AgilePulse™
Contents
1. Immune system reacts to viral vector inefficient when boosting Pain 2 out of 10 www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM USER MANUAL 800 272 2779 SOB 893 8999 AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM AGILEPULSE TUTORIAL 3 3 DNA DELIVERY INTO CELLS USING ELECTROPORATION 3 3 1 GENERAL ELECTROPORATION DISCUSSION Electroporation is the use of a transmembrane electric field pulse to induce microscopic pathways pores in a bio membrane Their presence allows molecules ions and water to pass from one side of the membrane to the other As the right side bar shows when the electric field is applied the ions inside and outside the cell membrane migrate As the charge builds up on either side of the membrane the membrane weakens and the pathways form permitting material outside of the cell to enter If the electric field is promptly removed the pathways close and the membrane reseals If the electric field duration is too long the pathways increase and the cell is killed Efficient electroporation depends on proper selection of electric field waveforms The electropores are located primarily on the membrane areas which are closest to the electrodes The pathways form in about a microsecond and seal in seconds to minutes The duration of the electric field is tens of microseconds to tens of milliseconds The use of electroporation was described by Neumann in the early 1980 The ro2. Maximum Number Duty Cycle of Pulses Pulse Interval Pulse Pulse Amplitude Width 50 0 050 0 2 50 10 to 300 V to 10 ms to 1000 ms 310 0 050 0 2 50 10 to 1000 V to1ms to 1000 ms Limited to 50 Duty Cycle i e Pulse Width Pulse Interval less than or equal to 0 5 Three groups may be used A group is 1 10 pulses with the same parameters The system software automatically concatenates each group USER MANUAL 800 272 2779 SOB 893 8999 AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM AGILEPULSE TUTORIAL 3 3 DNA DELIVERY INTO CELLS USING ELECTROPORATION CONTINUED 3 3 2 AGILEPULSE ELECTROPORATION CONTINUED Electrode Electrodes used in ex vivo and in vivo electroporation are quite different In ex vivo electroporation the cells are generally placed in an aqueous ionic medium in a chamber that has parallel plate metal electrodes This configuration produces very uniform electric fields For in vivo electroporation the electric fields must be established in human tissue in the body In this configuration parallel plates are not practical for vaccine delivery For in vivo use two parallel rows of needles are used This configuration was first published in an abstract form by Dr Julie Gehl in 1997 Herlev Hospital Denmark Parallel row arrays must be carefully designed to produce electric field intensities as close to uniform as possible to ensure as many
3. 2006 The AgilePulse system is typically used to deliver cancer vaccines in doses of 50 ug or more The AgilePulse system includes a AgilePulse voltage waveform generator and an Intradermal Array IDA electrode used to convert the voltage to electric fields which deliver the vaccine directly into cells in the skin 3 2 DNA VACCINE DELIVERY METHODS There are several methods used to del iver DNA vaccines System Name Company Mechanism Target Factors all pain ratings are estimates AgilePulse BTX Inject vaccine in dermis with hypodermic needle insert needle array apply electric field to deliver into cells Dermis e Large dose e Reduced pain compared to other electroporation systems Pain 3 out of 10 High cell transfection PowderMed Microscopic particles coated with DNA delivered into cells using compressed helium Epidermis Small doses Possible tattooing Pain 3 out of 10 Low Medium cell transfection Biojector 2000 Bioject Medical Technologies Inject using CO pressure with spacer subcutaneous or w o spacer to muscle Skin or Muscle Large doses Pain 3 out of 10 Medium cell transfection Many muscle electroporation Inject into muscle insert electrode and apply electric field Muscle Large dose Significant pain Pain 5 6 out of 10 High cell transfection Deliver viral vector with hypodermic needle into muscle
4. Kakorin S amp Toensing K 1999 Fundamentals of electroporative delivery of drugs and genes Bioelectrochem Bioenerg 48 3 16 Neumann E Toensing K Kakorin S Budde P amp Frey J 1998 Mechanism of electroporative dye uptake by mouse B cells Biophys J 74 98 108 Sukharev S l Klenchin V A Serov S M Chernomordik L V amp Chizmadzhev Y 1992 Electroporation and electrophoretic DNA transfer into cells The effect of DNA interaction with electropores Biophys J 63 1320 1327 www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM USER MANUAL 800 272 2779 SOB 893 8999 AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM AGILEPULSE TUTORIAL 3 3 DNA DELIVERY INTO CELLS USING ELECTROPORATION CONTINUED 3 3 2 AGILEPULSE ELECTROPORATION In section 3 3 1 general concepts of electroporation were presented This section presents the types of waveforms and electrodes available for use in the AgilePulse system Waveforms Traditional in vivo electroporation uses four to six rectangular pulses that are 100 microseconds in duration at a rate of one per second Thus the total treatment time is four to six seconds More advanced waveforms such as the PulseAgile have been found to be more effective in eliciting T cell and antibody responses simultaneously when delivering DNA vaccines Roos et al 2006 Vertuani et al 2009 Brave et al 2009 A PulseAgile waveform consists of
5. Check for obvious signs of exterior damage If damage is User name admin Change noted contact BTX Customer Service before proceeding Password Change Place the Waveform Generator on the top of a sturdy table Connect the electrode cable into the connector at the bottom right of the front panel Connect the mains power cord into the back panel at the bottom right Connect the foot switch if applicable into one of the USB Figure 4 3 Login Screen ports at the bottom left corner of the Waveform Generator visible about 10 seconds after power on front panel PLEASE CONTACT BTX IF YOU ARE INTERESTED IN AN ON Experiment Welcome to SITE TRAINING Agile Pulse II Electrode 4 4 1 INITIAL SYSTEM TEST Bee ohh We perator admin This section will describe the process to verify mains line cable s has been installed properly and the computer boots up BS 1 Connect the system power cord to the mains Plug in the device Turn the rocker switch Line Main Power on the front panel to the on position I The rocker switch labeled Power should illuminate and the star Bee awe dT Login Screen should appear within 10 seconds If the Figure 4 4 System OK Screen Operating Correctly Waveform Generator power switch fails to illuminate then return it to the OFF position O Verify the power cord is properly plugged into the wall Verify the wall socket is a Welcome to functional If necessary you may need
6. Electroporation and electrophoretic mediated transformation isolation and characterization of a DNA transfer into cells The effect of DNA interaction with TK subclone Biochem Biophys Res Commun 129 611 618 electropores 1992 Biophys J 63 1320 1327 Nickoloff Jac A ed 1995 Plant Cell Electroporation and Electrofusion Protocols Methods in Molecular Biology Volume 55 Humana Press Totowa New Jersey E A Disalvo and S A Simon eds 1995 Permeability and Stability of Lipid Bilayers CRC Press Boca Raton p 105 121 Chang D C Chassy B M Saunders J A and Sowers A E eds 1992 Guide to Electroporation and Electrofusion Academic press San Diego 581 pp Neuman E Sowers A E and Jordan C A eds 1989 Electroporation and Electrofusion in Cell Biology Plenum Press New York 581 pp Bartoletti D C Harrison G 1 amp Weaver J C 1989 The number of molecules taken up by electroporated cells quantitative determination FEBS Lett 256 4 10 Djuzenova C S Zimmermann U Frank H Sukhorukov V L Richter E amp Fuhr G 1996 Effect of medium conductivity and composition on the uptake of propidium iodide into electropermeabilized myeloma cells Biochim Biophys Acta 1284 143 152 Klenchin VA Sukharev SM Chernomordik LV Chizmadzhev YA Electricaly induced DNA uptake by cells is a fast process involving DNA electrophoresis 1991 Biophys J 60 804 81 1 Neumann E
7. IN VIVO GENE DELIVERY SYSTEM USER MANUAL 800 272 2779 508 893 8999 AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM BTX HARVARD APPARATUS LIST OF FIGURES 3 1 Electroporation 3 2 Transmembrane Voltage 3 3 Two Needles per Row Field 3 4 Electrical Field Coverage 3 5 Bleb Formation 4 1 Waveform Generator 4 2 AgilePulse Front Panel 4 3 Login Screen 4 4 System OK Screen 4 5 Self Check Not Successful Screen 5 1 IDA Electrode Handle 5 2 IDATip 5 3 Attaching IDA Tip to Handle 5 4 User Login Screen 5 5 Opening Screen 5 6 Delivery Screen Initialization 5 7 Delivery Screen Load Estimation 5 8 Delivery Screen High Voltage Power Supply Charge 5 9 Delivery Screen Pulse Waveform Active 5 10 Delivery Screen Shutdown 5 11 Text Log Screen 5 12 Sample Amplitude Analysis 5 13 User Account Maintenance 5 14 Log Retrieval Screen 5 15 Pulse Protocol Modification www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM USER MANUAL 800 272 2779 508 893 8999 AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM Caution Notice This instrument contains a high voltage power supply adjustable to 1 000 volts High voltage power supplies present a serious risk of personal injury if not used in accordance with design and or use specifications if used in applications on products for which they are not intended or designed or if they are used by untrained or unqualified personnel The user must read this manual c
8. Skin Electroporation Effects on Transgene Expression DNA Persistence and Local Tissue Environment PLoS ONE 2009 Sept 30 AGILEPULSE IN VIVO GENE DELIVERY SYSTEM USER MANUAL 800 272 2779 SOB 893 8999 AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM BTX HARVARD APPARATUS AgilePulse System Components and Set Up 4 1 INTRODUCTION The AgilePulse system provides the capability of delivering DNA vaccines to the dermis The system is designed for easy set up and operation Specifications are presented in Appendices A and B Tabs 7 and 8 The AgilePulse system is composed of three subsystems e Computer controlled AgilePulse Waveform Generator e Parallel row needle Intradermal Array ID electrodes e User Manual Optional equipment available includes e Foot Pedal 47 0420 4 2 THE AGILEPULSE Figure 4 1 Waveform Generat r WAVEFORM GENERATOR The Waveform Generator Figure 4 1 is composed of a computer and high voltage pulsing circuits The computer runs mobile Windows and is operated by a touch screen On the front panel there is a power switch a touch screen an emergency stop switch an electrode connector and two USB ports All User inputs are entered via the touch screen The system may be operated by one health care professional in a sterile protocol by using an optional foot pedal On the back panel there is a line mains power connector The pulsing protocol is not accessible by the healthcare professi
9. 6 SOFTWARE MAINTENANCE MODE CONTINUED 5 3 6 2 LOG RETRIEVAL All previous therapeutic delivery logs can be retrieved and saved onto a USB Key or deleted from internal memory The interface consists of a series of chronological sub folders from which the user is allowed to double tap on the touch screen to descend through the hierarchy The standard naming sequence is Year Month Day followed by special folders with the time of day These time folder icons show a hand underneath meaning they contain all files generated from a single vaccine delivery The folders allow the user to return to the previous level of hierarchy Figure 5 14 Log Retrieval Screen Special time folders display basic information on the right hand side of the screen when selected Identification numbers time of day and status completion indicators help the user find the correct vaccine delivery run to download When selected by a single tap on the touch screen icon any level of hierarchy can be saved to a USB Key inserted in the front panel by pressing the SAVE button The hierarchy structure will be maintained and copied to the USB Key The DELETE button is also available to remove the hierarchy from internal memory This delete is permanent and the lost files cannot be recovered A maximum of 20 000 delivery logs can be stored on the internal system memory DEPENDING ON THE NUMBER OF FILES TO BE DELETED THIS PROCESS CAN BE SLOW www btxonli
10. Volume No approx V d V d 1000 V cm 1500 V cm 47 0050 8 7 mm x2 2 mm 50 90 ul 400 600 47 0060 15 6 mm x212 mm 100 ul 600 900 AGILEPULSE IN VIVO GENE DELIVERY SYSTEM There is a significant tradeoff between wider row spacing and larger delivery volume and pulse amplitude As pulse amplitude increases so does pain of the applied pulse voltage USER MANUAL 800 272 2779 508 893 8999 Pulse Voltage Pulse Voltage AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM AGILEPULSE TUTORIAL 3 3 3 SPECIFIC REFERENCES There are ten specific references for this technique Gehl Julie Thesis Copenhagen May 2002 Roos A K Moreno S Leder C Pavlenko M King A Pisa P Enhancement of cellular immune response to a prostate cancer DNA vaccine by intradermal electroporation Molecular Therapy 2006 13 320 327 Biragyn A Schiavo R Olkhanud P Sumitomo K King A McCain M Indig FE Almanzar G Baatar D Tumor associated embryonic antigen expressing vaccines that target CCR6 elicit potent CD8 T cell mediated protective and therapeutic antitumor immunity J Immunol 2007 Jul 15 179 2 1381 8 Roos A K King A Pisa P DNA vaccination for prostate cancer Electroporation protocols Experimental and Clinical Medicine Editor S Li Humana Press Inc Totowa NJ Methods Mol Biol 2008 423 463 72 Vertuani S Triulzi C Roos A K Pisa P Charo J Lemonnier F Nishimura M Sel
11. administrator The administrator may grant a standard user the ability to modify an already defined protocol but not to add remove protocols AgilePulse is factory configured with only the admin account available By default no password is defined so the user can enter the system with parameters matching that of Figure 5 4 on first login The password may then be changed from the User account screen in Setup section The most recently logged in user will be displayed in the User Name field on startup To enter User names and Passwords touch the Change buttons to activate a virtual keyboard User name Enter the user s name to login Password Enter the password associated with the user account Password entry is masked with characters in order to provide increased security When the User name and Password have been entered touch OK to log in 5 3 2 WELCOME SCREEN IDENTIFICATION INPUT An opening screen Figure 5 5 to allow input of key experimental information after the initial system log in Information is entered by a virtual keypad The purpose of this screen is for the USER Researcher to enter the Experiment ID Transfectant and Electrode ID www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM BTX HARVARD APPARATUS Experiment Welcome to Agile Pulse II Therapeutic Electrode Protocol Default Operator admin Figure 5 5 Opening Screen All three parameters are set
12. can be removed at any time after the saving data message disappears The USB key may be removed without performing any additional procedures The data logs that result from a experiment are automatically stored on the Key 5 2 INTRADERMAL ARRAY ID ELECTRODE The IDA Electrode consists of two parts The Handle and the Tip 5 2 1 ID ELECTRODE HANDLE The Handle consists of an assembly into which the tip is placed and a permanently attached cable The cable is connected to the Waveform Generator The Handle is made of a durable plastic It is not sterile and may be cleaned with alcohol The Handle is reusable www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM USER MANUAL 800 272 2779 SOB 893 8999 AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM SYSTEM OPERATION 5 2 INTRADERMAL ARRAY ID ELECTRODE CONTINUED 5 2 2 ID ELECTRODE TIP The ID tip is a single use sterile and disposable device The tip is made of medical grade plastic with surgical steel needles There are three tips that may be used with the Handle The tips are shipped in individual Tyvek pouches A cover is included to protect the needles during shipping and handling When ready to use the electrodes open the Tyvek Pouch and remove the tip Insert the tip into the handle by aligning the arrow on the tip with the arrow on the handle Side grips have been added to the tip for easier application Figure 5 2 IDA Tip Once the vaccine is
13. on 50 seconds off INDEPENDENT LABORATORY TESTING EN 60601 1 Medical equipment Part 1 EN 60601 1 1 Medical electrical equipment Part 1 1 UL 60601 1 and CSA 601 1 EN 60601 1 2 Electromagnetic compatibility Part 1 2 www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM USER MANUAL BOO 272 2779 SOB 893 8999 Declaration of Conformity Manufacturer BIX 84 October Hill Road Holliston Massachusetts 01746 1388 U S A Phone 508 893 8999 We herewith declare that the following product Product Name BTX AgilePulse and AgilePulse II Systems Model No Catalog 47 04xx To which this declaration relates is in conformity with the applicable EC Directives harmonized standards and other normative requirements Standard s to which conformity is declared Safety EN61010 1 2 edition Emissions Immunity IEC 6060 1 2 edition CISPR 11 2003 edition IEC 61000 4 2 2001 edition IEC 61000 4 3 2006 edition IEC 61000 4 4 2004 edition IEC 61000 4 5 2005 edition IEC 61000 4 6 2006 edition IEC 61000 4 11 2004 edition IEC 61000 3 2 2005 edition IEC 61000 3 3 2005 edition Over Voltage Category CAT II Local level mains applications and portable equipment Report F22Q2608B 01E and F22Q2608B 06S EMC compliance was evaluated by F squared Laboratories I the undersigned hereby declare that the equipment specified above conforms to the above Directive s and Standard s Place United S
14. ready for administration remove the protective cover from the IDA Tip taking care not to disconnect the tip from the handle www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM BTX HARVARD APPARATUS Figure 5 3 Attaching IDA Tip to Handle 5 3 WAVEFORM GENERATOR 5 3 1 LOGIN SCREEN USER IDENTIFICATION INPUT After power is turned on a Login Screen is presented that requires the user to enter a user name and password combination The Login system is designed to prevent unauthorized users from accessing the system and confine authorized users to permissible activities Two types of users are defined system administrator and standard user There is only one system administrator of the system who has the ability to add remove pulse waveform protocols add remove standard user accounts and assign pulse waveform protocol access privileges to standard users The administrator always operates under the admin account Welcome to Agile Pulse II User name admin Password Figure 5 4 User Login Screen USER MANUAL 800 272 2779 SOB 893 8999 AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM SYSTEM OPERATION 5 3 WAVEFORM GENERATOR CONTINUED 5 3 1 LOGIN SCREEN USER IDENTIFICATION INPUT CONTINUED Standard users are intended to be everyday users of the system who only have the ability to run pre defined pulse waveform protocols from an access list as determined by the
15. to check the fuse on the ated Agile Pulse II back panel of the Waveform Generator If it is faulty replace it with the exact type fuse 240V 5A slo blo 5x20 mm Protocol Default Operator admin If the power is properly applied the screen should appear as that shown in Figure 4 3 After entering the user login information and tapping OK the software continues its initialization procedure and the NEXT button will have a white fill If the internal system checks are successfully completed the NEXT button will turn GREEN indicating the system is ready for use Figure 4 4 If the NEXT button turns RED then the internal checks failed Figure 4 5 The system cannot be used if the button is red Contact BTX Customer Service for assistance Figure 4 5 Self Check Was Not Successful www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM USER MANUAL 800 272 2779 SOB 893 8999 p AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM BTX 4 AGILEPULSE SYSTEM COMPONENTS AND SET UP HARVARD APPARATUS 4 4 SETTING UP THE SYSTEM CONTINUED 4 4 2 TOUCH SCREEN CLEANING If the surface of the touch screen display needs to be cleaned use a standard non ammonia glass cleaner or mild detergent with warm water and a soft lint free paper or cloth towel Do not apply the cleaning solution directly to the screen to avoid liquid running into other parts of the cabinet Put a small amount of cleaner on the towel and gently rub t
16. 0 IMPORTANT The changes made on this screen will only be permanently saved when the HOME button is pressed to go back to the Welcome Screen If modifications are made and system power is turned off while still viewing Caution the screen all changes will be lost www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM USER MANUAL 800 272 2779 SOB 893 8999 p AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM BTX 6 HARVARD APPARATUS Customer Service 6 1 LIMITED WARRANTY The terms of the warranty are covered in the BTX warranty 6 2 CUSTOMER SERVICE If the user believes that there is a defect in the BTX product the customer should contact BTX Technical Support at 800 272 2775 or email techsupport btx harvardapparatus com If the system needs to be returned to BTX please contact Technical Support for a Return Material Authorization RMA number www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM USER MANUAL 800 272 2779 508 893 8999 gt AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM BTX A HARVARD APPARATUS Appendix A Agile Pulse Specifications OPERATION DELIVERY ELECTRODE Vaccine Delivery Volume 2 blebs x 40 uleach IDA 4 6 DELIVERY 2 blebs x 45 uleach IDA 4 6 TOUCH SCREEN 2 blebs x50 uleach IDA 6 6 Opening Screen for parameter entry Delivery Target Skin dermis Experiment entry Electrode Transfectant entry Handle Reusable with alcohol cleaning Electrode
17. 2 2779 508 893 8999 2 AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM BTX 5 SYSTEM OPERATION HARVARD APPARATUS 5 3 WAVEFORM GENERATOR CONTINUED 5 3 4 EXPERIMENT DELIVERY LOGS CONTINUED 5 3 4 2 CVS DATA Detailed pulse information is exported from raw data and formatted to a standard comma separated file primarily meant to be imported into Microsoft Excel for further user manipulation As an example in Figure 5 12 a user chart was made to display the monitored amplitude samples of three 100 microsecond pulses run at 5 kHz In this case each pulse contains eight amplitude samples Time vs Amplitude T 2 5 2 lt Time ms Figure 5 12 Sample Amplitude Analysis www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM USER MANUAL 800 272 2779 508 893 8999 AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM BTX SYSTEM OPERATION HARVARD APPARATUS 5 3 WAVEFORM GENERATOR CONTINUED 5 3 5 DELIVERY COMPLETION RETURN CODES Return codes are available following system shutdown describing how it took place Return Code Description Successful completion Normal Operation Emergency Flash memory storage failure Waveform protocol data integrity verification error Waveform protocol mode not recognized Protocol voltage below minimum specification Protocol voltage above maximum specification Protocol pulse width below minimum specification Proto
18. 8 73 provides the equation that relates the transmembrane voltage TMV to electric field intensity As the charge accumulates at the membrane which is a capacitance the voltage across the membrane increases voltage eiaie capacitance As the voltage increases from its quiescent value of a few tenths of a volt to more than 0 5 volts pathways begin to form The TMV is given by TMV Er cosal where E electric field intensity in volts cm r the cell radius in cm a angle off the center line www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM BTX HARVARD APPARATUS To produce a TMV of 1 volt across the membrane of a cell with 7 um radius the required electric field intensity is 950 volts cm Membrane Electrode 1 Electrode 2 Figure 3 2 TRANSMEMBRANE VOLTAGE USER MANUAL 800 272 2779 508 893 8999 AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM BTX AGILEPULSE TUTORIAL HARVARD APPARATUS General References Dimitrov D S and Sowers A E 1990 Membrane Wolf H Rols M P Boldt E Neumann E amp Teissie J electroporation fast molecular exchange by electroosmosis 1994 Control by pulse parameters of electric field mediated Biochimica et Biophysica Acta 1022 381 392 gene transfer in mammalian cells Biophys J 66 524 531 Sukharev SI Klenchin VA Serov SM Chernomordik LV and Zerbib D Amalric F amp Teissie J 1985 Electric field Chizmadzhev YA 1992
19. AgilePulse In Vivo Gene Delivery System USER S MANUAL HARVARD APPARATUS The Electroporation Experts AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM AgilePulse In Vivo Gene Delivery System Research Only BTX Harvard Apparatus 84 October Hill Rd Holliston MA 01746 USA phone 1 508 893 8999 fax 1 800 429 5732 www btxonline com BTX WARRANTY Harvard Apparatus warranties the AgilePulse In Vivo Gene Delivery System for a period of two years from the date of purchase At its option BTX Harvard Apparatus will repair or replace the unit if it is found to be defective as to workmanship or materials This warranty does not extend to any instrumentation which has been a subjected to misuse neglect accident or abuse b repaired or altered by anyone other than BTX HARVARD APPARATUS without BTX HARVARD APPARATUS express and prior approval c used in violation of instructions furnished by BTX HARVARD APPARATUS This warranty extends only to the original customer purchaser IN NO EVENT SHALL BTX HARVARD APPARATUS BE LIABLE FOR INCIDENTAL OR CONSEQUENTIAL DAMAGES Some states do not allow exclusion or limitation of incidental or consequential damages so the above limitation or exclusion may not apply to you THERE ARE NO IMPLIED WARRANTIES OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR USE OR OF ANY OTHER NATURE Some states do not allow this limitation on an implied warranty so the ab
20. Current monitored during pulse RMon Equivalent load resistance calculated from 5 3 4 1 TEXT LOG voltage and current monitors R V I The text log contains the same information that appears on the DurSet Pulse duration programmed to waveform generator vaccine delivery process screen after completion Text log data DurMon Pulse duration monitored by independent is Summarized in an easy to read format and is designed to time auditor system provide quick feedback regarding the delivery completion An intSat example Log printout is given below in Figure 5 11 Pulse interval programmed to waveform generator IntMon Pulse interval monitored by independent time auditor system Date Start 8 14 2011 6 00 09 AM 8 14 2011 1 00 09 PM GMT Experiment Pl Transfectant Plasmid A Electrode IDA 2001 Operator BTX Final Completion Code Code 0 Success HHHHHHEHH H H H H H H H H H H H Start 8 14 2011 6 00 09 AM 8 14 2011 1 00 09 PM GMT Process Completion Return Code 0 Success Pulse vSet Durset 450 0 050 450 0 050 L10 000 000 000 000 000 000 000 000 PO DA NI DH FF WwW NY PB bi i a fe ee e O oO O Oo O O oO O Load Reading 204 Date Stop 2 12 2007 2 16 40 PM 2 12 2007 10 16 40 PM GMT Time Elapsed 00 00 18 Figure 5 11 Text Log Screen www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM USER MANUAL 800 27
21. ID entry Tip Sterile Single packaged Disposable Operator ID entry Pulse Screen LOAD measure skin resistance every second IDA 4 4 2 IDA 4 6 2 IDA 6 6 2 and display Row Spacing 4mm 4mm 6mm READY Turn on high voltage power supply Needles row 4 6 6 START Start pulsing Needle Spacing 1 5 mm 1 5mm 1 5mm DONE vaccination completed Needle Diameter 0 3 mm 0 3 mm 0 3 mm Needle Length 2mm 2mm 2mm V d Maximum 2500 v cm 2500 v cm 1667 v cm MODE 2 SETUP BY TRAINED IT SPECIALIST PULSE PARAMETERS DOWNLOAD DATA FILES www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM USER MANUAL BOO 272 2779 SOB 893 8999 AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM A AGILEPULSE SPECIFICATIONS AGILEPULSE WAVEFORM GENERATOR PULSING RESISTANCE PULSING 4s at5 volts every second PULSE PROTOCOL PARAMETERS Parameters in a Group Pulse Width 50 us to 1 ms 50 us to 10 ms 50 to 300 volts 50 to 1000 volts Pulse Current Trip 26 amps Load Range 15 to 1500 ohms Number of Pulses 1 to 10 Maximum Duty Cycle 50 Interval 200 ps to 1 sec pulse start to pulse start Number of Groups 3 PULSE MEASUREMENT INTERNAL DIGITIZER Levels 12 bit Samples Pulse width 8 minimum 100 us DATA STORED INTERNALLY AND ON EXTERNAL USB KEY DATA TYPES Raw Data DV lt Date gt xml Log Data DV lt Date gt txt CS
22. V Data DV lt Date gt csv All data automatically stored in internal memory and may be downloaded to an external USB Key Maximum Data Logs stored and retrievable from internal flash memory gt 20 000 www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM BTX HARVARD APPARATUS FRONT PANEL Computer Operating System Windows Mobile 6 0 Interface Touch screen Line Mains Switch with illumination Emergency Stop Button resets computer to ready state Touch Screen USB Ports 2 Electrode connector Fischer Series 4032 BACK PANEL Power Entry IEC 320 Ethernet RJ45 NOT CURRENTLY INSTALLED ELECTRICAL AND MECHANICAL AgilePulse Cabinet with handle 32 cm w x 20 cm h x 40 cm 12 6 in wx 7 9 in hx 15 7 inl Weight 25 pounds 11 3 kg Operating temperature 10 to 40 oC Mains Voltage 100 to 250 VAC Fuse 5 Amp Slo Blo 5 mm x 20 mm USER MANUAL 800 272 2779 SOB 893 8999 2 AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM BTX B HARVARD APPARATUS Appendix B Compliance Summary THE AGILEPULSE SYSTEM IS CLASSIFIED MDD Class llb CFR 820 Class II THE AGILEPULSE SYSTEM IS CLASSIFIED UNDER IEC 60601 1 Type of protection against electric shock Class Degree of protection against electric shock Type BF by applied part Degree of protection against ingress of water IPX0 Mode of operation Intermittent 10 seconds
23. ading will appear in the System message box If the researcher wants to abort the process the electrode should be removed and the DONE button pressed In this case an early termination message will appear in the data log and the system reset Figure 5 6 Delivery Screen Initialization The purpose of the delivery screen is to initiate pulses to deliver vaccine to tissue The procedure is as follows 1 The researcher injects the vaccine forming a bleb under the skin if the animal and inserts the electrode according to the approved protocol The researcher determines if the electrode is inserted properly by touching the LOAD button or by depressing the foot pedal A number will appear in the LOAD Reading message box and the LOAD button will change to a green color This number is the effective resistance of the animal s skin and will continue to be updated every second www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM USER MANUAL 800 272 2779 SOB 893 8999 AGI LE PU LSE IN VIVO GENE DELIVERY SYSTEM BTX SYSTEM OPERATION HARVARD APPARATUS 5 3 WAVE FORM G EN ERATOR All run history log data are automatically stored in CONTI NU ED internal Flash Memory as well as a USB Key if one is inserted 5 3 3 EXPERIMENT DELIVERY SCREEN Experiment FUNCTIONS AND OPERATION CONTINUED eee Electrode Protocol Experiment Pi System 49 volts Operator Therapeutic Plasmid A Est Load 5000 Electrode IDA 2001 Protoc
24. arefully before the instrument is placed into operation Removing the cover may void the warranty Do not connect or disconnect the high voltage cable with the high voltage enabled To connect or disconnect the cable turn line power off and unplug line mains cord Do not touch the electrode tip while the waveforms are being applied If a problem occurs during a run push the STOP RESET button on the front panel If there are any questions about the operation of this instrument call BTX Customer service at 1 508 893 8999 www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM USER MANUAL 800 272 2779 508 893 8999 BTX HARVARD APPARATUS AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM BTX HARVARD APPARATUS Introduction The AgilePulse system was developed to deliver DNA vaccines using pulsed electric fields electroporation The system has been designed for research purposes The system has three components the AgilePulse Waveform Generator the in vivo Parallel 4 and 6 needle Intradermal Array IDA electrodes and the User Manual Using other components may damage the system will certainly provide degraded performance and invalidate the warranty Note The AgilePulse Waveform Generator contains a high voltage power supply and was designed with safety features to protect the user and the equipment If used properly the AgilePulse Waveform Generator is a safe and reliable instrument Front and Back Pane
25. by user selected input Touching a text field box highlights it blue and allows modification Use the available touch screen keypad to enter up to 15 characters for each Alternatively a USB keyboard may be plugged into one of the front panel USB connectors and used to edit the ID fields manually This screen cannot be operated from the foot pedal ID fields cannot be selected or edited The following pulse delivery screen may be operated by either the touch screen or by the optional foot pedal depending on the specification After verifying the identifications are correct go to the delivery screen by touching NEXT USER MANUAL 800 272 2779 508 893 8999 AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM SYSTEM OPERATION BTX HARVARD APPARATUS 5 3 WAVEFORM GENERATOR CONTINUED 5 3 3 EXPERIMENT DELIVERY SCREEN S System Q volts FUNCTIONS AND OPERATION PRE See Est Load 5000 _ ohms Electrode Protocol IDA 2001 Default Experiment Therapeutic Electrode Protocol Pi System 0 volts Plasmid A Est Load ohms Operator admin IDA 2001 Default Operator admin E ser Al Figure 5 7 Delivery Screen Load Estimation If the LOAD Reading is within range Less than 3500 ohms for skin then the next step is to turn on the high voltage power supply by touching the READY button or depressing the foot pedal The READY button will flash green until fully charged and then will stay solid green The high voltage re
26. cells as possible are exposed to the same field The key parameters are Number of needles in each row Diameter of the needles Spacing of the needles in the row Space between the two rows In general the more needles per row and the greater the spacing between the rows the closer the electric field approaches that of a parallel plate As an example the calculated electric field for two needles per row is presented below Figure 3 3 Two Needle per Row Field www btxonline com BTX HARVARD APPARATUS As the space between the rows increase the electric field rapidly falls off and cold spots form pores in the cells and vaccine delivery does not occur The AgilePulse needle electrodes are specifically designed to produce near uniform electric fields in the treatment volume There are currently three ID electrodes available Needle Length 2mm Needle Diameter Model Row Use No Space 47 0040 4mm 4 Needle Row Animal Only Animal Only Animal 0 3 mm 47 0050 4mm 0 3 mm 2mm 47 0060 6 mm 0 3 mm 2mm The electric field coverage is presented in Figure 3 4 COOOL Gre ere oe Figure 3 4 IDA 4 6 2 and IDA 6 6 2 electric field coverage The recommended bleb placement for the IDA 4 6 2 and IDA 6 6 2 electrodes is shown in Figure 3 5 Figure 3 5 Bleb Formation ID 4 6 2 and ID 6 6 2 The total bleb volume for each is approximately Model Area Depth
27. col pulse width above maximum specification OPN AD M BR WIN O Protocol pulse interval below minimum specification Protocol pulse interval above maximum specification Protocol pulse number below minimum specification Protocol pulse number above maximum specification Protocol has a zero pulse group in between two non zero pulse groups Protocol pulse settings exceed maximum duty cycle Low voltage power supply failure Hard system turn off High volt power supply voltage above maximum voltage High volt power supply maximum current draw Pulse voltage monitor detected off zero voltage while no pulse was active Pulse voltage monitor below maximum droop specification Pulse voltage monitor above maximum overshoot specification Pulse current monitor below minimum specification Pulse current monitor above maximum specification Independent time audit system failed to respond to query Independent time audit system detected less pulses than expected Independent time audit system detected more pulses than expected Independent time audit system exceeded maximum measurable pulse number Independent time audit system detected excessive pulse duration variance Independent time audit system detected excessive pulse interval variance Independent time audit system data integrity verification failure Reset button on front panel triggered Control system watch
28. dog timeout High voltage timeout Load estimation timeout Delivery process was terminated prematurely Control system experienced a system reset of unknown source Load estimate outside of allowable range www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM USER MANUAL 800 272 2779 SOB 893 8999 AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM BTX SYSTEM OPERATION 5 3 WAVEFORM GENERATOR CONTINUED 5 3 6 SOFTWARE MAINTENANCE MODE The authorized system administrator is provided with the means to set system access privileges add and delete pulse protocols and access delivery log files Non administrator standard users can modify pulse protocols if given access by the administrator and retrieve previous therapeutic delivery logs To enter this mode press the Setup button on the startup screen 5 3 6 1 USER ACCOUNT MAINTENANCE The user account maintenance screen is accessed from the Welcome screen Touch SETUP Touch USER User accounts and privileges are managed from this screen While standard users will only see the Active User section and thus will only be able to change their password the system administrator has access to account properties for all standard users As an administrator a new user account can be adding by pressing the Add New User button Once the account is created the new user is assigned read only access to all of the pulse protocol waveforms on t
29. e interval www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM BTX HARVARD APPARATUS Before Field Applied Membrane Field Removed Membrane Seals Figure 3 1 Electroporation USER MANUAL 800 272 2779 508 893 8999 AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM AGILEPULSE TUTORIAL 3 3 DNA DELIVERY INTO CELLS USING ELECTROPORATION CONTINUED 3 3 1 GENERAL ELECTROPORATION DISCUSSION CONTINUED Research in the late 1980s and early 1990s showed that certain experimental conditions and parameters of electrical pulses may be capable of causing many more molecules to move per unit time than simple diffusion There is also good evidence Sukharev et al 1992 that DNA movement is in the opposite direction of the arrow in the sidebar An additional important consideration is when the voltage pulse is applied to the cells and medium that the amount of current that flows is dependent on the conductivity of the material in which the cells are located Some material is quite conductive and severe heating will occur if the pulse duration is too long Therefore long duration fields will kill cells by destroying the membrane and heating The electric field in which the cells are located is produced by two system components The first is the voltage waveform generator and the second is the electrode which converts the voltage into the electric field Neumann Sowers and Jordan 1998 pages 6
30. ed system and runtime data a text log with basic runtime data and a comma separated CSV file containing pulse monitor data The directory structure is constructed in a AgilePulse Log lt Year gt lt Month gt lt Day gt lt Time gt hierarchy A maximum of 20 000 delivery logs can be stored in the internal system memory BTX HARVARD APPARATUS Raw Data CCEP lt Date gt lt Time gt xml Text Log CCEP lt Date gt lt Time gt log CSV Data CCEP lt Date gt lt Time gt csv Start and Stop Date Start and Stop Time Experiment Transfectent Electrode ID Process Completion Type Set Pulse Parameters Average monitor pulse voltage Average monitor pulse current Average monitor pulse resistance All monitor pulse voltage samples All monitor pulse current samples Monitor width of each pulse Monitor interval of each pulse Pre pulse Load Reading System Serial Numbers System Software Versions www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM USER MANUAL 800 272 2779 SOB 893 8999 AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM BTX SYSTEM OPERATION HARVARD APPARATUS 5 3 WAVEFORM GENERATOR Field Description CONTINUED Pulse Pulse number VSet Voltage programmed to high voltage power supply 5 3 4 EXPERIMENT DELIVERY LOGS VMon Voltage monitored during pulse CONTINUED IMon
31. er is required to read the User Manual and undergo training before use Certain concepts and processes are covered by patents patents pending know how and trade secrets The IDA electrode tip is disposable single use and sterile unless otherwise indicated on the wrapper label Do not under any circumstances turn the power switch off while the system is pulsing if an emergency occurs push the emergency button on the front panel Do not connect or disconnect any electrode from the waveform generator when the pulse waveforms are being applied Do not put your fingers or any other object on the ID electrode tip while pulse waveforms are being applied This system produces voltages as high as 1000 volts and may cause injury or death if used improperly The system shall not be used in the presence of flammable anesthetic BTX welcomes any comments from the users to improve this manual www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM USER MANUAL BOO 272 2779 SOB 893 8999 AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM BTX 3 HARVARD APPARATUS AgilePulse Tutorial 3 1 APPLICATIONS The AgilePulse system is used to deliver DNA vaccines to study the effects and potential of this application for research purposes only The AgilePulse system has been shown to increase DNA delivery to cells resulting in 100 1000 times higher protein expression compared to hypodermic needle injection Roos et al
32. ey if one is inserted The other USB port may be used for the optional Foot Pedal Appendix C Tab 9 The Emergency Stop button switch is used to stop system operation This switch immediately stops all pulsing and turns off the high voltage power supply The Line Mains power switch should never be used to turn off the system in an emergency situation if pulsing is in progress Figure 4 2 AgilePulse Front Panel 4 2 2 BACK PANEL On the back panel there is an IEC Power Entry module and provisions for an Ethernet port Ethernet connectivity is not currently available 4 3 INTRADERMAL ELECTRODE ID The ID consists of two parts the handle and the tip The handle is connected to the waveform generator The tip contains needle electrodes used to deliver the DNA vaccine into the target cells The needle arrays are disposable but can be used for multiple experiment provided proper cleaning and care of electrodes are followed and sterile Available needle configurations are described in Appendix A www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM USER MANUAL 800 272 2779 SOB 893 8999 p AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM BTX 4 AGILEPULSE SYSTEM COMPONENTS AND SET UP HARVARD APPARATUS 4 4 SETTING UP THE SYSTEM Experiment Welcome to The system will be set up in the following sequence eer Agile Pulse II Unpack the contents of the shipping box Protocol Te Log On to Age Pulse II x lt
33. he screen Avoid hard rubbing abrasives or harsh solvents like alcohol or ammonia This completes the set up and testing of the AgilePulse System If there is a problem contact Technical Support for assistance Phone 508 893 8999 or Toll Free 800 272 2775 www btxonline com www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM USER MANUAL BOO 272 2779 SOB 893 8999 2 AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM BTX 5 HARVARD APPARATUS System Operation This chapter describes the procedure to operate the Waveform Generator and to use the Intradermal Electrode 5 1 EXPERIMENT DELIVERY The user operates the system via the front panel touch screen When the power is turned on the system boots up and displays the opening screen The system is immediately ready for use All data logs produced for a vaccine delivery will be automatically placed on a USB Key if one is inserted into an open slot on the front panel The user must ensure the USB Key is inserted before the Protocol Delivery Screen is displayed as the protocol pulsing is started If the user does not have a USB Key inserted at run time all run history log files can be retrieved at a later time from the log files 5 1 1 USB KEY MEMORY STICK Figure 5 1 IDA Electrode Handle The USB key can be inserted into either of the two USB ports that are located on the front panel of the waveform generator It should be inserted before delivering pulses and
34. he system The administrator may then select individual protocols and provide more access by giving both read and write privileges or they may restrict access to a protocol altogether by giving the user no access at all When a user is not given any protocol access they will not be able to see or select it from the Protocol screen when they login under their own user name When protocols are added through the Protocol screen after a standard user account has already been created all user accounts in the system will receive read only access to the new protocol The administrator also has the ability to change a user s password This is ideal for situations in which the user forgets their password and thus need the password to be reset in order to login again HARVARD APPARATUS Active User U Password Accounts U P Pi Caution www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM ser Name _ admin ser Name assword rotocol Gx Figure 5 13 User Account Screen IMPORTANT The changes made on this screen will only be permanently saved when the HOME button is pressed to go back to the Welcome Screen If modifications are made and system power is turned off while still viewing the screen all changes will be lost e USER MANUAL 800 272 2779 SOB 893 8999 AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM BTX SYSTEM OPERATION HARVARD APPARATUS 5 3 WAVEFORM GENERATOR CONTINUED 5 3
35. iger S Kiessling R HER 2 neu mediated down regulation of MHC class antigen processing prevents CTL mediated tumor recognition upon DNA vaccination in HLA A2 transgenic mice 2008 Cancer Immunol Immunother 2009 May 58 5 653 64 Epub 2008 Sep 27 Lundberg K Roos A K Pavlenko M Wehrum D Pisa P A modified epitope identified for generation and monitoring of PSA specific T cells in patients on early phases of PSA based immunotherapeutic protocols Vaccine 2009 Mar 4 27 10 1557 65 Brave A Hallengard D Gudmundsdotter L Stout R Walters R Wahren B Hallermalm K Late administration of plasmid DNA by intradermal electroporation efficiently boosts DNA primed T and B cell responses to carcinoembryonic antigen Vaccine 2009 Jun 8 27 28 3692 6 Epub 2009 May 3 Roos A K Eriksson F Walters D Pisa P King A Optimization of skin electroporation in mice to increase tolerability of DNA vaccine delivery to patients Molecular Therapy 2009 Sep 17 9 1637 42 Epub 2009 Jun 16 www btxonline com BTX HARVARD APPARATUS Lladser A Ljungberg K Tufvesson H Tazzari M Roos A Quest FG Kiessling R Intradermal electroporation with a survivin DNA vaccine induces CTLs against a self epitope suppresses angiogenesis and confers long term protection against mouse melanoma Cancer Immunol Immunother 2009 Jun 14 Epub ahead of print Roos A K Eriksson E Timmons J Gerhardt J Nyman U Gudmundsdotter L Brave A Wahren B Pisa P
36. ights which vary from state to state AGILEPULSE IN VIVO GENE DELIVERY SYSTEM USER MANUAL 800 272 2779 508 893 8999 5 AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM BTX HARVARD APPARATUS Table of Contents SUBJECT 1 Introduction 2 Items of Particular Interest 3 AgilePulse Tutorial 3 1 APPLICATION 3 2 DNA DELIVERY METHODS 3 3 DNA DELIVERY INTO CELLS USING ELECTROPORATION 3 3 1 General Electroporation Discussion 3 3 2 AgilePulse Electroporation 3 3 3 Specific References 4 AgilePulse System Components and Set up 4 1 INTRODUCTION 4 2 THE AGILEPULSE WAVEFORM GENERATOR 4 2 1 Front Panel 4 2 2 Back Panel 4 3 INTRADERMAL ELECTRODE ARRAY ID 4 4 SETTING UP THE SYSTEM 4 4 1 Initial System Test 4 4 2 Touch Screen Cleaning 5 System Operation 5 1 VACCINE DELIVERY MODE 5 1 1 USB Key Memory Stick 5 2 INTRADERMAL ARRAY IDA ELECTRODE 5 2 1 Handle 5 2 2 Electrode Tip 5 3 WAVEFORM GENERATOR 5 3 1 User Identification Input 5 3 2 Identification Input 5 3 3 Delivery Screens Functions and Operations 5 3 4 Experiment Delivery Logs 5 3 4 1 Text Log 5 3 4 2 CVS Data Delivery Completion Return Codes Software Maintenance Mode oe ot ot d Au wu s _ et as OM OO O WONN DD DD N N oO 5 3 6 1 User Account Maintenance 5 3 6 2 Log Retrieval 5 3 6 3 Pulse Protocol 6 Customer Service 7 Appendix A AGILEPULSE SPECIFICATIONS B COMPLIANCE SUMMARY Declaration of Conformity www btxonline com AGILEPULSE
37. l Symbols IEC 60417 50032 60417 5333 60878 ISO 7000 1641 7010 W001 7000 1051 ik A A A V Type BF Caution Risk of Operating Caution Emergency Applied Part Electric Shock Instructions Stop D off On Do Not Reuse Fuse AC Mains The AgilePulse system is classified MDD Class IIb CFR 820 Class Il The AgilePulse system is classified under IEC 60601 1 Type of protection against electric shock Class Degree of protection against electric shock by applied part Type BF Degree of protection against ingress of water IPXO Mode of operation 10 seconds on 50 seconds off Intermittent This product should not be used in the presence of a flammable atmosphere such as an anesthetic mixture with air oxygen or nitrous oxide The following material is contained in this User Manual Chapter 2 System Items of Particular Interest Chapter 3 AgilePulse Tutorial Chapter 4 AgilePulse System Components and Set up Chapter 5 AgilePulse Operation Chapter 6 Customer Service www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM e USER MANUAL 800 272 2779 508 893 8999 p AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM BTX P HARVARD APPARATUS Items of Particular Interest The AgilePulse DNA Vaccine Delivery System is a sophisticated system designed for delivery of DNA vaccines to the skin The following are important 1 The system shall only be used as described in this User Manual the us
38. ne com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM USER MANUAL 800 272 2779 SOB 893 8999 AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM BTX SYSTEM OPERATION HARVARD APPARATUS 5 3 WAVEFORM GENERATOR CE Add New Remove Group 1 Group2 Group 3 CONTINUED i 450 Guso Himo Evy Duration 0 050 0 050 10 000 ms 5 3 6 SOFTWARE MAINTENANCE MODE Interval o2 500 20 0 ms CONTINUED Number 1 oe k l8 Sound F Enable 5 3 6 3 PULSE PROTOCOL Multi Cycle r Enable 1 The pulse protocol to be delivered at run time is set by pressing CISTA the PROTOCOL button and adjusting parameters via the touch Protoc screen interface Parameters for three pulse groups are i modifiable by selecting a box and scrolling through the wee ana available values Subsequent groups are activated by selecting more than one pulse in the number field and conversely deactivated by selecting zero pulses in the number field Figure 5 15 Pulse Protocol Modification The adjusted protocol is saved to internal memory when the HOME button is pressed Minimum and maximum parameter values are as follows Protocol Parameter Boundaries Amplitude volts Duration ms Interval ms Pulse Pulse Groups Minimum 50 0 050 0 200 5 kHz 1 1 Maximum 1000 10 1000 1 Hz 10 3 For Pulse Amplitudes greater than 300V the maximum allowable Pulse Width is 1ms Pulse duty cycle is limited to 5
39. ol Default ohms Operator admin Figure 5 10 Delivery Screen Shutdown m N 7 Press the color filled DONE button to return to the opening screen Figure 5 8 Delivery Screen High Voltage Power Supply Charge 4 Ifthe researcher determines that the system voltage is correct then pulsing is initialized by touching START or depressing the foot pedal The START button turns a solid blue while pulsing is active and solid green when pulsing is completed Experiment Pi System 449 volts Therapeutic Plasmid A Est Load 5000 Electrode IDA 2001 ohms Protocol Default Operator admin Figure 5 9 Delivery Screen Pulse Waveform Active When the system reaches a shutdown state either by successful waveform delivery or by error all buttons will turn solid green and pulse delivery data will be displayed in the System message text box The load message box will change from the estimated load value to the actual high voltage pulse monitored value www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM USER MANUAL 800 272 2779 SOB 893 8999 AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM SYSTEM OPERATION 5 3 WAVEFORM GENERATOR CONTINUED 5 3 4 EXPERIMENT DELIVERY LOGS All collected data from the therapeutic vaccine delivery are automatically stored in the internal Flash Memory and an inserted USB key under a unique chronological directory structure Included items are an XML file containing raw detail
40. onal The protocol is set by a trained operator via the touch screen The high voltage pulsing circuits are controlled by a microcontroller The microcontroller accepts user inputs from the touch screen and produces the pulse waveforms A second microcontroller is used as an independent audit of the waveforms produced If the audit function detects a deviation from the desired pulse protocol the system immediately terminates pulsing The system also digitizes the output of the pulse voltage and pulse current monitors and calculates the skin resistance of the tissue on each pulse This is a significant quality control function that indicates the vaccine is being delivered properly After the vaccine is delivered a log of all parameters is saved in Internal Memory and on a USB memory key if one is inserted www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM USER MANUAL 800 272 2779 SOB 893 8999 AGI LE PULSE IN VIVO GENE DELIVERY SYSTEM BTX AGILEPULSE SYSTEM COMPONENTS AND SET UP HARVARD APPARATUS 4 2 THE AGILEPULSE WAVEFORM GENERATOR CONTINUED B f e oin 4 2 1 FRONT PANEL The following can be found on the front panel the Mains Line power switch illuminated when on the Emergency Stop button switch two USB ports the ID Electrode Connector and the User Touch Screen Figure 4 2 One USB port can be used for a Memory Key The data logs that result from a vaccination are automatically stored on the K
41. ove limitation may not apply to you Without limiting the generality of the foregoing BTX HARVARD APPARATUS shall not be liable for any claims of any kind whatsoever as to the equipment delivered or for non delivery of equipment and whether or not based on negligence www btxonline com BTX HARVARD APPARATUS Warranty is void if the AgilePulse instrument is changed in any way from its original factory design or if repairs are attempted without written authorization by BTX HARVARD APPARATUS Warranty is void if parts connections or cell fusion chambers not manufactured by BTX HARVARD APPARATUS are used with the Hybrimune instrument If a defect arises within the warranty period promptly contact BTX Harvard Apparatus 84 October Hill Road Building 7 Holliston Massachusetts USA 01746 1388 using our toll free number 1 800 272 2775 US Only or 508 893 8999 E mail techsupport btx harvardapparatus com Goods will not be accepted for return unless an RMA Returned Materials Authorization number has been issued by our customer service department The customer is responsible for shipping charges Please allow a reasonable period of time for completion of repairs replacement and return If the unit is replaced the replacement unit is covered only for the remainder of the original warranty period dating from the purchase of the original device This warranty gives you specific rights and you may also have other r
42. tates of America Date August 3 2011 C Signature HARVARD ae APPARATUS VP Engineering Operations Position
43. utine use of electroporation became very popular with researchers through the 1980s because it was found to be a practical way to place drugs or other molecules into cells In the late 1980s scientists began to use electroporation for applications in multi cellular tissue In the early 1990 s Lluis Mir of the Institute Gustave Roussy was the first to use electroporation in a human trial to treat external tumors Research has shown that the induction of pathways is affected by three major factors First cell to cell biological variability causes some cells to be more sensitive to electroporation than other cells Second for pathways to be induced the product of the pulse amplitude and the pulse duration has to be above a lower limit threshold Third the number of pathways and effective pathway diameter increases with the product of amplitude and duration Although other factors are involved this threshold is now understood to be largely dependent on a fourth factor the reciprocal of cell size If the upper limit threshold is reached pore diameter and total pore area are too large for the cell to repair by any spontaneous or biological process The result is irreversible damage to the cell or cell lysis Because the mechanism of electroporation is not well understood the development of protocols for a particular application has usually been achieved empirically by adjusting pulse parameters amplitude duration number and inter puls
44. various pulse groups with different characteristics from group to group For example the study by Dr Roos found the optimum waveform for induction of high gene expression and high induction of antigen specific T cells to be Pulse Width 0 05 ms 300 ms Pulse Group Pulse Interval Interval Number 500 ms 2 Pulse Amplitude 450 V 1125 V cm 110 V 275 V cm Group 10ms_ 300 ms 500 ms The total treatment time for this waveform is 2 98 seconds Waveforms for clinical use must be optimized in pre clinical studies Another more recent study by Roos et al Mol Ther 2009 developed a variant of the PulseAgile waveform described above This waveform is called Fast PulseAgile PAfast and differs only in the length of the pulse intervals Pulse Group Pulse Interval Interval Number Pulse Width Pulse Amplitude Group 450 V 50ms 2 1125 V cm 0 05 ms 0 2 ms 110 V 50 ms 275 V cm 10ms_ 20 ms www btxonline com AGILEPULSE IN VIVO GENE DELIVERY SYSTEM BTX HARVARD APPARATUS The total treatment time for this waveform is 0 27 seconds this waveform is currently tested in three clinical studies When the pulses are delivered in less than half a second there is only one muscle contraction and tolerability is highly improved The AgilePulse system has the following waveform settings available
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