinse 2x with sterile distilled water Let MEAs air dry over night on a sterile workbench laminar flow with UV light turned on Coating with Fibronectin Fibronectin is a more biological coating alternative especially used for heart tissues The adhesion tends to be very stable which allows longer cultivation times Materials Fibronectin BD BioCoat Fibronectin Cellware BD Biosciences Fibronectin solution Prepare a stock solution of 1 mg ml fibronectin in distilled water or PBS and store it at 4 C The stock solution is diluted with water or PBS to a final concentration of 10 ug ml before use Procedure Cover the MEA surface w
6. This is considered a normal behavior Possible causes The insulation layer is too thin As a result the MEA gets the behavior of a low pass filter This means that the signal frequency may be shifted to a lower frequency and spikes are missing Optically control the MEA with a microscope If concentric colored rings Newton rings are visible due to light interference the insulation layer is too thin and you should use a fresh MEA The insulation layer has been abraded and is missing in parts This will result in a short circuit between the electrode tracks and the bath You will still see signals but as an unspecific smear over the complete array Use a fresh MEA 41 Appendix 10 Appendix 10 1 Contact Information Local retailer Please see the list of official MCS distributors on the MCS web site User forum The Multi Channel Systems User Forum provides an excellent opportunity for you to exchange your experience or thoughts with other users worldwide Mailing list If you have subscribed to the NeuroElectronics Mailing List you will be automatically informed about new software releases upcoming events and other news on the product line You can subscribe to the list on the MCS web site www multichannelsystems com 43 MEA User Manual 10 2 Ordering Information Please see the MEA data sheet for more information about available MEA types Please contact your local retailer for pricing and orde
7. 2 5 3 5 4 Table of Contents Introduction About this Manual Important Information and Instructions Operator s Obligations Guaranty and Liability Important Safety Advice Microelectrode Arrays MEAs Overview Extracellular Recording with Microelectrode Arrays MEA Design and Production Electrodes Tracks and Insulation MEA Types and Layouts Standard Electrode Numbering Standard MEAs HighDenseMEAs HexaMEAs ThinMEAs 3 D MEAs EcoMEAs FlexMEAs MEA Handling Hydrophilic Surface Treatment 5 1 1 5 1 2 5 1 3 Plasma Cleaning Protein Coating Preculturing Sterilization 5 2 1 5 2 2 5 2 3 5 2 4 Sterilization with Ethanol and UV Light Steam Sterilization Autoclavation Dry Heat Sterilization Sterilization with Hot Water MEA Storage MEA Coating 5 4 1 5 4 2 5 4 3 5 4 4 5 4 5 5 4 6 5 4 9 Coating with Nitrocellulose Coating with Polyethyleneimine PEI plus Laminin Coating with Polyornithine plus Laminin Coating with Poly D Lysine plus Laminin Coating with Poly D Lysine plus Fibronectin Coating with Fibronectin Coating with Collagen N wu wm ui Ui o 00 11 11 12 12 13 13 14 15 16 17 17 17 17 18 18 18 18 18 18 19 19 19 20 21 22 22 23 24 5 5 6 6 1 6 2 6 3 7 2 8 1 8 2 8 3 8 4 9 1 9 2 9 3 9 4 9 5 10 10 1 10 2 10 3 10 4 10 5 Cleaning of Used MEAs 5 5 1 General Recommendations for Cleaning MEAs 5 5
8. 2 Cleaning of 3 D MEAs 5 5 3 Cleaning of EcoMEAs 5 5 4 Removing Nitrocellulose Coating 5 5 5 MEA Cleaning with EDTA Collagenase Culture Chamber Options Sealed MEA Culture Dish MEA Culture Chamber with Lid Removable Recording Chamber Recording with MEAs Mounting the MEA 7 1 1 Cleaning the Contact Pads 7 1 2 Positioning the MEA 7 1 3 Grounding the Bath General Performance Noise Level Stimulation Using MEA Electrodes for Stimulation Capacitive Behavior of Stimulating Electrodes Aspects of Electrode Size and Material Recommended Stimulus Amplitudes and Durations Troubleshooting About Troubleshooting Technical Support Noise on Single Electrodes Overall Noise Unsteady Baseline Missing Spikes or Strange Signal Behavior Appendix Contact Information Ordering Information 10 2 1 MEA Systems 10 2 2 MEA Amplifiers 10 2 3 Accessories MEA Data Sheet MEA Layouts Safe Charge Injection Limits 25 25 25 25 26 17 26 27 27 29 29 29 29 29 30 33 33 34 34 35 37 37 37 38 40 41 43 43 44 44 44 45 46 47 48 1 1 2 1 2 2 Important Information and Instructions Introduction About this Manual The MEA User Manual comprises all important information about the microelectrode arrays MEA for use with the MEA System from Multi Channel Systems The MEA User Manual focuses on general information on the MEA design use and handling and more specific information on different MEA types It
9. 200 250 300 time us safe charge injection limits 60 50 40 30 20 10 300 400 500 600 700 800 900 1000 time us Safe Charge Injection Limits of Multi Elctrode Arrays with TiN Electrodes diameter 30um max pulse amplitude HA max pulse amplitude uA max pulse amplitude HA safe charge injection limits 2000 1500 1000 500 0 50 100 150 200 250 300 350 400 450 500 time us safe charge injection limits 400 r r r r 350 300 250 200 150 100 50 500 1000 1500 2000 2500 3000 time us safe charge injection limits 0 1 1 1 1 1 3000 4000 5000 6000 7000 8000 9000 10000 time us
10. 63 73 45 55 48 58 show a high noise level 5 Standard Noise with Shielding 525000sec Aus 1000 p ms gt oo ER 500 525th sweep YAsis 20 p WY 200 J 200 p 200 400 go e 200 Op 200400 goo OP goo so 200 sop goo 40 4 lor G Possible causes The electrode or the contact pin of the amplifier may be defective To test this do the following Open the amplifier and turn the MEA by 90 degrees Close the amplifier again and start the recording If the same electrode in the MEA layout is affected the amplifier s contact is not ok If another electrode is now affected and the previously affected electrode is ok now the MEA electrode is not ok but the amplifier is fine The following screen shot shows the same MEA than above that has been turned clockwise by 90 You see that different channels are now affected which indicates that the amplifier is fine but some electrodes on the MEA are defective imi Turning MEA by 90 degrees 1278 000sec XAxis 1000 p me oo gt 1278thsweep YAsis 220 p WW ao 0 goo 400 200 400 200 Moo 200 P go B 200 sep goo sop goo so oll AY A ES EL aaa au A ab Ls AAA GE Use the model test probe to test the amplifier If the noise level is fine without the MEA bad MEA electrodes cannot be the cause 38 Troubleshooting MEA is defective MEAs wear out after multiple uses or over a longer time of use for example for
11. Attach the provided silver wire or Ag AgCl pellet to the amplifier s ground and place it into the bath OR If you use an MEA with internal reference electrode connect the ground to the reference electrode socket pin 15 with the provided connector Please see the user manual of the respective MEA amplifier for more information about mounting MEAs and grounding 29 7 2 MEA User Manual General Performance Noise Level You can test an MEA before use by filling it with a standard saline buffer for example PBS and recording the noise level of the MEA and the amplifier MEA amplifiers have a maximum noise level of 8 UV The noise level on the MEA depends on the electrode size and material The smaller the electrode the higher is the noise level TiN electrodes have a larger surface area due to their microfold structures and therefore they have generally a lower impedance and a lower noise level than electrodes of the same size that are made from other materials for example Pt electrodes The total maximum noise level for an MEA and the amplifier should be about 40 uV peak to peak for 10 um TiN electrodes and 10 uV for 30 um TIN electrodes The larger Pt electrodes of the 3 D MEAs generally show a noise level comparable to the 30 um TiN electrodes The initial noise level may be higher if the MEAs are hydrophobic New MEAs should be made hydrophilic before use Typical noise level of a used standard
12. Most problems are caused by minor handling errors Contact your local retailer immediately if the cause of trouble remains unclear Please understand that information on your hardware and software configuration is necessary to analyze and finally solve the problem you encounter Please keep information on the following at hand Description of the error the error message text or any other useful information and of the context in which the error occurred Try to remember all steps you had performed immediately before the error occurred The more information on the actual situation you can provide the easier it is to track the problem The serial number of the MEA You will find it on the MEA case The amplifier type and serial number You will find it on the device The operating system and service pack number on the connected computer The hardware configuration microprocessor frequency main memory hard disk of the connected computer This information is especially important if you have modified the computer or installed new hard or software recently The version of the recording software On the Help menu click About to display the software version 37 9 3 MEA User Manual Noise on Single Electrodes The noise level on single electrodes is significantly higher than expected or you see artifact signals In the following example 200 30 MEA filled with PBS silver pellet as bath electrode shielded electrodes No 53
13. b The next pictures show the same MEA with bath electrode silver pellet but without shielding The baseline is very unsteady and oscillates with a frequency of 50 Hz E 50 Hz Hum 365000sec Ass 1000 ms gt 386th sweep YAris 20 ef EV gt 0 1000 1000 sasali iia nn 1000 1000 LL 00 f 200 Mos PR Lo 10 413 000 sec 413th sweep Y Axis 1 1000 gt 20 gt 40 50 60 1 1 oa CELA lof 00 E 1400 MM I I 0 go go too to 10130 1 1 L L 1 I L L 1 1 LOMA SCR NAA E17 AO UA AM NATI Ji PAULUM PRA 40 9 5 Troubleshooting The next screen shot shows the effect of shielding The noise level is neglectible and the baseline is steady The shielding has been achieved with a metal plate connected to the metal part of the 68 pin MCS High Grade cable connector and placed above the amplifier You could also use aluminum foil or a Faraday cage for the same effect for example E Standard Noise with Shielding 514000sec xs 1000 ms gt ao H SMhsweep Vans 220 p WY gt 200 2 150 200 250 300 350 Lai Missing Spikes or Strange Signal Behavior MEAs wear out after multiple uses or over a longer time of use for example for long term cultures The insulation layer gets thin over time
14. before use to improve the cell attachment and growth please see MEA Coating on page 19 Spike activity can be detected at distances of up to 100 um from a neuron in an acute brain slice Typically signal sources are within a radius of 30 um around the electrode center The smaller the distance the higher are the extracellular signals The higher the spatial resolution the lower the numbers of units that are picked up by a single electrode that is the less effort has to be put into the spike sorting Multi Channel Systems provides MEAs with the highest spatial resolution in the market HighDenseMEAs have electrodes with a diameter of only 10 um arranged in a distance of only 30 um center to center The challenge of manufacturing very small electrodes and at the same time keeping the impedance and the noise level down has been met by introducing a new electrode material titanium nitride TiN The NMI in Reutlingen Germany www nmi de produces MEAs from very pure fine quality and highly biocompatible materials The NMI is a research institute with which Multi Channel Systems has collaborated in many projects and over many years 3 D MEAs with platinum electrodes are produced for Multi Channel Systems by Ayanda Biosystems in Lausanne Switzerland www ayanda biosys com Quality controls and production processes have been improved over the last years so that MEAs are always of a fine consistent quality at very reasonable prices
15. for applications where a high spatial resolution is not important but which emphasize on cheap consumables They have proven to be especially useful for recordings from established cardiomyocyte cultures They are not useful for establishing a new cell culture as the cell performance cannot be monitored Multi Channel Systems recommends to use standard 200 30 MEAs for establishing the cell culture first then switch to ECOMEAs Standard EcoMEAs are provided in the typical 8 x 8 layout Custom layouts following your personal specifications are possible at very reasonable prices Please ask your local retailer for details Electrodes tracks and contact pads are made of pure gold Due to the soft gold material of the contact pads the contact to the amplifier pins is excellent Using EcoMEAs Like standard MEAs EcoMEAs are very robust and heat stable They can be autoclaved and coated with different procedures for cell and tissue cultures The electrodes are very robust too and are the only MEA electrodes that will endure more severe cleaning methods New EcoMEAs are very hydrophobic They should be coated with nitrocellulose or treated with a Plasma cleaner before use 15 4 8 MEA User Manual FlexMEAs FlexMEAs are made of flexible polyimid material perfect for in vivo and special in vitro applications Only 12 um thick and weighing less than 1 g the FlexMEA biosensor is very thin and lightweight FlexMEAs have 32 electrod
16. foreign bodies or acts of God Those parts in this user manual that refers to the applications and not to the product itself for example coating of MEAs is only a summary of published information from other sources see references and has the intention of helping users finding the appropriate information for setting up their experiments Multi Channel Systems MCS GmbH has not tested or verified this information Multi Channel Systems MCS GmbH does not guarantee that the information is correct Multi Channel Systems MCS GmbH recommends to refer to the referenced literature for planning and executing any experiments Important Information and Instructions Important Safety Advice Warning Make sure to read the following advice prior to install or to use the device and the software If you do not fulfill all requirements stated below this may lead to malfunctions or breakage of connected hardware or even fatal injuries Warning Obey always the rules of local regulations and laws Only qualified personnel should be allowed to perform laboratory work Work according to good laboratory practice to obtain best results and to minimize risks The product has been built to the state of the art and in accordance with recognized safety engineering rules The device may only be used for its intended purpose be used when in a perfect condition Improper use could lead to serious even fatal injuries to the user or third parties and damage to the
17. long term cultures This is considered a normal behavior MEAs are also easily damaged by mishandling for example if wrong cleaning solutions or too severe cleaning methods are used or if the recording area is touched If you observe a bad long term performance of MEAs consider a more careful handling Possible causes The contact pads are contaminated Clean the contact pads carefully with a swab or a soft tissue and pure 100 96 alcohol The contact pads or the electrodes are irreversibly damaged You could have a look at the electrodes under a microscope If they appear shiny golden the TiN is gone and the electrode is irreversibly damaged Electrodes may be damaged without changing their visual appearance though Pick one of the bad channels after the other and ground it See the MEA amplifier s user manual for more information on grounding channels In most cases only one of the electrodes that appear bad is actually defective and the other ones are only affected by the single defective electrode Ground as many electrodes as you need for a good general performance In the following example all defective electrodes have been grounded imi Defective electrodes on ground lol 1036 000 sec Ass 1000 p ms gt 00 J H 5000 1036th sweep YAxis 20 p WY gt 200 20 200 400 200 400 200 400 200 400 200 400 200 400 200 400 200 EA oc acri Lir oti l an 1050000sec Ass 1000 00 ER 1050th sw
18. the MEA System for extracellular recording from Multi Channel Systems MCS GmbH FlexMEAs may be used with components of the ME System from Multi Channel Systems MCS GmbH FlexMEAs are designed for use in in vitro or in vivo studies Several MEA geometries are provided for a wide variety of applications Almost all excitable or electrogenic cells and tissues can be used for extracellular recording in vitro for example central or peripheral neurons cardiac myocytes whole heart preparations or retina There are various applications for MEAs and the MEA System in the fields of neurobiology and cardiac electrophysiology Typical neurobiological applications are lon channel screening drug testing safety pharmacology studies current source density analysis paired pulse facilitation PPF long term potentiation LTP and depression LTD I O relationship of evoked responses circadian rhythm neuroregeneration developmental biology microencephalograms EEG and microelectroretino grams ERG Typical applications in the cardiac field are Activation and excitation mapping measuring of the conduction velocity longterm characterizations of cell types especially stem cells culture pacing drug testing safety pharmacology studies monitoring of QT Prolongation and arrhythmias cocultures and disease implantation model For more information on published applications or procedures for biological preparations please see the application not
19. 0 1 r 500 30 i r Electrode layout ser EE EE EIE Electrode 11 H2 K2 22 63 H3 H4 K4 PAN systems H5 K6 Ox 90900900 Oo a 60606026020 Or 200000 e 200000 Ss TIKI Oe e 6 6 G A 200000 Oa 200000 8666660966066 066606 The letter of the electrode number code refers to the row number and the digit is the column c4 72 H6 82 65 73 G6 83 Fa 64 rs 74 F6 84 E6 85 E4 65 pe 86 os 76 ce 87 pa 77 number The specified MEA1060 amplifier pin numbers are the MEA System channel numbers that are used in the MC_Rack program The substrate integrated reference electrode i r is connected to pin 15 of the MEA1060 amplifier 2005 Multi Channel Systems MCS GmbH multichannel MEA 200 30 Stim systems with 16 additional stimulating electrodes 8 30 um 8 250 x 50 um STIM STIM N o e E y3 000000 AA 2 200 um Q m o 250 x 50 um NET sm O 6000000 900000 Es 9 2450 pm STIM STIM 6006060 00 un un zi d z lt Multi Channel Systems Fon 49 7121 9 09 25 O O 2002 2005 Multi Channel Systems MCS GmbH Fax 49 7121 9 09 25 11 GmbH Aspenhaustrasse 21 72770 Reutlingen info multichannelsystems com German y www multich Isystems com Prod form bj hang multicha
20. 00 um interelectrode distance and 10 um electrode diameter EcoMEA 0 C to 125 C 49 mm x 49 mm x 1 mm 8g Gold 100 um 700 um Planar Gold approx 30 kQ 8x8 60 Date of Print 05 07 2005 FlexMEA 10 C to 40 C 12 um height 1g Polyimid 2611 Gold 30 um 200 um 16 electrode version 300 um 36 electrode version 600 to 700 um 72 electrode version Planar Titanium nitride approx 50 kQ 4x4 14 electrode version 6 x 6 32 electrode version 9 x 8 72 electrode version Versions with 14 32 and 72 electrodes available 14 and 32 electrode versions with 2 reference electrodes 32 electrode version with 2 ground electrodes Culture chambers for MEAs Inner diameter ID Outer diameter OD Height Page 1 of 1 Glass ring gr 20 mm 24 mm 6 mm Plastic ring without thread for Plastic ring with thread for a a lid pr 26 mm 30 mm 6 mm removable lid pr T 26 mm 30 mm 6 mm with screw thread for lid No ring for custom use w o O 2003 Multi Channel Systems MCS GmbH multichannel Standard MEA Layout 8x8 systems Standard MEAs with TiN electrodes in the 8x8 grid are available in versions MEA 100 10 200 10 i r or 200 30 i r with optional ITO tracks Dimensions refer to standard MEAs ThinMEAs 3 D MEAs and EcoMEAs also follow the 8x8 grid For other specifications like electrode material diameter and spacing please refe
21. 0075 6 Fi 10 um Bm FSR 76 17 Fat FAR 87 27 ex E3R 77 The first letter of the electrode number code refers to the row number the digit is the column number and the second letter refers to the electrode field left or right of the HighDenseMEA The specified MEA1060 pin numbers are the MEA System channel numbers that are used in the MC_Rack program 2005 Multi Channel Systems MCS GmbH HexaMEA Electrode layout multichannel systems ide A AS EE EE EP Electrode B7 B10 C5 C4 C3 de C5 15 a10 14 24 gt A9 I I on e 25 MEA1060 pins 31 C4 44 B7 33 A7 35 C2 C6 C8 Cl C8 52 30 um C6 C9 42 54 20 um C3 C7 D3 e e O 43 53 63 10 um C2 c0 p e 0 8107 c1 67 p1 7 pio e e 0 B2 32 gi p41 8 E1 84 ph e e o e e 1351022 4138 F1 77 g19 86 e e o e 15 aa 16 p4947 p2 67 o 0 9 2 30 um A3 F7 F3 e e e 36 48 56 46 57 NI NI 4 gt 60pm F7 F1 F6 F2 C7 D7 64 E7 66 D4 C9 C10 D5 D5 62 D8 6 73 82 D9 74 E4 E3 e e 65 75 e 76 x 87 F5 68 F4 F5 E10 E5 85 D2 72 D6 os D1 D7 D9 D10 E5 E4 75 E3 E2 E6 E8 E1 7 The letter digit code is the electrode identifier and refers to the position of the electrode in the hexa grid The specified MEA1060 amplifier pin numbers are the MEA System channel numbers that are used in the MC Rack program 2005 Multi Channel Systems MCS GmbH multichannel MEA 500 1
22. 130 0 5 mM EDTA Phosphate buffered saline PBS Gibco Invitrogen 14190 144 Collagenase solution Dissolve collagenase type in PBS at 20 U ml Method Fill the MEA culture chamber with 0 5 mM EDTA and incubate for 30 min Rinse the chamber 3x with PBS Fill the MEA with collagenase solution and incubate for at least 30 min at 37 C Discard the collagenase solution and rinse the MEA with distilled water at least 3x Air dry the MEA preferably under a laminar flow hood Culture Chamber Options You have several options regarding culture chamber interface rings without ring glass ring plastic ring without and with thread and culture chambers which are especially useful for long term cultures or experiments For more details or pricing information please ask your local retailer Sealed MEA Culture Dish In order to allow long term cultivation and recording Multi Channel Systems recommends the use of teflon membranes fluorinated ethylene propylene 12 5 microns thick developed by Potter and DeMarse 2001 The ALA MEA MEM membrane is produced in license by ALA Scientific Instruments Inc and distributed via the world wide network of MCS distributors The sealed MEA culture chamber with transparent semipermeable membrane is suitable for all MEAs with glass ring A hydrophobic semipermeable membrane from Dupont that is selectively permeable to gases O CO but not to fluid and H O vapor keeps your culture c
23. 200 30 MEA 1828000sec Aus 1000 p ms gt 00 10000 18288 sweep YAsis 250 wV gt soo 500 500 500 d 500 Y 500 p 500 500 p 500 0 21 Ml 51 61 2 3 3 n 4 Figure 1 This picture shows the typical noise level of a standard 200 30 MEA on most electrodes recorded with a MEA1060 BC amplifier Electrodes 43 52 53 and 84 show an increased noise level after a longer cycle of use The bath was grounded with the internal reference electrode 15 Time axis 1000 ms voltage axis 50 UV You should ground some of the electrodes if you want to use this MEA for recording E Standard Noise with Shielding CECI 514000sec Ass 1000 ms gt 00 E 500 514th sweep Ys 220 p WY gt 200 250 as rl ee rl VEN Pi Figure 2 Same MEA zoom to single channel 22 Time axis 500 ms voltage axis 20 pV 30 imi Continuous Data A i FI nj xj 1890 000 sec 1890th sweep 500 0 500 1 21 22 23 XAwis 1000 p ms gt oo 10000 Yaris 100 uY 000 J d 500 p 500 o 500 4 500 0 500 0 31 41 51 61 71 pe_p pasala li io DEDO RS PRA AAA PNE NEUE EP S S EI 2 E DJ E E E 3i EJ EJ E a 3 ES Y Ej E EE EE 100 0 500 Appendix Figure 3 Same MEA after grounding defective electrodes Time axis 1000 ms voltage axis 100 uv 31
24. 30 i r hexagonal Hexa 10 20 30 60 i r with internal reference electrode Optional with 8 stimulating electrodes TiN electrodes SiN isolator and ITO contact pads 100 10 ITO 200 30iR ITO HexaMEA ITO 10 20 30 0 C to 125 C 49 mm x 49 mm x 1 mm 8g Glass Indium tin oxide As specified 10 20 and 30 um available As specified 100 and 200 um available not applicable to Hexa 10 20 30 ITO MEA layout Planar Titanium nitride Silicon nitride 500 nm PEVCD 30 kO to 400 kO depending on electrode type and diameter 8 x 8 100 10 200 30 i r hexagonal Hexa 10 20 30 60 i r with internal reference electrode ThinMEA ThinMEA 180 pm 100 10 ITO ThinMEA 180 pm 200 30iR ITO 0 C to 125 C 49 mm x 49 mm x 180 um glass part 8g Glass on a ceramic carrier Titanum As specified 10 and 30 um available As specified 100 and 200 um available Planar Titanium nitride Silicon nitride 500 nm PEVCD 30 kO to 400 kO depending on electrode type and diameter 8x8 60 i r with internal reference electrode multichannel systems 3 D MEA gr 0 C to 80 C 49 mm x 49 mm x 1 mm 8g Glass Platinum 40 um at base 200 um Approx 50 um to 70 um Platinum SU 8 approx 400 kO range from 250 to 500 8x8 60 The product type specifies the interelectrode distance and the electrode diameter that is 100 10 means 1
25. 34 Greiner Bio One Chamber Non Toxic Silicon area 3 1 cm diameter 2 cm volume 4 ml 27 7 1 7 1 1 7 1 2 7 1 3 Appendix Recording with MEAs Mounting the MEA Cleaning the Contact Pads You should always clean the contact pads with alcohol before placing it into the MEA amplifier Even if you do not see any contaminations a very thin grease layer from touching the pads with bare fingers for example may be present and results in a bad contact between the pads and the amplifier pins A bad contact will result in an increased noise level on the affected channel This is the most prominent handling error Carefully wipe the MEA contact pads with a clean and soft tissue moistened with pure alcohol Positioning the MEA Warning 3 D MEAs are sensitive to distortions and deflections Always put the provided yellow plastic plate first into the MEA amplifier and then place the 3 D MEA on top of it Otherwise the pressure applied by the MEA amplifier will irreversibly damage the MEA When placing an MEA into the amplifier please make sure that the orientation of the MEA is correct The writing NMI should be on the right side viewed from the front with the sockets of the amplifier in the back For 3 D MEAs The writing BOT ME60 V4 should be on the right bottom Otherwise the MEA layout will not match with the pin layout Grounding the Bath Make sure that the bath is connected to the amplifier s ground
26. As have also a fine noise level but bigger electrodes and a lower spatial resolution They are used in experiments that emphasize on the higher surface area of a 3 D MEA rather than a high spatial resolution Gold electrodes EcoMEAS are only available with a low spatial resolution and are useful for medium throughput screening where costs are a limiting factor All planar TiN electrodes are positioned on a round pad with a diameter of 40 uim 3 D MEAs feature tip shaped electrodes with a base of 40 um If you like to check the electrodes with a light microscope you will need an upright microscope to see the MEA from above With an inverse microscope you are only able to see the bigger pad from below not the electrode itself The electrodes are embedded in a carrier material usually glass Standard tracks made of titanium or indium tin oxide ITO are electrically isolated with silicon nitride PEVCD The contact pads are of the same material as the tracks are except 3 D MEAs ITO contact pads and tracks are transparent for a perfect view of the specimen under the microscope 10 4 1 MEA Types and Layouts MEA Types and Layouts Various types of MEA biosensors are available for all kind of extracellular multichannel recordings Typical MEAs for in vitro applications have 60 microelectrodes arranged in an 8 x 8 layout grid embedded in a transparent glass substrate You can cultivate the tissue or cell culture directly on the MEA
27. PEI plus Laminin Polyethyleneimine PEI has been successfully employed for dissociated cell cultures and proven to enhance cell maturation in culture compared to polylysine coated plates Polyethyleneimine is a positively charged polymer and thus changes the charge on the glass surface from negative to positive The tissue sticks even better with this method than with the nitrocellulose method but the polyethylenimine forms a uniform layer that can get more easily detached from the surface for example by the perfusion This coating method can optionally be combined with laminin Materials Poly ethyleneimine solution PEI Sigma Aldrich Inc P3143 Boric acid crystalline Fisher Scientific A73 500 Borax sodium tetraborate Sigma Aldrich Inc B0127 1 N HCl Laminin 1mg ml Sigma Aldrich Inc L2020 Borate buffer 3 10 g boric acid 4 75 g borax Dissolve in 11 distilled water Adjust pH to 8 4 with 1 N HCI PEI stock solution 0 05 0 1 96 PEI dissolved in borate buffer Laminin solution 20 ug ml laminin in plating medium Procedure Note It is necessary to thoroughly rinse off unbound PEI from the plates before use as dried PEI is toxic 20 5 4 3 Appendix Pipette 500 ul PEI solution onto the MEA The recording field should be completely covered Incubate at RT for 1 h or at 4 C over night Remove the PEI solution and thoroughly rinse 4 x with distilled water Air dry the MEA Steril
28. ally for established cardiomyocyte cultures large slices or whole heart preparations FlexMEAs made of flexible polyimid material perfect for in vivo and special in vitro applications for example whole heart preparations Standard Electrode Numbering The numbering of MEA electrodes in the 8x8 21 Gr G1 71 grid standard MEAs ThinMEAs 3 D MEAs ecoMEAs follows the standard numbering scheme for square grids The first digit is the 12 22 2 2 2 column number and the second digit is the row number For example electrode 23 is 13 2 83 a3 3 63 3 83 positioned in the third row of the second O E au These numbers are the same numbers that are used as channel numbers in the MC Rack 15 25 35 65 program Please make sure that you have selected the two dimensional MEA layout as the Channel Layout in MC_Rack For more details please refer to the MC_Rack user manual or 2 0900 Other electrode grids are described in the Appendix 11 4 2 4 3 MEA User Manual Standard MEAs The following standard MEAs are available 100 10 200 10 200 10 i r 200 30 200 30 i r 500 10 500 10 i r Standard MEAs have 60 electrodes in an 8 x 8 layout grid with electrode diameters of 10 um or 30 um and interelectrode distances of 100 um 200 pm or 500 um Versions 200 10 200 30 500 10 and 500 30 are available with an internal reference electrode as indicated by the abbreviation i r You ca
29. also includes recommendations on sterilization coating and cleaning procedures from scientifical papers or from recommendations of other MEA users For more details on issues that refer to the amplifier like grounding or mounting the MEA please refer to the user manual for the MEA amplifier you use You will find more information about the MEA System and its components in general especially the data acquisition card in the MEA System User Manual For more details on the data acquisition and analysis program MC_Rack please refer to the MC_Rack User Manual It is assumed that you have already a basic understanding of technical terms No special skills are required to read this manual The components and also the user manual are part of an ongoing developmental process Please understand that the provided documentation is not always up to date Please check the MCS Web site www multichannelsystems com from time to time for downloading up to date manuals Important Information and Instructions Operator s Obligations The operator is obliged to allow only persons to work on the device who are familiar with the safety at work and accident prevention regulations and have been instructed how to use the device are professionally qualified or have specialist knowledge and training and have received instruction in the use of the device have read and understood the chapter on safety and the warning instructions in this manual and confi
30. aped electrodes are intended to penetrate dead cell layers Using conventional flat electrodes the electrodes may interface with the damaged cell layer rather than with the healthy cells The 3 dimensional electrode of a 3 D MEA may be able to penetrate this cell layer and contact the healthy cells above better The tip shaped electrode results in a larger surface area The spatial resolution is limited 60 electrodes are aligned in an 8 x 8 grid with interelectrode distances of 200 um The platinum electrodes are 50 to 70 um high and have a diameter of about 40 um at the base ending in a fine small tip Tracks and contact pads are made of platinum insulation material is SU 8 3 D MEAs are produced for Multi Channel Systems by Ayanda Biosystems in Lausanne Switzerland www ayanda biosys com Using 3 D MEAs Due to the production process there may be more variations in the electrode impedance in comparison with TiN electrodes which is important for stimulation experiments especially with current 3 D MEAs consist of several layers that are glued together This leads to the fact that these MEAs are very sensitive to distortions and deflections A yellow plastic plate is provided to stabilize the form of 3 D MEAs 3 D MEAs should always be used in combination with these plates These MEAs are only stable to temperatures of up to 80 C they are not suited for autoclavation because of the high temperature and press
31. ble and will be irreversibly damaged 5 2 1 Sterilization with Ethanol and UV Light 1 Rinse MEAs with 70 ethanol 2 Let MEAs air dry over night on a sterile workbench laminar flow hood with UV light turned on 5 2 2 Steam Sterilization Autoclavation Autoclave MEAs at 134 C for 3 min 5 2 3 Dry Heat Sterilization Thermally sterilize MEAs in an oven at 121 C for 15 min Thermally sterilize 3 D MEAs in an oven at 56 C for 8 hours 5 2 4 Sterilization with Hot Water Expose MEAs to hot water 90 C for 1 min 18 Appendix 5 3 MEA Storage To maintain a hydrophilic surface after hydrophilization it is recommended to store the MEAs filled with water until use Dry MEAs will get hydrophobic again after some time Store MEAs filled with sterile distilled water at 4 C in the dark that is in the fridge to prevent microbiological contaminations to maintain a hydrophilic surface 5 4 MEA Coating 5 4 1 Coating of MEAs with various materials is used for improving the attachment and growth of cell cultures or cultured slices Coating is generally not required for recordings from acute slices Coating of MEAs has the same purpose than coating of other culture dishes Therefore you can generally use the same standard protocols that you have established for coating culture dishes for your cell cultures provided that the involved chemicals are not aggressive and damage the electrodes see rec
32. device itself or other material damage Warning The device and the software are not intended for medical uses and must not be used on humans Malfunctions which could impair safety should be rectified immediately Regard the technical specifications of the various MEA types especially the temperature range and the safe charge injection limits for stimulation Do not autoclave or expose 3 D MEAs or FlexMEAs to heat Do not touch the electrode field in any way Always put the provided yellow plastic plate beneath a 3 D MEA before placing it into the MEA amplifier Avoid any mechanic pressure or stress when handling 3 D MEAs Do not use any liquids or cleaning solutions with a high pH gt 7 for a longer period of time on MEAs of a silicon nitride insulation type Basic solutions will damage TiN electrodes 3 1 MEA User Manual Microelectrode Arrays MEAs Overview Extracellular Recording with Microelectrode Arrays A microelectrode array MEA is an arrangement of several typically 60 electrodes allowing the targeting of several sites in parallel for extracellular recording and stimulation Cell lines or primary cell preparations are cultivated directly on the MEA Freshly prepared slices can be used for acute recordings or can be cultivated as organotypic cultures OTC on the MEA Recorded signals are amplified by a filter amplifier and sent to the data acquisition computer All MEAs except FlexMEAs are only for use with
33. e shorter is the maximum duration of the pulse Do not apply pulses with a higher amplitude or for a longer time than is recommended for the electrode type As a consequence of the points discussed above Multi Channel Systems recommends using negative monophasic voltage pulses to make sure that the voltage level of the stimulating electrode is zero and thus the electrode is discharged at the end of the pulse According to the experience of MEA users voltage pulses should be lt 1 V 100 mV to 900 mV for neuronal applications to avoid damage to electrode and cells Generally pulse durations between 100 500 us are used See also Potter S M Wagenaar D A and DeMarse T B 2005 Closing the Loop Stimulation Feedback Systems for Embodied MEA Cultures Advances in Network Electrophysiology Using Multi Electrode Arrays M Taketani and M Baudry Springer Wagenaar D A Madhavan R Pine J and Potter S M 2005 Controlling bursting in cortical cultures with closed loop multi electrode stimulation J Neurosci 25 3 680 8 For pacing cardiomyocytes higher voltages and durations are generally required for example 2 V for 2 ms As these pulses are not supported by standard MEA electrodes the use of larger stimulating electrodes is recommended A special MEA with four pairs of large 250x50 um stimulating electrodes MEA 200 30 stim and a special stimulation adapter is provided for such applications by Multi Channel Syst
34. easily damaged except EcoMEA electrodes 5 1 Hydrophilic Surface Treatment 5 1 1 5 1 2 The surface of new MEAs is hydrophobic and even hydrophilic MEAs tend to become hydrophobic again during storage A hydrophobic surface prevents attachment and growth of the hydrophilic cells The first step in preparing an MEA for use is therefore to ensure that the surface is hydrophilic enough for coating and cell adhesion To test this without contaminating the surface place a small drop of water on the MEA surface outside the culture chamber If the drop does not wet the surface you likely need to perform one of the following steps in particular when using new arrays Literature Ulrich Egert Thomas Meyer Heart on a Chip Extracellular multielectrode recordings from cardiac myocytes in vitro Methods in Cardiovascular Research S Dhein and M Delmar eds 2004 Plasma Cleaning Laboratories with access to electron microscopy facilities are likely to have a sputter device or a plasma cleaning chamber for example PDC 32G from Harrick Plasma Ithaca NY United States MEAs can be treated in these chambers with low vacuum plasma for about two minutes The MEA surface is exposed to a gas plasma discharge which will make the surface polar and thus more hydrophilic The treatment gives a very clean and sterile surface that can be coated readily with water soluble molecules Note that the effect wears off after a few days Protei
35. eep YAsis 220 200 If too many electrodes are defective use a new MEA Contact pin is defective Please see the user manual for the respective MEA amplifier 39 MEA User Manual Overall Noise Unsteady Baseline The baseline is unstable signals are jumping or drifting Possible causes Bath electrode is not connected to ground Connect the internal or external bath electrode to one of the ground inputs of the amplifier AgCI bath electrode needs is not well chlorided Rechloride the electrode or use a new one 50 Hz hum 50 Hz is the frequency of mains power in Europe If the shielding and grounding of the setup is not sufficient electrical signals are picked up from the environment Use a proper shielding For example you can place aluminum foil over the amplifier that is connected to any metal part of the MEA amplifier You can also use special shielding equipment like a Faraday cage The following screen shot shows a recording of an MEA 200 30 without bath electrode and without shielding You see that the signals are so high that the amplifier gets saturated and you see a very strong 50 Hz hum fm 50 Hz Hum without groun 145 000 sec Aus 1000 n ms p 145th sweep Ys 21000 uV 310000 1000 1000 1000 aan mm 1000 00 salad aala asala aada aa ad fF
36. ems Warning When using MEA electrodes of TiN material use only negative voltages pulses or biphasic current pulses applying the negative phase first Always regard the safe charge injection limits as described in the appendix of this manual Otherwise electrodes can be irreversibly damaged by electrolysis 35 9 1 9 2 Troubleshooting Troubleshooting About Troubleshooting The following hints are provided to solve special problems that have been reported by users Most problems occur seldom and only under specific circumstances Please check the mentioned possible causes carefully when you have any trouble with the product In most cases it is only a minor problem that can be easily avoided or solved If the problem persists please contact your local retailer The highly qualified staff will be glad to help you Please inform your local retailer as well if other problems that are not mentioned in this documentation occur even if you have solved the problem on your own This helps other users and it helps Multi Channel Systems to optimize the instrument and the documentation Please pay attention to the safety and service information chapter Important Safety Advice on page 7 Multi Channel Systems has put all effort into making the product fully stable and reliable but like all high performance products it has to be handled with care Technical Support Please read the Troubleshooting part of the user manual first
37. ends on the stimulus and on the electrode type When stimulating via MEA electrodes and CEE zin xd with standard MEA amplifiers you will see 453838sec XAWs 200 ms gt 300 100 F a stimulus artifact on all amplifier Th sweep Y s 21000 uW 4600 f 3980 M channels during stimulation due to the cca N 61 high charge that is injected into the circuit and the following saturation of the filter amplifiers The time constant of the stimulus artifact depends on the amplifier bandwidth if the lower cutoff frequency is quite low for example 1 Hz the stimulus artifact will be longer than with e g 10 Hz In most cases it will not be possible to record true signals that are close to the stimulus pulse This can be avoided by using a MEA amplifier with blanking circuit The stimulating electrode can generally not be used for recording in parallel to stimulation because the injected charge is so high and the time constant for discharging so low Figure 4 The screen shot shows a prominent stimulus artifact on all channels followed by a response The stimulating electrode No 61 has been grounded The next pictures demonstrate the blanking feature On the left screen shot you see the stimulus artifacts on a non stimulating electrode without blanking On the right you see the same electrode and stimulation pattern but with blanking The stimulus artifacts have been comp
38. er TCO2 ALA MEA PPORT2 and accessories 10 2 2 MEA Amplifiers Product Product Number Description MEA amplifier for inverted MEA1060 1 Probe interface and 60 channel pre microscopes and filter amplifier with custom gain MEA amplifier for upright MEA1060 2 ena PERAWAN microscopes MEA amplifier with blanking circuit for inverted microscopes MEA amplifier with blanking circuit for upright microscopes MEA1060 1BC MEA1060 2BC Probe interface and 60 channel pre and filter amplifier with custom gain and bandwidth The blanking circuit prevents the amplifier from getting saturated and thus prevents stimulus artifacts 10 2 3 Accessories Product MEA culture chamber MEA culture chamber lid Sealed MEA culture dish MEA perfusion insert Product Number CCIR CCL ALA MEA MEM ALA MEA SHEET ALA MEA MEM5 ALA MEA MEM TOOL ALA MEA INSERT Appendix Description Suitable for all MEAs with plastic ring and thread Simply screw the culture chamber onto the plastic holder on the MEA An o ring ensures that the chamber fits tightly and is leakproof Autoclavable Complete with lid Additional or replacement lid for MEA culture chamber MEA culture chamber with transparent semipermeable membrane suitable for all MEAs with glass ring Simply slide the culture chamber over the glass ring A hydrophobic semipermeable foil from Dupont that is selectively permeable to gases O CO but not to fluid
39. es on the MCS web site http www multichannelsystems com support applicationnotes htm 3 2 Introduction MEA Design and Production A standard MEA biosensor has a square recording area of 700 um to 5 mm length In this area 60 electrodes are aligned in an 8 x 8 grid with interelectrode distances of 100 200 or 500 um Planar TiN electrodes are available in sizes of 10 20 and 30 um and three dimensional Pt electrodes have a diameter of 40 um at the base with a very fine tip Standard MEAs are useful for a wide variety of applications Different geometries match the anatomical properties of the preparation Most MEAs are available with a substrate integrated reference electrode replacing the silver pellet in the bath All electrodes can either be used for recording or for stimulation Several other MEA types and layouts that are dedicated to special applications are also available please see MEA Types and Layouts for more details The biological sample can be positioned directly on the recording area the MEA serves as a culture and perfusion chamber A temperature controller controls the temperature in the culture chamber Various culture chambers are available for example with leak proof lid or with semipermeable seal An incubator is not necessarily required long term recordings in the MEA culture chamber are possible over several weeks or even months For cell or slice cultures MEAs have to be coated with standard procedures
40. es plus two indifferential reference electrodes and two ground electrodes More layouts can be provided on request The flexible base is perforated for a better contact with the surrounding tissue The electrodes have a diameter of about 30 um with an interelectrode distance of 300 uim Conducting material is pure gold Using FlexMEAs FlexMEAs are usually connected to a headstage preamplifier that is connected to a filter amplifier or programmable gain amplifier see also the ME System product line of Multi Channel Systems FlexMEAs can be directly connected to a 32 channel miniature preamplifier from Multi Channel Systems for in vivo experiments FlexMEAs are stable at a temperature range from 10 C to 40 C Warning Do not autoclave or sterilize FlexMEAs by heat These MEA types are not heat stable and will be irreversibly damaged if the temperature is too high 16 Appendix 5 MEA Handling A A Warning If possible use only liquids or cleaning solutions with a neutral pH 7 on MEAs Do not expose MEAs with a silicon nitride insulation or TiN electrodes to basic liquids pH gt 7 or aggressive detergents for a longer period of time Basic or aggressive liquids may damage TIN electrodes irreversibly Warning Do not to touch the electrode field in any way during the coating or cleaning procedure Keep all instruments tissues pipette tips and similar at a safe distance from the recording area The electrodes are
41. ghly with distilled water Rinse the 3 D MEA with 70 ethanol for a few minutes Rinse the 3 D MEA with distilled water for 1 minute to remove the ethanol Air dry the MEA preferably under a laminar flow hood Cleaning of EcoMEAs The gold electrodes of EcoMEAs are very robust and are the only MEA electrodes that will endure more severe cleaning methods You can check the need for cleaning under a stereo microscope The electrodes should be shiny and look golden If they are gray or if they show a film you should clean them Carefully clean the electrodes with a swab and distilled water under microscopic control Removing Nitrocellulose Coating Note It is very important that you clean MEAs that have been coated with nitrocellulose and remove all biological material first before removing the coating If you applied methanol on an uncleaned MEA you would rather fix the cell debris on the MEA than actually remove the coating Directly after usage biological material is rinsed off under running water and the MEA is cleaned with pH neutral cleaning agents or enzymatically if necessary Rinse the MEA 2x with methanol If nitrocellulose is not sufficiently removed by rinsing incubate the MEA filled with methanol for 15 to 30 min to dissolve the cellulose nitrate Rinse the MEA with distilled water 25 5 5 9 6 1 MEA User Manual MEA Cleaning with EDTA Collagenase Materials Collagenase Type Sigma Aldrich Inc C0
42. glass pipette The solution can be reused Incubate the MEA in a CO incubator for 30 min Rinse the MEA with sterile distilled water Fill the MEA with culture medium and keep it sterile in a CO incubator until use for up to one week Check for contaminations before use 24 Appendix 5 5 Cleaning of Used MEAs 5 5 1 5 5 2 5 5 3 5 5 4 General Recommendations for Cleaning MEAs The cleaning procedure depends on the kind of coating and on the kind of biological preparation In the following a few general considerations are listed If you have recorded from an acute slice without coating you can simply rinse the MEA with distilled water and the MEA should be fine If necessary the MEA can then be cleaned with any pH neutral cleaning agent for example a standard dish washing detergent When cleaning coated MEAs parts of the coating may go off You have to recoat an MEA when the coating is not sufficient anymore that is when you observe problems with cell attachment or recording If more severe methods are needed the MEA can also be cleaned in an ultrasonic bath for a short moment But this method is a bit dangerous because there are ultrasonic baths that are too strong and will destroy the MEA The behavior should be tested with an older MEA first EcoMEAs are easier to clean because the golden electrodes are not so easily damaged Cleaning of 3 D MEAs Rinse the culture chamber of the 3 D MEA thorou
43. ith 300 ul fibronectin solution and incubate the MEA at 37 C for at least Th Aspirate the solution and rinse the MEA 2x with PBS Plate the cells onto the MEA immediately after coating Literature Ulrich Egert Thomas Meyer Heart on a Chip Extracellular multielectrode recordings from cardiac myocytes in vitro Methods in Cardiovascular Research S Dhein and M Delmar eds in print 23 5 4 9 MEA User Manual Coating with Collagen Coating with collagen is useful for short term cultures It tends to detach from the surface if used for long term cultures Materials DMEM Dulbecco s Modified Eagle Media DMEM F12 Gibco Invitrogen 21331 020 0 01 N Hydrochloric acid pH 3 0 Acid soluble type collagen solution 3mg ml pH3 0 Cellmatrix Type I A Nitta Gelatin Inc Preparation buffer 200 mM HEPES in 0 08 N NaOH Collagen solution Add 1 ml of 10x DMEM F 12 medium to 8 ml Cellmatrix Type I A and stir gently Add 1 ml of preparation buffer and stir gently Incubate the mixture at 4 C for 30 min to remove any air bubbles if necessary Store at 4 C until use Procedure Sterilize the MEA before the coating with collagen and perform all following steps under sterile conditions Incubate the MEA at 4 C for at least 1h Fill the MEA with collagen solution until the bottom of the culture chamber is completely covered Immediately remove the collagen solution with a
44. ize with UV light for at least 1 h after coating Place a drop of sterile laminin solution onto the MEA and incubate for 30 min Aspirate do not rinse and directly seed your cells Alternatively mix the cells with laminin solution before plating Literature Ulrich Egert Thomas Meyer Heart on a Chip Extracellular multielectrode recordings from cardiac myocytes in vitro Methods in Cardiovascular Research S Dhein and M Delmar eds in print Lelong IH et al 1992 J Neurosci Res 32 562 568 Coating with Polyornithine plus Laminin Poly D lysine can be used as an alternative for polyornithine Materials Polyornithine Laminin 1mg ml Sigma Aldrich Inc L2020 Polyornithine solution 500 ug ml polyornithine in distilled water Laminin solution 5 ug ml laminin in plating medium or PBS Procedure Incubate the MEA with polyornithine solution at RT for 2 3 hours or overnight at 4 C Aspirate the polyornithine solution and rinse the MEA 3x with distilled water before direct use or before the following coating with laminin MEAs coated with polyornithine can be stored at 4 C for several weeks Incubate pre coated MEA with laminin solution for at least 1 h Aspirate the laminin solution and directly plate cells Literature Cellular Neurobiology A practical approach ed By Chad and Wheal IRL Press Oxford 21 5 4 4 3 MEA User Manual Coating with Poly D Lysine plus Laminin Po
45. lean and sterile preventing contaminations by airborne pathogens It also greatly reduces evaporation and thus prevents a dry out of the culture Reference Reference Potter S M and DeMarse T B 2001 A new approach to neural cell culture for long term studies J Neurosci Methods 110 1 2 17 24 26 6 2 6 3 Appendix MEA Culture Chamber with Lid Another possibility is to use a MEA culture chamber with lid available from Multi Channel Systems which is suitable for all MEAs with plastic ring and thread It can be adapted by inserting metal perfusion cannulas for setting up a continuous perfusion Removable Recording Chamber As an alternative to the fixed culture chambers you can use silicone rings that adhere to a dry MEA surface and can be removed without leaving any residue This is especially useful for acute experiments The removable recording chamber should be stored immersed in distilled water simply put it into a bottle filled with distilled water for best adhesion properties You dry the silicone chamber with a clean tissue put it onto the dry MEA with no rings and fill the chamber with your recording buffer Then you can mount the slice onto the recording field and perform your experiments After the experiment you simply remove the chamber from the MEA and rinse off the slice Sources of supply Product Product No Supplier flexiPERM conA Single Well Removable amp Reusable TC 960774
46. letely avoided making it possible to detect signals shortly after the stimulus E Blanking Off ord Em Blanking On 101 1858030sec Mass 500 p ms gt 4100 p H 600 28830sec Mawis 500 lm gt 100 p 600 1887th sweep Yis 100 V gt 1000 1000 30th sweep YAxis 100 p uY 41000 1000 5 15 20 25 30 35 40 45 50 55 10 15 20 25 30 35 40 45 50 55 1 33 8 2 8 3 MEA User Manual Capacitive Behavior of Stimulating Electrodes Regarding the generally used stimulus pulses stimulating electrodes behave as plate capacitors The charge cannot flow back to the stimulus generator due to the high output resistance and thus is kept in the electrode The electrode needs a quite long time to discharge itself after stimulation As a result stimulus artifacts interfere with the recording and electrodes deteriorate over time due to electrolysis You can avoid that by choosing an appropriate stimulus protocol that actively discharges the electrode after the pulse When using voltage driven stimulation the electrodes are discharged when the voltage level is set to zero at the end of the monophasic pulse Not so in current mode When applying a negative current pulse the electrode is charged and needs to be actively discharged by applying an inverted pulse with a matching product of current and time that is you need to stimulate with biphasic pulses for current driven stimulation to reduce both the
47. ly D lysine has been used by several groups Results seem to be equivalent to a coating with polyornithine Some users complained about cell clumping and resulting cell death when using poly D lysine and had better results when using polyethylenimine PEI Materials Poly D lysine 5 mg 10 mL 2 0 05 96 w v stock solution Sigma Aldrich Inc P7280 Laminin solution 1 mg ml Sigma Aldrich Inc L2020 Laminin solution 20 ug ml laminin in plating medium or PBS Procedure Incubate the MEA with poly D lysine solution and incubate at 4 C over night Rinse MEA with sterile distilled water 3x to remove toxic unbound lysine and let the MEAs air dry under sterile conditions laminar flow before plating the cells or before the following coating with laminin MEAs can be stored at 4 C for up to two weeks Incubate pre coated MEA with laminin solution at 4 C over night 4 Aspirate the laminin solution and directly plate the cells 5 4 5 Literature Goslin et al 1988 Nature 336 672 674 Maeda et al 1995 J Neurosci 15 6834 6845 Gross et al 1997 Biosensors amp Bioelectronics 12 373 393 Coating with Poly D Lysine plus Fibronectin This coating method is used for example for culturing dissociated suprachiasmatic nucleus SCN neurons on standard 200 30 MEAs It is very stable and therefore especially useful for long term cultures Materials Poly D lysine 5 mg 10 mL 0 05 96 w v stock solution
48. multichannel systems Microelectrode Array MEA User Manual n 0 0 90906909909 999999 99999299 999 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 2004 2005 Multi Channel Systems MCS GmbH All rights reserved Printed 2005 11 04 Multi Channel Systems MCS GmbH AspenhaustraBe 21 72770 Reutlingen Germany Fon 49 71 21 90 92 5 0 Fax 49 71 21 90 92 5 11 info multichannelsystems com www multichannelsystems com Microsoft and Windows are registered trademarks of Microsoft Corporation 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 2 1 2 2 2 3 3 1 3 2 3 3 4 1 4 2 4 3 4 4 4 5 4 6 4 7 4 8 5
49. n Coating If protein coating is acceptable in the planned experiments there is another quick and simple way to render the surface hydrophilic Sterilize the MEAs as described below Place approximately 1 ml of a concentrated sterile protein solution for example albumin fetal calf serum or similar onto the culture region for about 30 min Wash the culture chamber thoroughly with sterile water afterwards The MEA can then be directly used for cell culture 17 MEA User Manual 5 1 3 Preculturing Another pragmatic method is to coat the hydrophobic MEAs and to plate the cell cultures on the MEA and let it grow for some days up to weeks until the cells have transformed the surface so that it is sufficiently hydrophilic The preculture will generally show very bad growth and viability and needs to be discarded before plating the culture that will be used for experiments Please note that the MEA and the electrode performance may suffer under cell culturing Therefore the above mentioned methods are preferable 5 2 Sterilization Sterilization of MEAs is not necessary for acute slices Silicon nitride MEAs with TiN electrodes can be sterilized with standard methods for cell culture materials using either 70 alcohol UV light about half an hour depending on the intensity vapor autoclavation or dry heat sterilization Warning Do not autoclave or sterilize 3 D MEAs or FlexMEAs by heat These MEA types are not heat sta
50. n connect the internal reference electrode directly to the amplifier s ground and will not need silver pellets for grounding the bath anymore Please refer to the MEA1060 user manual delivered with your MEA amplifier for more information The flat round electrodes are made of titanium nitride TiN MEAs with TiN electrodes are very stable Therefore the MEAs can be reused several times and are perfect for long time experiments up to several weeks and even months The electrode impedance ranges between 30 kQ and 400 kO depending on the electrode diameter Generally the smaller the electrode the higher is the impedance Tracks and contact pads are made of titanium or ITO insulation material is silicon nitride ITO contact pads and tracks are transparent for a perfect view of the specimen under the microscope Using standard MEAs Standard MEAs can be used for a wide variety of applications They are robust and heat stable They can be autoclaved and coated with different procedures for cell and tissue cultures Generally they can be used for acute experiments as well as long term cultures HighDenseMEAs 10 um electrodes are arranged in two recording field with 5 x 6 electrodes each The interelectrode spacing is only 30 um center to center The very high electrode density of the two recording fields on a HighDense MEA is only possible by the special TiN electrode material and production process This MEA type is especially useful fo
51. nitrocellulose can be stored for a few days Nitrocellulose coating has to be removed after use Main advantages of this method are that nitrocellulose is cheap coating is fast and easy and it is also easily removed after use Note Nitrocellulose solutions cannot be stored for a longer period of time The solution forms a visible gelatinous precipitate after extended storage of at least half a year and will not produce satisfactory adhesive coatings anymore Prepare a fresh solution if there are visible precipitates Materials Protran or other standard nitrocellulose membrane Whatman PerkinElmer 100 Methanol Nitrocellulose solution For preparing a stock solution dissolve a piece of 1 cm nitrocellulose membrane in 10 ml methanol Stock solutions may be stored at room temperature in polystyrene tubes For the working solution dilute the stock solution 10 1 with methanol You can adjust the concentration to meet your requirements 19 5 4 2 MEA User Manual Procedure Directly before use pipet 3 5 ul of the working solution onto the recording field and let it air dry The recording field should be completely covered It takes just a few seconds for the methanol to evaporate Literature Ulrich Egert Thomas Meyer Heart on a Chip Extracellular multielectrode recordings from cardiac myocytes in vitro Methods in Cardiovascular Research S Dhein and M Delmar eds 2004 Coating with Polyethyleneimine
52. nnel FlexMEA systems 36 Electrodes 32 recording electrodes 2 indifferent reference electrodes 2 large ground electrodes for use with miniature preamplifier MPA32I FLEX 9 909090 9000 The letter digit code is the electrode identifier and refers to the position of the electrode in the grid Below are the recording channel numbers in italics that refer to the channel numbers in the MC_Rack program Please make sure that you have selected the linear channel layout in the MC_Rack program See the MC_Rack user manual or help for details If you use more than one MPA32I FLEX and a ME64 System the signal collector SC2x32 leads the output channels of the second amplifier to channel number 33 64 Please see the SC2x32 data sheet for details Multi Channel Systems Fon 49 7121 9 09 25 O O 2002 2005 Multi Channel Systems MCS GmbH Fax 49 7121 9 09 25 11 MCS GmbH Aspenhaustrasse 21 72770 Reutlingen info multichannelsystems com Germany www multichannelsystems com Product information is subject to change without notice Safe Charge Injection Limits of Multi Elctrode Arrays with TiN Electrodes diameter 10um max pulse amplitude HA max pulse amplitude HA max pulse amplitude HA safe charge injection limits 2000 r r r 1500 1000 500 0 5 10 15 20 25 30 35 40 45 50 time us safe charge injection limits 400 350 300 250 200 150 100 50 A 0 1 1 1 1 50 100 150
53. ommendations for the various MEA types In the following some standard coating procedures are shortly described You should try out which coating procedure proves best for your application The listed materials are only recommendations you may use any equivalent equipment Most coatings are stable for several uses of the MEA and do not have to be removed after use except nitrocellulose Please note that the materials and procedures described in the following are only a summary of published information from other sources see references or from personal communications with MEA users and has the intention of helping users finding the appropriate information for setting up their experiments Multi Channel Systems MCS GmbH has not tested or verified this information and therefore cannot guarantee that the information is correct Please refer to the referenced literature for planning and executing any experiments Coating with Nitrocellulose Coating with nitrocellulose is a fast procedure that works with several cell types and tissues and that is also successful with slightly hydrophobic MEAs This method has the advantage that the cells stick well to the surface Nitrocellulose does not form a uniform layer on the MEA The coating leaves patches of nitrocellulose which serve as a glue for the tissue on the MEA surface The tissue is not likely to get detached even under severe mechanical disturbance by perfusion for example MEAs coated with
54. r applications where a high spatial resolution is critical for example for multitrode analysis For example the very high spatial resolution of the HighDense MEAs is very useful for recording from retina ganglia cells The double recording field can also be used for coculturing two slices each on one recording field The flat round electrodes are made of titanium nitride TiN Tracks and contact pads are made of transparent ITO insulation material is silicon nitride Using HighDenseMEAs The same material is used for standard MEAs and HexaMEAs Therefore they are equally robust and heat stable They can be autoclaved and coated with different procedures for cell and tissue cultures 12 4 4 4 5 MEA Types and Layouts HexaMEAs HexaMEAs feature a hexagonal layout perfect for recording from retina 9 d j Gum Y e 60 electrodes are aligned in a special configuration Bron e O e with varying electrode diameters 10 20 30 um o and interelectrode distances see picture The a specific layout resembles ideally the regularity of s E s T e the retina s architecture The density of neurons is 0 so 0 more important in the center than in the a ge sonde y 6 y T peripheral This is matched by the density of A electrodes on the MEA which is also higher in the O o center that in the peripheral e eo o o The flat round electrodes are made of titanium nitride TiN 9 e Tracks and contact pad
55. r to the MEA data sheet mm MEA Border 0 2 54 22t BERS 100 um or 200 um 1 Electrode grid 0060000 00000000 00000000 00000000 00000000 00000000 00000000 900000 10 or 30 um 49 0 The numbering of MEA electrodes in the 8x8 grid follows the standard numbering scheme for square grids The first digit is the column number and the second digit is the row number For example electrode 23 is positioned in the third row of the second column These numbers are the same numbers that are used as channel numbers in the MC_Rack program Please make sure that you have selected the two dimensional MEA layout as the Channel Layout in MC_Rack For more details please refer to the MC_Rack user manual or help Multi Channel Systems Fon 49 7121 9 09 25 O O 2002 2005 Multi Channel Systems MCS GmbH Fax 49 7121 9 09 25 11 MCS GmbH Aspenhaustrasse 21 72770 Reutlingen info multichannelsystems com Germany www multichannelsystems com Product information is subject to change without notice multichannel HighDenseMEA systems Electrode layout meai pins 33 27 G2 O G amp WO amp OO Electrode A3L C3L A4L A5L B4L B5L C4L C5L CIR C2R B1R B2R A1R A2R C3R A3R 22 B3L B3R 72 12 A2L Left electrode field Right electrode field AAR 82 23 At ASR 73 0000 3 B2L 23 12 33 32 13 MEA1060 pins CAR 83 Ge O 00006 o 60 606606070 5 02022 Heeeee g Ex 666 9 2 OOO OO 000
56. ring information 10 2 1 MEA Systems Product MEA recording system for inverted microscopes 60 electrode channels MEA recording system for upright microscopes 60 electrode channels MEA recording system for inverted microscopes with advanced perfusion 60 electrode channels MEA recording system for upright microscopes with advanced perfusion 60 electrode channels MEA recording system for inverted microscopes 120 electrode channels MEA recording system for upright microscopes 120 electrode channels Product Number MEA60 1System MEA60 2System MEA60 1SystemE MEA60 2SystemE MEA120 1System MEA120 2System Description Complete with 5 MEAs data acquisition computer with MC_Card and IPS10W MEA1060 1 amplifier TCO1 ALA MEA PPORT2 and accessories Complete with 5 MEAs data acquisition computer with MC_Card and IPS10W MEA1060 2 amplifier TCO1 ALA MEA PPORT2 and accessories Complete with 5 MEAs data acquisition computer with MC_Card and IPS10W MEA1060 1 amplifier TCO2 PHO1 ALA MEA PPORT2 and accessories Complete with 5 MEAs data acquisition computer with MC_Card and IPS10W MEA1060 2 amplifier TCO2 PHO1 ALA MEA PPORT2 and accessories Complete with 5 MEAs data acquisition computer with MC Card and IPS10W 2 x MEA1060 1 amplifier TC02 ALA MEA PPORT2 and accessories Complete with 5 MEAs data acquisition computer with MC_Card and IPS10W 2 x MEA1060 2 amplifi
57. rmed this with their signature It must be monitored at regular intervals that the operating personnel are working safely Personnel still undergoing training may only work on the device under the supervision of an experienced person Guaranty and Liability The General conditions of sale and delivery of Multi Channel Systems MCS GmbH always apply The operator will receive these no later than on conclusion of the contract Multi Channel Systems MCS GmbH makes no guaranty as to the accuracy of any and all tests and data generated by the use of the device or the software It is up to the user to use good laboratory practice to establish the validity of his findings Guaranty and liability claims in the event of injury or material damage are excluded when they are the result of one of the following Improper use of the device Improper installation commissioning operation or maintenance of the device Operating the device when the safety and protective devices are defective and or inoperable Non observance of the instructions in the manual with regard to transport storage installation commissioning operation or maintenance of the device Unauthorized structural alterations to the device Unauthorized modifications to the system settings Inadequate monitoring of device components subject to wear Improperly executed and unauthorized repairs Unauthorized opening of the device or its components Catastrophic events due to the effect of
58. s are made of opaque Ti or transparent ITO insulation material is silicon nitride Electrodes in the center have a diameter of 10 um with an interelectrode distance of 20 um where the peripheral electrodes have a diameter of 20 um and 30 um Using HexaMEAs The same material is used for standard MEAs and HexaMEAs Therefore they are equally robust and heat stable They can be autoclaved and coated with different procedures for cell and tissue cultures ThinMEAs ThinMEAs are only 180 um thick ideally suited for high resolution imaging ThinMEAs are like standard MEAs but the electrodes are embedded in a very thin and delicate glass substrate on a robust ceramic carrier The thin glass allows the use of oil immersion objectives with a high numerical aperture Like standard MEAs 60 electrodes are arranged in an 8 x 8 layout grid with electrode diameters of 10 uim and 30 um and interelectrode distances of 100 uim or 200 um The flat round electrodes are made of titanium nitride Tracks and contact pads are made of transparent ITO insulation material is silicon nitride Using ThinMEAs ThinMEAs are heat stable and can be autoclaved They can also be coated with different procedures for cell and tissue cultures They should be handled with great care because of the thin and delicate recording area 13 4 6 MEA User Manual 3 D MEAs 3 D MEAs are the ideal solution for acute slices because the three dimensionally sh
59. stimulus artifact and to avoid an electrode damage The easiest way is to use the same signal amplitude and the same duration with an inverse polarity For voltage driven stimulation monophasic pulses are fine The following illustration shows the effect of a biphasic current pulse on the discharge of the stimulating electrode As you can see the first monophasic pulse is followed immediately by a pulse of the opposite polarity and the same product of current and time Effect of a bipolar pulse on the electrode voltage Monopolar pulse Bipolar pulse U U Electrode Electrode voltage voltage Stimulus Stimulus t t Aspects of Electrode Size and Material Titanium nitrite TiN electrodes are generally more robust than electrodes from other materials for example platinum Pt In the Appendix you find safe charge injection limit curves that document maximum current and stimulus durations for standard TiN electrodes Please note that these curves document the limits Stimulus pulses should be kept safely below these limits The safe charge injection limit of platinum 0 4 mC cm is much smaller than for TiN 23 mC cm This fact results in a considerably lower charge that you can inject into the electrode before faradic reactions occur that will lead to electrolysis of the electrode For more information on safe charge injection limits of 3 D MEAs please contact Ayanda Biosystems Please note that when using voltage driven stimula
60. tion the current flow to the electrode depends on the electrode impedance The lower the impedance the higher is the current Please make sure to obey the safe charge injection limits always Generally TiN electrodes have lower impedances than Pt electrodes and larger electrodes also have lower impedances than smaller 34 8 4 Stimulation When using TIN electrodes it is extremely important to not charge the electrodes positively as this will lead to electrolysis This is not an issue for Pt electrodes Therefore when using voltage driven stimulation it is important to apply negative voltages only Positive voltages will shortly charge the electrodes positively even though the electrode is discharged at the end of the pulse As a consequence biphasic voltage driven stimulation is not recommended When using current stimulation it is required to use biphasic stimulation and to apply the negative phase first to avoid a positive net charge on the electrode Recommended Stimulus Amplitudes and Durations The higher the amplitude and the longer the stimulus the higher is the impact on the electrode performance Therefore the amplitude and duration should be as low as possible It is advisable to start with a low amplitude and duration and then increase it slowly until responses are evoked The safe charge injection limits in the appendix describe the relationship between maximum pulse amplitude and time The higher the amplitude th
61. ure that is applied during the autoclavation procedure Warning 3 D MEAs are sensitive to distortions and deflections Always put the provided yellow plastic plate beneath a 3 D MEA before placing it into the MEA amplifier Do not autoclave 3 D MEAs or sterilize 3 D MEAS by heat Avoid rapid temperature changes even if they are within the recommended temperature range Distortions of the MEA due to mechanic pressure or temperature will lead to bad contacts and irreversibe damage the MEA 3 D MEAs are generally used for acute slices and therefore do not need to be coated When stimulating with 3 D MEA electrodes please note that the safe charge injection limit of Pt electrodes is much lower than of TiN electrodes See also Recommended Stimulus Amplitudes and Durations 14 4 7 MEA Types and Layouts EcoMEAs EcoMEAs are a very cheap variant for medium throughput applications like small screens where material costs play a bigger role than in more scientific MEA applications New production processes and the use of new materials made it possible to create this high quality MEAs at very low prices EcoMEAs are opaque and are therefore useful only for applications where you do not need a visual control under a microscope for example for established cell cultures Due to the special production process electrodes of EcoMEAs are available only with a diameter of 100 um and an interelectrode distance of 700 um Thus ecoMEAs are useful

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