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1. Hybridoma Production System User s Manual toll free 800 272 2775 508 893 8999 www btxonline com 30 E Hybrimune System Components amp Set Up 4 10 The Waveform Generator Interface Software cont d 4 10 1 The Toolbar Across the top left of the main window is a list of standard Windows pull down menus File Pull down Menu e Open Opens a previously saved protocol e Save Saves the current protocol overwriting the file if it had been previously saved e Save Protocol As Saves the current protocol The user is prompted for a filename e Log Save As Saves the protocol Log The user is prompted for a filename e Print Log Prints the Last Protocol Log e Exit Exits the program Tools Pull down Menu e Electric Field Converter Application to help convert between absolute voltages and electric field e Standard Calculator The Microsoft Windows calculator e Quick Pulse Runs the open protocol without additional user input Pressing F12 achieves the same result The software activates Turn HV ON and START May be used to run the same protocol repeatedly A protocol must be open for this to work e Options General options to set sound mode auto save provide communications check etc Help Pull down Menu e About Shows the BTX logo software version and phone numbers 4 10 2 Tools Area The Tools area is found on the left side of the screen and contains a set of frequently used function
2. e User s Manual EY Producing Fusion Products Using Hybrimune 5 4 Cell Handling After Electrofusion cont d 5 4 3 Method Culture of Cells 5 4 4 Culture of Cells Centrifuge cells Select one of the above cloning media Dilute cells to 30 000 G ml in Cell cloning medium plus HAT Mix well and add 100 ml well to individual wells of a U bottom 96 well plate Incubate 37 C 5 CO2 5 4 5 Feeding Cells Optionally replace half of the medium daily for 4 days not done by everyone At one week note wells that are showing signs of metabolism by the medium turning yellow Take 50 ml aliquots of supernatant for antibody screening Re feed with 150 ml Complete medium plus HAT 5 4 6 Cloning Cells Choose selected positive wells Mix cells in the wells Count cells Dilute cells to 3 cells 900 ml 3 3 cells ml in cloning medium with HAT Add 100 ml well to individual wells of a 96 well plate At one week note wells that are showing signs of metabolism by the medium turning yellow Take 50 ml aliquots of supernatant for antibody screening Re feed with 150 ml complete medium with HAT medium 5 4 7 Weaning Clones Clones are selected based upon immunoassays Expand clone to T 25 flask After growth in T 25 flask separate out aliquot for freezing Pass cells in HT medium for one passage Gradually reduce the HT concentration to wean cells off HT Hybrimune Hybridoma Production System toll free 800 272 2775 5
3. may be set from 0 1 to 20 in steps of 0 1 This function is used for slow alignment and then compress If k 1 then the ramp is a constant Non linear Step This function steps the AC Amplitude instead of continuously changing it Up to 10 groups or steps can be programmed T Hybrimune Hybridoma Production System User s Manual Ak toll free 800 272 2775 508 893 8999 e www btxonline com 21 3 3 2 Waveforms for Electrofusion continued 3 3 2 3 Electrofusion Pulse The function of this waveform is to form pathways in the cell membranes to allow the cytoplasm to mix and the cells to fuse One or more high electric field pulses are needed This process is electroporation and is illustrated in Figure 3 15 An electric field is applied that is high enough to induce a voltage across the cell membrane that induces pore formation The minimum transmembrane voltage is approximately one volt This requires applying electric fields of hundreds of volts cm to achieve the at least one volt across the cell membrane Small diameter cells require higher electric fields than do large diameter cells Theoretically the formation of a pore in one cell increases the likelihood that a pore will form in an adjacent cell membrane in the same location as the original pore This is because pores in cell membranes conduct electricity through the pore Whatever the actual mechanism of formation pores in cells whose membranes are touching can re seal i
4. E 0 a r 2p medium The larger the force the faster the cells will move Although the equation is complex much can be learned by looking at the terms in the equation rc is the radius of the cell The equation says that the force is proportional to the radius of the cell cubed As a practical matter when the radius of the cell gets below 3 or 4 micrometers the force falls below that of other forces and the process becomes ineffective Thus attempting to electrofuse bacteria is not a productive endeavor This parameter is also of practical interest when attempting to fuse a large cell with a smaller cell The forces applied to the large cells are larger causing them to move faster THE CELL RADIUS IS NOT UNDER USER CONTROL medium IS the permittivity of the medium Permittivity also called electric permittivity e is a constant of proportionality that exists between electric displacement and electric field intensity This constant is equal to approximately 8 85 x 10 12 farads per meter F m in free space a vacuum In other materials it can be much different often substantially greater than the free space value which is symbolized eo The medium used in electrofusion is close to high purity water which has a permittivity of 80 x eo The permittivity of BTX Cytofusion medium used in the electrofusion process is about 78 5 x eo This term is constant over all conditions specified in this User Manual THE PERMITTIVITY IS NOT UNDER USER CONT
5. bl This shows the E term has two distinct terms the first is the amplitude of the AC waveform the second is the geometric configuration of the chamber Chamber Geometry The chamber geometry is fixed by the BTX design A significant amount of effort was expended to determine an optimum dimension The dimensions of the Fusion chambers are Optimization Batch Production paramere Chamber Chamber Volume 2 ml 9 ml Outer Inside Radius 22 86 mm 22 86 mm b Inner Outside 19 05 mm 19 05 mm Radius a Gap 3 81 mm 3 81 mm Well Height 5mm 18 mm a b 0 8333 0 8333 The two chambers are electrically equivalent As shown in the above height is not a factor only the radius of the inner and outer electrodes An image of cell alignment in the 2 ml Optimization Chamber is presented in Figure 3 9 Hybrimune Hybridoma Production System toll free 800 272 2775 508 893 8999 e www btxonline com 16 Figure 3 9 Cell Alignment in Optimization Chamber The direction of the force on the cells in the fusion chamber is from the outer to inner electrode The field is non uniform from outer to inner electrode The force does not change as a function of chamber height which is a very significant advantage over other electrode configurations User s Manual 3 3 1 Cell alignment Dielectrophoresis continued AC Waveform Amplitude The AC waveform amplitude and duration are the only
6. 1 5 0 1 4 50 50 ee ee System Message gt Normal Completion Auto Saved Log Filepath Log C My Documents Harvard Apparatus Hybrimune log PulseAgile 2006 06 27 15 52 44 txt ee eee System Message Ps System v5 81 Figure 4 6 The Last Protocol Log for the TEST pro Run E Hybrimune Hybridoma Production System User s Manual Ak toll free 800 272 2775 508 893 8999 e www btxonline com 29 E Hybrimune System Components amp Set Up 4 9 Elementary Verification Tests There are two elementary tests that can be run to determine If the AC waveforms are operating properly TEST 1 Fill the CHNF 9R chamber with 9 ml of medium Allow the medium to reach room temperature 25 C Set the AC start and stop to 70 volts the duration to 10 seconds and the frequency to 1 MHz The LED bar segment display should show between one and four bars This test shows e The cable between waveform generator and chamber is OK e The conductivity in the chamber is OK e The AC waveform is working properly TEST 1 TEST 2 1 4 bars lit All bars lit TEST 2 Set the AC waveform to start and stop 70 volts the duration 10 seconds and the frequency to 2 MHz All bars should be illuminated This test demonstrates the effects of frequency Although the conductivity of the medium in the chamber is still the same as Test 1 since the chamber is also a capacitor at 2 MHz it begins to look like a short ci
7. Appendix A System Specifications Chamber Optimization Chamber Production Chamber Volume gt 2 ml gt 9 ml Outer ID 45 72 mm 45 72 mm Inner OD 38 10 mm 38 10 mm Gap 3 81 mm 3 81 mm Well Height 5 mm 18 mm r1 r2 018333 0 8333 Overall Dimensions 13 5 mn x 7 7 cn xX 2 7 11 cm x 10 cm x 5 cm Weight 210 g 7 oz 270 g 9 oz Operating temperature 20 10 30 Storage temperature O to 50 C Cytofusion Medium Formula C Parameter Tolerance Sterility Sterile filtered no growth on random samples using tour bacterial test media pH Td TO Conductivity 80 uS cm 5 uS cm Endotoxin lt 0 2 IU ml Osmolarity 270 290 milliOsmoles Storage 4 to 8 C Packaging 500 ml bottle T Hybrimune Hybridoma Production System User s Manual ae toll free 800 272 2775 508 893 8999 e www btxonline com 48 Hybridoma Production E Fusion vs PEG Medarex Results The data described in this document was produced by Marco Coccia Ph D in the Platform Development Group Medarex Inc Milpitas CA using the BTX Hybrimune Commercial Electrofusion System BTX has permission from Medarex to release this information In preparation for the fusion mice were immunized intraperitoneally or subcutaneously every 2 4 weeks with an antigen plus Ribi adjuvant The mice were bled periodically to determine if an adequate antibody titer had developed Mice wer
8. Hybrimune System uses an alignment technique called dielectrophoresis that applies a force to overcome the surface charge From basic physics we know that the force on a charged particle in an electric field is FORCE ELECTRIC FIELD X CHARGE Therefore if a cell which has a net charge of zero is placed between two parallel flat electrodes which produce a uniform field the force applied to the cell is zero However there are charges inside the cell such as salt ions A force is applied to these charges and they will move until stopped in this case by the cell membrane an insulator creating a dipole inside the cell In a uniform field the force applied is equal and opposite and the cell will not move However if a non uniform field is used the forces are not equal and opposite and the cell will move in the direction of the highest field intensity see Figure 3 5 If there are a number of cells in the non uniform field then the cells will line up due to the attraction of unlike charges as Uniform Field Non Uniform No Movement Field Movement Figure 3 5 Uniform and Non Uniform Fields shown in Figure 3 6 Hybrimune Hybridoma Production System Toll free 800 272 2775 508 893 8999 e www btxonline com Figure 3 6 Cell Alignment User s Manual Cell Electrofusion Tutorial 3 3 1 Cell alignment Dielectrophoresis continued The actual force applied is a complex formula see Pohl Chapter 4 K e s w r
9. Quantitation of Cell Cell Electrofusion Products by Flow Cytometry Analytical Biochemistry 216 271 275 6 Hui SW Stenger DA 1993 Electrofusion of cells Hybridoma production by electrofusion and polyethylene glycol Methods in Enzymology 220 212 227 7 Neil GA Zimmerman U 1993 Electrofusion Methods in Enzymology 220 174 196 8 Schmidt et al 2001 CD19 B lymphocytes are the major source of human antibody secreting hybridomas generated by electrofusion J Immunological Methods 255 93 102 9 Darveau A et al 1993 Efficient preparation of human monoclonal antibody secreting heterohybridomas using peripheral B lymphocytes cultured in the CD40 system J Immunol Meth 159 139 143 10 Niedbala W G Scott D I 1998 A comparison of three methods for production of human hybridomas secreting autoantibodies Hybridoma 17 299 304 11 Shirahata S et al 1998 Cell hybridization hybridomas and human hybridomas Meth Cell Biol 57 111 145 12 Zimmerman U et al 1995 Genreation of a human monoclonal antibody to hepatitis C virus JRA1 by activation of peripheral blood lymphocytes and hypoosmolarelectrofusion Hum Antibodies Hybridomas 6 77 80 Hybrimune Hybridoma Production System Toll free 800 272 2775 508 893 8999 e www btxonline com 43 EY Producing Fusion Products Using Hybrimune James K Bell G T 1987 Human monoclonal antibody production J mmunological Met
10. The table below shows the relationship between the various definitions Also included is the standard USA mains voltage Note the 120V 240 in many other countries rating on appliances is actually rms The bottom of the table shows that peak to peak at the wall socket power mains is actually 340 volts Vpk to pk Vpk Vrms 10 5 35 20 10 7 1 30 15 10 6 40 20 14 1 50 25 17 7 60 30 21 2 70 35 24 7 80 40 28 3 90 45 31 8 100 50 354 110 55 38 9 120 60 42 4 130 65 46 0 140 70 49 5 150 75 53 0 340 US mains 170 120 680 EU mains 340 240 Hybrimune Hybridoma Production System User s Manual Toll free 800 272 2775 508 893 8999 e www btxonline com 19 Cell Electrofusion Tutorial 3 3 2 Waveforms for Electrofusion continued The voltage range of the Hybrimune voltage waveform generator can be set from 5 to 75 volts peak in 5 volts peak steps using several algorithms These algorithms and their names are presented in Figure 3 13 The method of programming these functions is described in Chapter 4 Constant Amplitude This function is the classic electrofusion waveform and has appeared on a number of vendors systems Generally the amplitude is set and the duration Is set This function is available on the BTX laboratory instrument system Linear Ramp Amplitude set by Ramp k 1 This function appeared on the original Zimmerman system a number of years ag
11. What should happen The screen should appear like that as shown in Figure 4 5 The CommLink window should show OK This display indicates that the computer is communicating with the internal microprocessor of the waveform generator Additionally the Electrofusion Chamber window should display OK the High Voltage window should display OFF and the Status window should say Ready The Power Supply Voltmeter window will display O volts Hybrimune Hybridoma Production System toll free 800 272 2775 508 893 8999 e www btxonline com 28 System Simus Comi Cremer Hah ecitaga chats kantara Perrot Sutil po Vower wom Last Protocol Log Figure 4 5 User Interface Application Opening Screen Troubleshooting tip If ERROR appears in the CommLink window then turn off the Waveform Generator and check the serial cable connection to the waveform generator and computer or USB to Serial adapter if applicable Once communication is established the test may proceed Then e Click on the file folder icon on the left side of the screen e Open the protocol file called HYBRIMUNE Test pro e Click Turn HV ON e User s Manual E Hybrimune System Components amp Set Up 4 8 System Test Continued What should happen The protocol settings are downloaded to the waveform generator microprocessor The green Charging Power Supply On LED will turn on and the High Voltage window should display On The inte
12. a small aliquot of the fused cells 1 2 in a LS A 10 11 phone 508 893 8999 fax 800 429 5732 complete media www btxonline com Allow the cells to rest 30 minutes Spin fused cells onto a slide at 350 RPM for 2 minutes using the cytospin machine method Allow slide to air dry a couple of minutes Dunk it into fresh 95 ethanol 5 seconds Allow to air dry Place the slide in the Wright s Giemsa for 1 minute Dunk the slide up and down in the first tube of DI water to remove most of the stain Repeat the dunking in the second tube of water keep dunking until most of those little precipitates of stain are no longer on the slide Knock the side of the slide on paper towels to get most of the water off Set the slide up so any water and ppt ed dye will flow off the slide and allow it to dry ie Hybrimune Hybridoma Production System User s Manual toll free 800 272 2775 508 893 8999 e www btxonline com 45 ES 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 T Hybrimune Hybridoma Production System User s Manual ae toll fre
13. and is f In a b non linear 2 V F2 varies with rg and force TE ain en PID is from the outer to inner f In a b electrode V The voltage applied from the inner electrode to the outer electrode a The radius of the outside of inner electrode b The radius of the inside of the outer electrode a b Always less than 1 rg The variable radial distance in between a and b Cell Electrofusion Tutorial Hybrimune Hybridoma Production System toll free 800 272 2775 508 893 8999 e www btxonline com 15 As a b gets small the gap gets large and E2 gets small and Vo must increase to compensate As a b approaches 1 the gap gets smaller and E2 gets large and less voltage is required However as the gap decreases so does the volume Increasing the chamber height to hold volume constant has practical limits It becomes more difficult to maintain a homogeneous cell suspension A simulation of E2 and the complete force equation and has been used to define the optimum radius of the inner electrode and the outer electrode in the BTX Fusion chamber to provide the optimum combination of parameters to 1 Have a quasi uniform force on the cells in the gap 2 Maximize volume 3 Minimize voltage required Figure 3 8 Chamber Definitions User s Manual Cell Electrofusion Tutorial 3 3 1 Cell alignment Dielectrophoresis continued The following is an equivalent presentation E V 0 2 E In a
14. buttons These functions can also be accessed from the Toolbar as described above Tool Buttons e Folder Button Opens a protocol e Disk Button Protocol Save As Saves a protocol under a user specified filename e Printer Button Prints the Last Protocol Log or the Log History user selectable to a file or printer e Electric Field Converter Button Opens the Electric Field Converter application e Calculator Button The Microsoft Windows Calculator e Notepad Button Saves the protocol Log under a user specified filename e Exit Button Exits the program 4 10 3 Status Area This area at the upper right corner of the screen displays up to date information about the system conditions Features Enabled This feature shows that there Is proper internal connection of the system when the box labeled Electrofusion is checked System Status This section shows the current system status These conditions frequently change during operation CommLink This box displays the status of the RS 232 serial communications link OK Communications is established and functional Error There is a problem with the communications link Hybrimune Hybridoma Production System User s Manual toll free 800 272 2775 508 893 8999 www btxonline com 31 E Hybrimune System Components amp Set Up 4 10 The Waveform Generator Interface Software cont d 4 10 3 Status Area continued Chamber This box displays
15. can be added to increase the chance that a B cell will fuse to a myeloma cell rather than another B cell Keep in mind though that this will reduce the total yield 5 2 4 Cell Preparation Process e Harvest cells e Optionally mix the cell populations at a ratio of 1 1 e Connect the chamber to the Hybrimune Waveform Generator with the provided cable e Wash cells 2 times 3 centrifugations in 20 ml Cytofusion Medium if the cell pellet is large a third wash will be needed It is recommended that the cells be centrifuged at 400g for 6 8 minutes to assure minimal cell loss during was steps e Re suspend the cells in Cytofusion medium at a cell density of 10 million cells ml or other desired cell density e Mix the cell populations at a ratio of 1 1 if not already mixed e Place cells in the electrofusion chamber e Important the fusion steps must be performed within 30 seconds e g as soon as possible of loading the cells into the chamber or cells will settle to the bottom and give poor alignment If allowed to sit more than 30 seconds gently pipette the cells to resuspend before proceeding e Place the cap on the chamber e Start the previously loaded fusion protocol User s Manual 5 3 Electrofusion The electrofusion process is done by applying a series of electrical waveforms These are described in this section The physics and theory of these processes are described in Chapter 3 Follow the set up procedures describe
16. designed or if they are used by untrained or unqualified personnel The user must read this manual carefully 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 1 800 272 2775 or 1 508 893 8999 ie Hybrimune Hybridoma Production System User s Manual Toll free 800 272 2775 508 893 8999 e www btxonline com 6 an Introduction The Hybrimune System was developed for large volume commercial applications using cell Electrofusion This includes hybridoma production and tumor dendritic cell immunotherapy The system has not been approved for clinical or in vitro diagnostic use The system can fuse up to 180 billion cells in one process run The Hybrimune System comprises four components The Hybrimune Waveform Generator the user interface software the large volume coaxial chambers and the low conductivity BTXpress Cytofusion Medium in which the electrofusion takes place NOTE The Hybrimune Waveform Generator contains a high voltage power supply and was designed with sa
17. is the 2 ml chamber top image in Figure 4 2 that holds a maximum volume of 2 mL This chamber permits light to enter in from the bottom so that the cell fusion process can be observed with a microscope The 2 ml chamber is used for process optimization The second chamber is the 9 ml chamber bottom image in Figure 4 2 that holds a maximum volume of 9 ml This chamber is used for large batch production The electrical characteristics of the two chambers are equivalent The electrodes are coated with MEDCOAT 2000 a chromium coating by Electrolyzing Corporation of Ohio EN Hybrimune System Components amp Set Up Hybrimune Hybridoma Production System toll free 800 272 2775 508 893 8999 e www btxonline com 25 BIX a division of Harvard Bioscience Inc Cytofusion Medium C Catalog No 47 0001 500 mi Do not use If temper proof seal is missing wew Dixontine com Figure 4 3 Cytofusion Medium 4 4 Cytofusion Low Conductivity Medium BTX has developed a proprietary formula cell fusion medium over several years that has under gone extensive testing The conductivity of this medium is low in order to minimize electrical current flow during the application of the intense alternating electric fields generated by the Waveform Generator By limiting current flow aqueous heating Is minimized which improves fusion efficiency and viability The medium is shown in Figure 4 3 The Hybrimune Waveform Generator
18. pulse parameter is changed but not Replaced an error dialog box will appear if the user tries to run the protocol The user will be prompted to replace the values first Remove or Alt M Deletes a group If a group is deleted the group numbers below the deleted group if any are renumbered accordingly Non linear step protocols Non linear step protocols containing multiple constant amplitude sine waves are programmed by the concatenation of multiple groups Typically the first group would only execute a single pre SINE parameter set followed by a second group that executes a second pre SINE parameter set with both fusion pulse and post SINE healing waveform A sine parameter set can be skipped by setting the duration to zero seconds Likewise a pulse parameter set can be skipped by setting the number of pulses to zero Hybrimune Hybridoma Production System Toll free 800 272 2775 508 893 8999 e www btxonline com 4 10 6 Running a Protocol The following function buttons are used to run the current protocol e Turn HV ON or Alt H After all of the electroporation parameters are set and the user Is ready start the protocol chamber in place then click Turn HV ON The internal high voltage power supply HVPS turns on and charges the reservoir capacitor to the level set in Group 1 After seven seconds the Start button is highlighted and the system is ready to deliver the pulse protocol e Start or Alt S
19. section describes various methods to program waveform protocols As stated previously there generally are three waveform components e Pre fusion Pulse AC e Fusion Pulse e Post fusion Pulse AC The waveform generator user interface uses the concept of Groups A group Is defined as a set of AC Pulse AC waveforms in which the parameters of the first AC the pulse and the last AC do not change The one exception is the change in AC amplitude The AC amplitude can be varies by changing the Ramp k parameter A group does not have to consist of all thee components only one of the three is required for a valid group e To inhibit or cause an AC waveform not to appear select zero for the duration e To inhibit or cause the pulse not to appear select zero for the number of pulses The software automatically concatenates all of the Groups in the proper order to be executed In series The programming of the recommended protocol will be demonstrated 4 11 1 Programming the Protocol This protocol requires two groups because the pre fusion pulse AC has two parts Note that setting pulse number equal to zero inhibits pulses and setting AC duration to zero inhibits AC GROUP 1 Pre sine Start 40 volts peak Stop 40 volts peak Function Linear Ramp k 1 Duration 15 seconds Frequency 0 8 Mhz Pulse Amplitude set at Group 2 value Width set at Group 2 value Interval set at Group 2 value Number 0 Post sine Start set at Group 2 value Stop
20. set at Group 2 value Function set at Group 2 value Duration 0 Frequency set at Group 2 value Hybrimune Hybridoma Production System Toll free 800 272 2775 508 893 8999 e www btxonline com Pre sine Start Stop Function Duration Frequency Amplitude Width Pulse Interval Number Post sine Start Stop Function Duration Frequency To program 75 volts peak 75 volts peak Linear Ramp k 1 20 seconds 0 8 Mhz 800 volts 40 us entered as 0 040 milliseconds 0 125 seconds 1 60 volts peak 5 volts peak Linear Ramp k 1 30 seconds 0 8 MHz e Enter the values for Group 1 in the user interface e Click Add e Enter the values for Group 2 in the user interface e Click Add e Save as BIX This can also be programmed as four groups e AC e AC2 e Pulse e AC User s Manual 34 This section describes a protocol to produce cell fusion using the Hybrimune System This is a starting protocol every lab has its own procedures which need to be considered The cleaning procedure and process optimization techniques are also described The protocol is presented in the following chapters 1 Cell Preparation before Electrofusion Day Section 5 1 2 Cell Preparation on Electrofusion Day Section 5 2 3 Electrofusion Section 5 3 4 Cell Handling After Electrofusion Section 5 4 The description in this section refers to Hybridoma production For immunotherapy refer to Trevor 2004
21. the Hybrimune System is the application of one 1000 Volt pulse for 40 microseconds 5 3 3 Post Fusion Pulse AC After the fusion pulse has been applied the cell fusion process starts The connections between cells are delicate until the fusion process begins to mature A post pulse AC waveform is applied for 30 seconds to 1 minute after the pulse to hold delicately connected cells together immediately after the pulse BTX recommends starting with a decreasing linear ramp see Figure 3 13 The start amplitude is lower than the ending amplitude of the pre fusion pulse sinewave amplitude If 70 was used then start at 50 or 60 volts End at 5 volts over 30 to 60 seconds Set frequency to 0 8 Mhz User s Manual EY Producing Fusion Products Using Hybrimune 5 4 Cell Handling After Electrofusion 5 4 1 Immediate Post Fusion Care of Cells After the electrofusion process cells begin to completely fuse Even though the pores that are responsible for the fusion were formed in a few microseconds the full fusion of cell membranes takes many minutes In a practical sense this fusion maturation occurs over the next 30 minutes During this time the cells should be handled gently The initial pores are very small nanometers While the pores are small they are fragile As the cell membranes finish their fusion the fused pores expand to the size of the cell The following is a recommended procedure e Allow the cells to rest 5 minute
22. 08 893 8999 e www btxonline com 41 5 5 Methods of Analysis The most common method of analysis is to determine the number of wells with clones 5 10 000 cells well that are secreting antibody and the number that are secreting antigen specific antibody If low numbers of clones are obtained it may be useful to analyze the cells immediately after the fusion to determine if there is a problem with the fusion itself A simple analysis is to use a Cytospin to place cells on a slide and stain the cells with a Wrights or Giemsa stain Simply count the percent of cells with two or more nuclei Another method for analyzing the fusion itself is flow cytometry For this cells are stained prior to the fusion with intracellular fluorescent dyes of different color A good method is published in Analytical Biochemistry 1994 Vol 216 271 275 User s Manual 5 6 Demonstration Experiment to Illustrate Turbulence A simple demonstration can be done in the lab to show what proper cell alignment looks like and what it looks like when it Is not working correctly e Wash 10 million myeloma cells twice in Cytofusion Medium C e Set up the Hybrimune System e Program a 70 V pre pulse sine wave for a 10 second duration other parameters can be set at minimum e Place the cells in the two ml chamber place the chamber on an inverted microscope and connect the chamber to the Hybrimune Waveform Generator e Click turn on HV Pulse then Star
23. 5 23 26 26 26 2 2 27 28 29 30 30 31 31 31 32 32 32 33 33 33 34 34 T Hybrimune Hybridoma Production System User s Manual ae toll free 800 272 2775 508 893 8999 e www btxonline com Table of Contents SUBJECT PAGE 5 Producing Fusion Products Using Hybrimune 35 5 1 Cell Preparation Before Electrofusion Day 25 5 1 1 Myeloma Cells 35 5 1 2 Available Myelomas or Heteromyelomas 36 5 1 2 1 Human 36 5 1 2 2 Mouse 36 5 1 2 3 Rat 36 5 1 2 4 Rabbit 36 5 1 3 B Cells 36 5 1 4 Immunization 36 5 1 5 Screening 36 5 1 6 Harvesting Spleen Cells eNi 5 1 7 In Vitro Activation 37 5 2 Cell Preparation on Electrofusion Day 37 5 2 1 Medium ey 5 2 2 Chamber Cleaning 38 5 2 3 Washes and Other Preparatory Needs 38 5 2 4 Cell Preparation Process 38 5 3 Electrofusion 39 5 3 1 Pre Fusion Pulse AC 39 5 3 2 Fusion Pulse 39 5 3 3 Post Fusion Pulse AC 39 5 4 Cell Handling After Electrofusion 40 5 4 1 Immediate Post Fusion Care of Cells 40 5 4 2 Materials Needed for Post Electrofusion Culture 40 5 4 2 1 Cloning Medium 1 40 5 4 2 2 Cloning Medium 2 40 5 4 2 3 Complete Growth Medium 40 5 4 2 4 HAT Stock 50X 40 5 4 2 5 HT Stock 50X 40 5 4 3 Method Culture of Cell and Clone Selection 41 5 4 4 Culture of Cells 41 5 4 5 Feeding Cells 41 5 4 6 Cloning Cells 41 5 4 7 Weaning Clones 41 5 5 Method of Analysis 41 5 6 Demonstration Experiment to Illustrate Turbulence 42 5 7 Optimization 42 5 8 Chamber Clea
24. 9 e www btxonline com 22 User s Manual 3 3 2 Waveforms for Electrofusion continued Cell Electrofusion Tutorial 3 3 2 6 Published Waveform Protocols Waveforms which have been published are as follows Parameter Medarex Appendix B Li J 2006 PNAS Trevor et al 2004 Cancer Imm Immoth AC Pre Pulse 40 to 60 volts peak 40 volts peak 40 volts peak 15 seconds 15 seconds 30 seconds 1 4 MHz Freq unknown 0 8 MHz 75 volts peak 75 volts peak 30 seconds 10 seconds Linear Step Step Pulse Amplitude 800 volts 800 volts 800 volts Pulse Width 40 us 40 us 40 us Pulse Number 1 1 4 Pulse Interval NA NA 0 125 seconds AC Post Fusion 60 to 5 volts peak 60 volt peak 45 volts peak 30 seconds 30 seconds 55 seconds 1 4 MHz Freq unknown 0 8 MHz AC Post Fusion Linear Constant Constant BTX recommends the following parameters as a starting protocol in the optimization process Waveform Parameter Value AC Before Fusion Pulse Amplitude Duration Step function 40 volts peak for 15 seconds 70 volts peak for 20 seconds Frequency 0 8 MHz Fusion Pulse Amplitude 1000 volts Width 40 us Interval 0 125 Number 1 AC After Fusion Pulse Amplitude Duration Linear decreasing Start 60 volts peak Stop 5 volts peak Duration 30 seconds Frequency 0 8 MHz The method of programming this protocol is shown in Section 4 7 These waveforms are cov
25. Clicking Start begins delivery of the protocol to the test sample The pulse groups are executed in sequential order A double beep signals the end of protocol execution if sounds were enabled If the Start button is not clicked within 80 seconds after turning on the HVPS the system will automatically reset itself e Reset or Alt R Clicking Reset stops the capacitor charging and or the delivery of pulses and can be used at any time e F12 Pushing F12 on the keyboard is the same as clicking Turn HV ON followed by Start Please note that the reservoir capacitor takes time to charge so there will be delay of approximately seven seconds before pulsing starts A double beep signals the end of protocol if sounds were enabled 4 10 7 File Management File management conforms to standard Windows conventions The protocols are saved to and retrieved from a folder created at the time of software installation or to any user created folder The file extension used for a protocol file is pro The software automatically applies this extension when a protocol is saved Log files are saved as plain text and are given the txt extension This completes the set up and testing of the Hybrimune System If there is a problem please contact Technical Support for assistance phone 508 893 8999 website www btxonline com User s Manual 33 E Hybrimune System Components amp Set Up 4 11 Programming Waveform Protocols GROUP 2 This
26. ER 3 3 Post fusion process cell processing CHAPTER 5 Hybrimune Hybridoma Production System User s Manual Toll free 800 272 2775 508 893 8999 e www btxonline com Cell Electrofusion Tutorial 3 3 Electrofusion The process of electrofusion has three steps 1 Cell alignment and compression 2 Fusion 3 Stabilization The first step is to align the cells and then compress using special electric fields The Hybrimune System uses an alignment and compression technique called dielectrophoresis The process of manipulating matter using electric fields has been discussed for hundreds of years The process of using non uniform electric fields has been used for a number of applications in chemistry physics biology and engineering for several decades Pohl Dielectrophoresis Cambridge University Press 1978 Cell electrofusion was described by Zimmerman ina 1982 paper Zimmerman and Vienken J Membrane Biology 67 165 182 1982 Section 3 3 1 covers the basic physics of dielectrophoresis which includes the chamber in which the cells are fused Section 3 3 2 presents more detailed information on the special waveforms used 3 3 1 Cell alignment Dielectrophoresis The first step is in electrofusion Is to bring the cells together and compress using special electric fields This is a critical and complex step Cells have a net zero charge but do have a local negative charge on the surface which acts as a repelling force The
27. EY Producing Fusion Products Using Hybrimune 5 1 Cell Preparation Before Electrofusion 5 1 1 Myeloma Cells Myeloma cells are B cell leukemia cells that have been selected for or engineered to have certain properties One is that the myeloma should not secrete antibody of its own Some myeloma cells produce a single heavy or light chain of their own but it is preferable to use one that does not produce either Another trait is that the myeloma cells should be susceptible to killing using selected reagents Ideally a hybridoma is immortal because the myeloma contributes immortality to the B cell and the B cell contributes resistance to the selection agent This leaves B cells to die on their own and myeloma cells to die because of the selection agent A common selection medium is hypoxanthine aminopterin thymidine HAT containing medium Aminopterin blocks the de novo biosynthesis of purines resulting in inhibition of cell growth Specifically the folic acid antagonist aminopterin interferes with the donation of methyl and formyl groups by tetrahydrofolic acid in the early stages of de novo synthesis of glycine purine nucleoside monophosphates and thymidine monophosphate Thus de novo synthesis of purines is blocked Normal cells but not myeloma cells have enzymes that allow production of the necessary nucleotides using purine bases and thymidine a salvage pathway Thus the chemicals thymidine and hypoxanthine are provided as sour
28. Mutagenicity Non mutagenic No evidence of change following repeated autoclave procedures Sterilization T Hybrimune Hybridoma Production System User s Manual ae toll free 800 272 2775 508 893 8999 e www btxonline com
29. ROL K e s w r This term is called the Clausius Mossotti function This function represents the time constants of the various ions medium in the cytoplasm medium external to the cell characteristics of the cell membrane and the cell radius The term w 2p f where f is the frequency of the applied alignment waveform T Hybrimune Hybridoma Production System User s Manual ae toll free 800 272 2775 508 893 8999 e www btxonline com 13 BTX Pr a division of Harvard Bioscience Inc Cell Electrofusion Tutorial 3 3 1 Cell alignment Dielectrophoresis continued A computer model of this formula is presented in Figure 3 7 K IS NOT UNDER USER CONTROL Clausius Mossotti Function cell radius 7 um 1 000 0 800 0 600 0 400 0 200 80 uS cm 200 uS cm x 0 000 0 200 0 400 0 600 0 800 1 000 0 001 0 01 0 1 1 10 100 1000 Frequency Mhz Figure 3 7 Clausius Mossotti Function A cell radius of 7 um is used in the model which is typical The model shows that within typical conductivities of 80 to 200 uS cm the Clausius Mossotti function is generally in the 0 90 to 0 95 range as long as waveform frequencies are kept within the range of 0 2 to 2 MHz red rectangle The Hybrimune System including waveform and medium was designed to operate in this range It should be noted that the nominal conductivity of Cytofusion medium is 80 uS cm After adding properly washed cell
30. Y k Ag 1 TT 336 96 ND ND 2 TT 170 40 ND ND 3 TT 208 20 0 0 4 TT 1400 10 150 0 5 TT lt 1100 lt 400 83 23 6 Ti 582 131 69 21 7 Ag 1 456 65 8 1 8 Ag 2 ND 166 ND 18 9 Ag 3 493 101 128 56 10 Ag 4 71 0 0 0 11 Ag 5 3 3 0 47 0 12 Ag 5 246 0 36 0 Hybrimune Hybridoma Production System User s Manual Toll free 800 272 2775 508 893 8999 e www btxonline com 50 9 Appendix C MEDCOAT 2000 Data MEDCOAT 2000 is a commercially available proprietary coating developed for stainless steel medical devices The coating increases resistance to wear prevents corrosion and provides a smooth surface to clean Physical Properties Wear Resistance Improved 1400 Biological Compatibility Excellent resistance to corrosion Crevice Corrosion USP Class VI Certification Microhardness Ro 72 Acute Systemic No systemic toxicity Toxicity Composition of Coating 100 Chrome Intracutaneous No localized tissue reaction Toxicity Meets the 200 hour requirement Embrittlement Relief Surgical Muscle No irritation to human Implantation tissue Resistivity Decreased 25 ISO Tripatite Testing Magnetic Characteristics Permeability reduced 20 Adhesion to Base Metal No separation occurred Cytotoxicity Non toxic to living cells Rabbit Pyrogen Non pyrogenic Hemolysis Non hemolytic Non sensitizer Sensitization Ames
31. and the large volume chambers were designed to work with low conductivity cell suspensions Do not use highly ionic buffers in the chambers If ionic buffers are used two catastrophic problems will occur First the cell suspension will rapidly heat causing cell death Second significant convection currents will be generated in the suspension that will inhibit cell alignment WARNING ONLY CYTOFUSION MEDIUM MAY BE USED IN THE LARGE VOLUME CHAMBERS User s Manual E Hybrimune System Components amp Set Up 4 5 Setting Up the Hybrimune System All of the cables necessary to install the equipment are included in the Hybrimune System They are 1 Serial Interface Cable contained within the cable set 2 Line Mains Power Cord contained within the cable set 3 Chamber Connection Cable Model No CS CHNF BEFORE PROCEEDING MAKE SURE THAT THE LINE MAINS SWITCH AT THE LOWER LEFT OF THE FRONT PANEL IS IN THE OFF 0 POSITION AND THAT THE POWER CORD IS NOT PLUGGED INTO THE LINE MAINS INPUT Serial Cable Connect the serial cable between the D Subminiature 9 position DB9 connector labeled Computer Control on the back of the unit and a serial port on the computer This is the communication link between the microprocessor within the waveform generator and the computer It is a standard RS 232 serial communication If only USB ports are available on the computer then a USB to Serial converter will be necessary These conver
32. ce material for the salvage pathway Myeloma cells do not survive in HAT medium because they lack the salvage pathway enzymes Fused B cells have limited growth potential and thus die in vitro within two weeks Myeloma B cell hybridoma cells survive because they have received salvage pathway enzymes trom the B cells and immortality from the myeloma cells Early preparation of myeloma cells is relatively simple The cells should be grown using good sterile technique and the recommended medium for that myeloma The cells should be in log phase of growth on the day of the fusion Passage level of the myeloma cells should be monitored Use low passage level cells when possible If cultured for extended passage levels the cells can lose the susceptibility to HAT medium selection Culture of the myeloma cells in either 8 azaquanine or 6 thioquanine will eliminate cells that are not sensitive to HAT T Hybrimune Hybridoma Production System User s Manual ae toll free 800 272 2775 508 893 8999 e www btxonline com 35 EY Producing Fusion Products Using Hybrimune 5 1 Cell Preparation Before Electrofusion cont d 5 1 2 Available Myelomas or Heteromyelomas Note This list is not exhaustive It was made from literature searches and a search of the ATCC and Coriell Institute s web sites Carefully research intellectual property issues relating to these cells before using them since some of these cell lines may be patented It does no
33. connecter on the converter Turn on the waveform generator Plug the USB connector from the converter into an available USB port on the computer The computer should recognize the connection User s Manual 4 8 System Test The following is a basic test of the waveform generator system using the preprogrammed protocol HYBRIMUNE Test pro located in the default protocol folder For this initial test the Chamber Cable and Chamber should be fully connected but it is not necessary to fill the Chamber First e Plug in the Line Mains power cord for the waveform generator e Turn the Line Mains Power rocker switch at the lower left front of the front panel to the ON I position What should happen H 11 The rocker switch Power Ready and Chamber Connected LED s should illuminate Troubleshooting tips If the rocker switch fails to illuminate then return it to the OFF position O and e Verify that the Line Mains cord is properly plugged into the receptacle on the back panel and into the source of electric power Check the fuse on the back panel If it is faulty replace it with the exact same type fuse 240V 2A slo blo If the problem persists contact Customer Service If only the rocker switch and Power LED are illuminated then connect the Chamber Cable and verify that the Ready and Chamber Connected LEDs turn on Then Start the Waveform Generator Interface software
34. d in Chapter 4 5 3 1 Pre Fusion Pulse AC The first step is to align the cells in a chain and then compress by increasing the force This is done by applying a high frequency sine wave with varying amplitude to the cells in Cytofusion Medium immediately prior to applying the fusion pulse Initially the cells are aligned using a lower amplitude waveform Once aligned additional force is applied to compress the cells This provides intimate contact of the cell membranes for optimum fusion There are two published approaches for this first step which are presented in the table on page 3 17 The optimum process will be dependent on cell type The user is encouraged to develop protocols for the specific cell line in use BTX recommends starting with the two level sinewave Use a low amplitude to align and a high amplitude to compress Figure 3 13 Non linear Step Reasonable values are 40 volts peak for 15 seconds at 0 8 Mhz 75 volts peak for 20 seconds at 0 8 Mhz The 2 ml Optimization Chamber supplied with the Hybrimune System has a clear bottom see Figure 3 9 This Is so a microscope can be used to observe the cell alignment Cell compression cannot be observed The cells should align in an orderly manner and migrate toward the inner electrode No turbulence or violent movements should be seen Electrically the 9 ml chamber and the 2 ml chamber are identical Protocols optimized using the 2 ml Optimization Chamber can be directly used in
35. e 800 272 2775 508 893 8999 e www btxonline com 46 Appendix A System Specifications Waveform Generator Pulse In a Group Pulse Amplitude 100 to 1000 volts in 5 volt steps Pulse Width 20 to 1000 us in 10 us steps Number of Pulses Pulse Interval O to 10 in steps of 1 0 125 to 10 sec in 0 001 sec steps Pre Pulse AC and Post Pulse AC In a Group Start AC Amplitude 5 to 75 vpk in 5 volt steps Stop AC Amplitude 5 to 75 vpk in 5 volt steps Frequency 0 2 to 2 0 MHz in 0 2 MHz steps Duration O to 60 sec in 1 sec steps Amplitude function k 0 1 to 20 in steps of 0 1 Inhibit Pre pulse AC Set AC Duration O Fusion Pulse Set Pulse Number O Post pulse AC Set AC Duration O Number of Groups Concatenation 1 to 10 groups Maximum Separation Pre Pulse 1 ms AC End to Pre Pulse AC start Maximum Separation Post Pulse 1 ms AC End to Post Pulse AC Start Maximum Separation Pre Pulse 10 ms AC End to Pulse Start Maximum Separation Pulse End 20 ms to Post Pulse AC Start Operating Temperature 20 to 30 C Storage Temperature O to 50 C Line Mains 100 240 vac rms Dimensions H x W x D 16 5 30 5 40 6 cm 6 5 x 12 x 16 In Weight 6 8 kg 15 lbs T Hybrimune Hybridoma Production System User s Manual ae toll free 800 272 2775 508 893 8999 e www btxonline com 47
36. e Installation The Hybrimune waveform generator interface software must be installed on a local computer with these minimum specifications Windows 98 or higher 2Mb free disk space and at least one RS232 Serial Port If only USB ports exist then a USB to Serial adapter will be required and should be installed first If the software needs to be installed use the following procedure 1 Close all other programs and insert the CD ROM 2 Click Start from the Windows lower menu bar 3 Select Run which will display a dialog box 4 Type CDRomDriveDesignation setup then click OK 5 The setup program will begin The default file locations are Program Files for the application and My Documents BTX for protocol and log files 4 7 1 Set up using a USB to Serial Converter This system requires an RS 232 interface However if only USB ports are available a converter will be required Please note that all converters types may not be compatible with the system The following procedure was developed using the Dynex USB PD Serial Adapter Cable Model DX UBDB9 not currently provided by BTX Use the manufacturer s instructions to install the converter s software on the computer After installation disconnect the converter from the computer s USB port Connect the serial cable between the D Subminiature 9 position DB9 connector labeled Computer Control on the back of the waveform generator and the DB9
37. e given an intravenous antigen boost 3 4 days before harvesting spleens to activate B cells and increase the number of antigen positive cells located in the spleen Spleens were collected from adequately immunized mice just prior to the electrofusion For the fusion mouse spleen cells and SP2 O mouse myeloma cells were washed twice in Cytofusion medium The cells were at a concentration of 10 million cells per ml A volume of 4 5 ml of each cell suspension was mixed then placed into a 9 ml volume electrofusion chamber Cells were aligned and fused using the following parameters 40 V to 60 V 1 4 MHz for 15 seconds Pulse Amplitude 800 volts width 40 ms one pulse Post pulse Sine Wave 60 V to 5 V 1 4 MHz for 30 seconds Pre pulse Sine Wave After electrofusion cells were left in the fusion chamber for 30 minutes Samples of cells from wells were then analyzed by Wrights stain of Cytospin preparations of the samples The bulk of the cells were cultured in HAT medium The cells were cultured in 96 well plates at 5000 cells ml Total clones were counted by screening the wells in 96 well plates by eye for hybridoma growth on day 7 to 9 The number of clones was mathematically calculated using a poison distribution analysis Wells were screened for presence of IgG antibody and antigen specificity using ELISA or an automated fluorescent screening system HTRF Data collected during the screening was normalized
38. ed Immediately after use rinse the chamber in reagent grade water Fill the chamber with 4 sodium hydroxide and soak for 5 minutes Empty the chamber Rinse in reagent grade water for 10 seconds Repeat rinse 10 times Rinse once in 70 ethanol Air dry Chemical Sterilization Fill chamber with 4 sodium hydroxide and soak for 10 minutes Empty chamber and fill with 70 isopropanol or ethanol and soak for 10 minutes Empty chamber and fill chamber with Spor Klenz Steris soak 10 minutes Rinse thoroughly in sterile reagent grade water Spor Klenz is a registered trade mark of Steris www steris com Other Sterilization Alternatively the chambers can be gas sterilized Do not autoclave the chambers Hybrimune Hybridoma Production System toll free 800 272 2775 508 893 8999 e www btxonline com 42 User s Manual 5 9 References 1 Kohler G Milstein 1975 C Continuous cultures of fused cells secreting antibody of predefined specificity Nature 256 195 497 2 Neil GA Zimmermann U 1993 Electrofusion Methods Enzymol 220 174 96 3 Vienken J Zimmermann U 1985 An improved electrofusion technique for production of mouse hybridoma cells FEBS Lett 182 278 80 4 Steenbakkers PG van Meel FC Olijve W 1992 A new approach to the generation of human or murine antibody producing hybridomas J Immunol Methods 152 69 77 5 Jaroszeski Mark J Gilbert Richard Heller 1994 Detection and
39. ely collect spleen and place into 10 ml cold DMEM e Pour spleens onto Cellector screen e With a 25 27 gauge needle inject 0 5 ml sterile PBS into spleen e Tease spleen apart using two syringes with 21 gauge needles Use the needle tips to cut the spleen into small parts e Express spleen cells through 100 mesh sterile screen using Cellector pestle or rubber end of a syringe plunger e Drop wise flush screen with 7 ml PBS e Pipette cells into 15 ml conical tube e Let settle for 5 minutes to remove large pieces e Collect cells in supernatant e Centrifuge 300 Xg for 7 minutes e Re suspend pellet in 5 ml red blood cell lysis buffer pre warmed to 37 C e Mix well and incubate 3 minutes e Add 5 ml PBS e Centrifuge cells and wash cells once in complete medium 5 1 7 In Vitro Activation Activated B cells fuse to produce more functional antibody producing myelomas than unactivated B cells The purpose of the Intravenous immunization 3 days before harvest is to increase the percent of activated B cells in the spleen A number of in vitro B cell activation methods are available as alternatives or as supplements to in vivo B cell activation See references 8 14 for additional methods The method for PHA L stimulation follows e Wash spleen cells in complete culture medium e Re suspend cells at 1 million cells ml in complete growth medium plus 2 4 ug ml PHA L e Place cells in T 75 flask and incubate at 37 C 5 CO for 3 da
40. er the zona pellucida and outside of the cell membrane Finally electrofusion is used to fuse the nucleus with the recipient cell The electrofusion step also serves to activate the cell An image furnished by an unidentified Chinese researcher is presented in Figure 3 4 Figure 3 4 Nuclear Transfer User s Manual Cell Electrofusion Tutorial 3 2 Cell Fusion Methods Cell fusion is the formation of a single hybrid cell containing the nuclei and cytoplasm from more than one cell It is accomplished by e Bringing cells together so that they touch e Compressing the cells to increase contact area e Disrupting the cell membrane e Allowing cell membranes to fuse and providing a good environment for growth of cells There are several methods to accomplish these steps The following table summarizes some of these Contact Fusion Centrifuge PEG Tissue Culture Virus Electrical Electrical Centrifuge Electrical Vacuum Electrical Any approach must overcome the natural negative surface charge on a cell The net charge on a cell as a whole is neutral Of these electrical methods to bring cells into contact and electrical methods to fuse cells are the methods used by the Hybrimune System and also tend to be the most efficient methods There are three distinct steps in cell fusion Each will be discussed in the sections Indicated 1 Pre fusion process cell processing CHAPTER 5 2 Electrofusion CHAPT
41. ered by BTX patents Hybrimune Hybridoma Production System Toll free 800 272 2775 508 893 8999 e www btxonline com e User s Manual Figure 4 1 Hybrimune System 4 1 Introduction The Hybrimune Hybridoma Production System is composed of four components a computer controlled waveform generator a large volume coaxial fusion chamber special low conductivity medium and user interface software A computer is required for the application software but is not included in the system The system provides the capability of fusing up to a 180 billion cells in a single protocol run The system is designed to be simple to set up and to operate The system is shown in Figure 4 1 EN Hybrimune System Components amp Set Up Hybrimune Hybridoma Production System toll free 800 272 2775 508 893 8999 www btxonline com 24 4 2 The Hybrimune Voltage Waveform Generator The waveform generator produces the voltage waveforms programmed by the user The desired waveform parameters and sequences are set by the user in the user interface application software When the Start button is clicked the software is downloaded from the computer to the microprocessor in the waveform generator The waveform Is generated and applied to the Electrofusion Chamber producing an electric field Depending on the user input instruction this microprocessor controlled waveform generator will generate e Pre Fusion Pulse AC Wave a l
42. ermine if they are producing polyclonal antibody to the antigen of interest Three days before harvesting spleen cells a final boost of antigen without adjuvant can be given intravenously to activate B cells and localize B cells to the spleen An alternate immunization procedure Is to use DNA vaccines delivered using intradermal inoculation of plasmid plus electroporation Mice are immunized every 3 4 weeks The immunization can be either a series of DNA vaccines or a DNA prime with protein boost A final boost is given 3 7 days prior to harvest For details consult BTX regarding the AgilePulse DNA vaccine delivery system 5 1 5 Screening Test Bleeds e Identify mice with ear tags or ear clips e Apply grease to tail an area of 3 4 inch from base toward tip e Use extremely sharp blades to make slice EM blades Be careful not to cut through the tail but make cut large enough to draw about 2 5 heparinized capillary tubes e Place blood into mouse clot tubes e Apply pressure to tail wound to stop the bleeding e Spin down the blood at max speed for 5 minutes in the microfuge e Draw off and store sera at 200 C until you can test on a blot or ELISA at 1 100 or at 1 50 Evaluating sera Use an ELISA or Western Blot to evaluate antibody production in mice User s Manual 5 1 Cell Preparation Before Electrofusion cont d 5 1 6 Harvesting Spleen Cells e Euthanize immunized mice e Immerse mice in alcohol e Steril
43. fety features to protect the user and the equipment If used properly the Hybrimune Waveform Generator is a safe and reliable instrument Front and Back Panel Symbols AN AJ Caution Risk of Off On Fuse AC Mains Electric Shock The following material is contained in this User Manual CHAPTER 2 Items of Particular Interest CHAPTER 3 Cell Electrofusion Tutorial CHAPTER 4 Hybrimune System Components and Setup and Use of System CHAPTER 5 Producing Fusion Products Using Hybrimune CHAPTER 6 Customer Service T Hybrimune Hybridoma Production System User s Manual ae toll free 800 272 2775 508 893 8999 e www btxonline com 7 EN Items of Particular Interest The Hybrimune Commercial Electrofusion System is the most sophisticated system available for fusing dissimilar cells The system is available only under license directly from Cellectis The following are important 1 2 SS A Y J 9 10 11 The System must remain in the possession of the licensee The System has four components the Hybrimune Waveform Generator the user interface software the fusion chambers and the Cytofusion medium These components were all designed to operate as a system Using other components may damage the system and will certainly provide degraded performance The System shall only be used as described in this User Manual Certain concepts and processes are covered by BTX patents and patents pendin
44. flue Hybrimune Hyoridoma Production system USER S MANUAL wrk nn mune nyot pae sysso e a ayeneo www btxonline com 015 100191 REV 3 a division of Harvard Bioscience Inc Hybrimune Hybridoma Production 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 BIX Warranty Harvard Apparatus warranties the Hybrimune Hybridoma Production 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 lim
45. g Other ionic medium shall not be used it will generate significant heating and kill cells The chamber must be cleaned after each use to prevent ionic build up which will cause heating and kill cells and reduce efficiency The cover must be used on the chamber while the waveforms are being applied Do not connect or disconnect the chamber from the waveform generator when the waveforms are being applied to the chamber Do not put your fingers or any other object in the chamber while the waveforms are being applied to the chamber This system produces voltages as high as 1000 volts and may cause injury or death if used improperly The user must read the User Manual If the user has any questions contact BTX BTX would welcome any comments from the users to improve this manual ie Hybrimune Hybridoma Production System User s Manual Toll free 800 272 2775 508 893 8999 e www btxonline com 8 3 1 Applications There are three common uses of electrofusion e Hybridoma production as one step in monoclonal antibody production e Producing therapeutic hybrids for immunotherapy e Nuclear transfer from one cell cytoplasm to another cell cytoplasm 3 1 1 Hybridoma Production One step in monoclonal antibody production is producing hybridomas The following are the basic concepts for making antibody producing hybridomas 1 Standard myeloma cells are B cell cancer cells that are immortal They have several important modif
46. hods 100 5 40 Larrick J W et al 1993 Characterization of human hybridomas secreting antibody to tetanus toxoid PNAS USA 80 6376 6380 Sugasawara RJ Cahoon BE Karu AE 1985 The influence of murine macrophage conditioned medium on cloning efficiency antibody synthesis and growth rate of hybridomas J Immunol Methods 79 2 263 75 Trevor K et al 2004 Generation of dendritic cell tumor cell hybrids by electrofusion for clinical vaccine application Cancer Immunology Immunotherapy 53 3 705 714 Li J 2006 Human antibodies for immunotherapy development generated via a human B cell hybridoma technology PNAS 103 10 3557 4562 Lee R et al 2008 A high throughput hybridoma selection method using Fluorometric Microvolume Assay Technology J Biomolec Screening 13 3 210 217 User s Manual This is a summary of the electrofusion protocol given in the Hybrimune user Manual Electrode Cleaning and Sterilization 1 Load 9 mL of 1N NaOH into the chamber incubate 5 minutes Dump out the NaOH into the sink and rinse thoroughly with running DI water Fill and dump the chamber 10x While under a sterile hood aspirate water from the chamber with a plastic pipette Spray the electrode inside and out with Sporklenze rinse with DI water and aspirate Spray the electrode with 70 ethanol and aspirate Place plastic cover on chamber until ready to load with cells Cell Preparati
47. ications One is that they have been made susceptible to being killed by specific chemicals that do not kill B cells Another is that they generally do not produce antibody on their own 2 In humans and animals there are millions of different types of B cells lymphocytes with different antibody specificities They are not immortal B cells expand in number in response to external events stimulation by antigen and T cells 3 A hybridoma is produced by fusing an immortal B cell line myeloma that does not secrete antibody with B cells that have been extracted from blood The fused cells take on characteristics of both cells the myeloma s immortality and the B cell s specific antibody 4 The resulting hybrid is cultured in a special medium that kills unfused myeloma cells while unfused B cells die naturally over time 5 The hybrids are then placed in a 96 well plate with a dilution where approximately one antibody secreting hybridoma exists per well After some growth antibody is detected in the culture supernatant Cells from positive wells are cloned A figure of merit is the number of productive wells with antibodies and the number of cells required to give on average a positive well The number of functional antibody secreting hybridomas is less than the number of fusions Cell Electrofusion Tutorial Hybrimune Hybridoma Production System Toll free 800 272 2775 508 893 8999 e www btxonline com 9 A
48. ists the temperature of the medium will rise to critical levels and kill the cells User s Manual EY Producing Fusion Products Using Hybrimune 5 2 Cell Preparation on Electrofusion Day cont d 5 2 2 Chamber Cleaning The chamber must be clean for two reasons to minimize the chance for contamination and to eliminate ions that increase conductivity of medium placed in the chamber See the chamber cleaning instructions in Section 5 8 There have been many cases where cell fusion has failed due to improperly cleaned chambers The chamber comes with two plastic caps One size fits the top snuggly the other loosely It is a matter of laboratory preference which one to use However the caps should be kept on the chamber when they are not in use and must be kept on the chambers when the waveforms are being applied The chamber electrodes will have as much as 1000 volts between them 5 2 3 Washes and Other Preparatory Needs It is important that all tissue culture medium be washed from the cells prior to electrofusion This requires a minimum of one centrifugation plus two washes in Cytofusion medium Residual ions from an incomplete wash will dramatically reduce hybridoma production For the cell washes it may be useful to wash the two cell populations separately This has two advantages One is that if there is differential cell loss during the washes compensation can be made during the mixing of the cells after the last wa
49. itation on an implied warranty so the above 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 Warranty is void if the Hybrimune 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 electrodes 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 rights which vary from state to
50. mpressed The solution to this problem is to use a non linear AC amplitude The first part of the AC sine wave will be low amplitude and then immediately increase the amplitude to compress while the cells are in contact The alignment chains are much less likely to break up at this point since they have cells touching on each side The Hybrimune voltage waveform generator will produce a number of these types of waveforms These are described below Care has to be taken with the amount of time the AC sinewave is applied If left on too long the current will heat up the medium like the heating element in a coffee maker Heat causes convection currents which in turn causes the chains to break apart 3 3 2 2 AC Sinewave Waveforms The definition of AC sinewave amplitude and frequency is shown in Figure 3 12 Electrical engineers use several definitions for amplitude As shown half of the sinewave is positive and half is negative The concept of average value is meaningless since that is zero There are three common definitions in use Peak Voltage is the amplitude from zero to the peak positive or negative value Peak to Peak Voltage is the value from the negative peak to the positive peak rms Voltage is square root of the mean value of the sinewave squared this is the power in the waveform root mean squared Figure 3 12 Sinewave Definition The Hybrimune voltage waveform generator uses the definition of Peak Voltage or Vpk
51. n example of E fusion versus PEG was produced by M Coccia Ph D Platform Development Group at Medarex Inc Milpitas CA Transgenic human Ab producing mice were used in experiments comparing efficiencies of E fusion to PEG fusion Each experiment used a portion of the same splenocyte preparation from mice immunized with tetanus toxoid TT for comparison of the E fusion and PEG fusion methods Results shown are the number of TT antigen specific clones generated by each method normalized to the same number of cells These data are representative of additional experiments not shown utilizing four different antigens Taken together all experiments showed E Fusion generated approximately ten fold more antigen specific antibody clones relative to PEG fusion Experiment Number Antigen Specific E Fusion 20 10 400 151 145 Figure 3 1 Medarex Fusion Example Figure 3 1 shows Cytospin prepared and Wrights Giemsa stained SP2 0 cells 30 minutes following large scale E fusion In these E fusion experiments more than 75 of cells in the final population were fused with 2 to 3 nuclei per cell Additional Medarex data Is presented in Appendix B Another process using human B cells is described in Li et al 2006 PNAS 103 10 3557 3562 User s Manual Cell Electrofusion Tutorial 3 1 2 Immunotherapy Another application is the ex vivo fusion of human dendritic cells and human tumor cells rem
52. n in Section 3 3 1 the force on the cell during alignment is a function of the cell radius cubed The radius of the cell varies within the same type and the mean radius of the two cell types to be fused varies This will affect the number of fusion products produced Some cell types are much more difficult to fuse than others This is inherent in the cell characteristics In the initial alignment process the cells will accelerate when the force Is applied If the acceleration is significant then the cells will move toward the center electrode and bounce off like a billiard ball It is possible to create such acceleration that the cells begin circulating in a race track pattern High acceleration is very detrimental to good cell alignment and must be avoided The principal use of the BTX 2 mL Optimization Chamber is waveform protocol optimization The alignment process must be optimized while observing the cell alignment User s Manual Cell Electrofusion Tutorial 3 3 2 Waveforms for Electrofusion continued D The initial alignment must be done slowly and deliberately so good alignments can be produced However if a small force is used to align as required then compression will not occur This still leaves the function of compression to increase the surface contact between adjacent cells to increase the probability of a successful fusion pair After alignment the cells will be in contact tangentially but not co
53. n one of two ways One is that the pore will simply close in the cell membrane Another is that adjacent pores in the membranes of two touching cells will fuse together combining the cell membranes of two cells Figure 3 15 Electric Field Pulse Forms Pathways Cell Electrofusion Tutorial 3 3 2 4 Post Fusion Pulse AC Sinewave The objective of this function is to hold in place the cells that have been fused together to allow them to mature Immediately stopping the AC sinewave causes the cells to break apart by thermal motion The same AC Amplitude functions described in Section 3 3 2 2 can be used Experience has shown that a simple decreasing Linear Ramp Amplitude k 1 has proven to be the most effective 3 3 2 5 Parameter Range of Hybrimune Voltage Waveform Generator The following parameters may be programmed in the waveform generator AC Before Amplitude 5 to 75 v peak 5 volts steps Fusion Pulse Amplitude Constant Linear increase or and function decrease k 1 AC After Step Ramp k 0 1 to 20 Fusion Pulse Frequency 0 2 to 2 0 MHz in 0 2 MHz steps Duration O to 60 seconds in 1 second steps Set zero inhibits AC Fusion Pulse Amplitude 100 to 1000 volts in 5 volt steps Width 20 to 1000 us in 10 us steps Interval 0 125 seconds in 0 001 second steps Number O to 10 in steps of 1 O inhibits pulse Hybrimune Hybridoma Production System toll free 800 272 2775 508 893 899
54. ndicates that the waveform generator is delivering an electrofusion waveform to an attached Chamber As the process runs the LED will alternate between a steady illumination and a brief flashing state This behavior indicates that the pre pulse AC the HV pulse s and the post pulse AC parts of the waveform are being delivered sequentially e Relative Load Indicator Multi Color Bar Graph This indicator is a series of 10 LED segments that provides an indication of the amount of electrical current flow being delivered to the coaxial chamber during waveform delivery The number of segments that illuminate is dependent on a number of factors buffer conductivity cell concentration cell solution temperature the volume of cell solution in the chamber the sine wave voltage and frequency being applied and the length of time the wave form is applied If many segments illuminate then significant heat and turbulence is being generated in the coaxial chamber which will have a negative effect on the fusion process During the factory calibration procedure the bar graph is set to display seven bars three green four yellow when a 70Vpk 1MHz sine wave is delivered to a CHNF 9R Chamber filled with 9ml of a 100uUS cm Conductivity Standard at 25 C E Hybrimune System Components amp Set Up Hybrimune Hybridoma Production System Toll free 800 272 2775 508 893 8999 e www btxonline com 27 4 7 Waveform Generator Interface Softwar
55. ning 42 5 9 References 43 Hbrimune Electrofusion Protocol Summary 44 45 6 Customer Service 46 S Hybrimune Hybridoma Production System User s Manual ae toll free 800 272 2775 508 893 8999 e www btxonline com 4 Table of Contents SUBJECT 7 Appendix A Specifications B Medarex Data C MEDCOAT 2000 Summary Test Results List of Figures Medarex Fusion Example Arizona Cancer Center Fusion Example Electrofusion Example Nuclear Transfer Example Uniform Field and Non Uniform Fields Cell Alignment Clausius Mossotti Function Chamber Definitions Cell Alignment in Optimization Chamber Force on a Cell Cell Alignment Sequence Sinewave Definition A Sinewave Amplitude Functions Ramp K Factor in Non linear Sinewave Amplitude Electric Field Pulse Forms Pathways Hybrimune System Large Fusion Chambers Cytofusion Medium Hybrimune Cable Connections User Interface Application Screen Last Protocol Log PAGE 47 48 49 50 51 10 10 10 11 12 iz 14 13 16 17 18 19 20 21 22 24 25 25 26 28 29 User s Manual T Hybrimune Hybridoma Production System ae toll free 800 272 2775 508 893 8999 e www btxonline com 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
56. o That system has not been sold for many years The linear ramp can be set to increase or decrease at a constant rate volts per second by setting the start voltage the stop voltage and the duration This function is also available on the BTX laboratory system Hybrimune Hybridoma Production System Toll free 800 272 2775 508 893 8999 e www btxonline com Stop AC amplitude and gt Start AC amplitude AC Duration lt ___ l Constant Amplitude Stop AC amplitude Start AC amplitude 3 Stop AC amplitude Start AC amplitude gt Start and Stop AC 2 amplitude a Start and Stop AC 1 amplitude AC Duration pe A Non linear Step using groups Figure 3 13 AC Sinewave Amplitude Functions e User s Manual 20 BTX a division of Harvard Bioscience Inc Cell Electrofusion Tutorial 3 3 2 Waveforms for Electrofusion continued Non linear Ramp k Amplitude This function is set by programming the start amplitude the stop amplitude the duration and the Ramp k factor The Ramp k factor determines how fast the amplitude becomes non linear These functions are presented in Figure 3 14 for a Start Amplitude of 20 volts a Stop Amplitude of 50 volts and duration of 10 seconds AC Amplitude t AC Duration k x Stop Amplitude Start Amplitude Start Amplitude Figure 3 14 Ramp k Factor in Non Linear Sinewave Amplitude Ramp k
57. on Prepare and count spleen cells Count myeloma cells Centrifuge splenocytes and SP2 0 cells together at a 1 1 ratio or separately at 400x g for 8 minutes Resuspend cells 5 10 mls of BTX Cytofusion media Spin at 400x g for 8 minutes Repeat wash After the second wash resuspend in less than the expected final volume resuspend fully by pipetting up and down several times to achieve a single cell suspension Count the cells mix the appropriate number of SP2 0 cells with the splenocytes if not done before and dilute cells at a final concentration of 1 x 107 cells per ml NOTE You should perform the fusion within 10 minutes after the final wash Suspension in fusion media any longer than 30 minutes will alter the electro physical properties of the cells and will reduce the fusion efficiency Hybrimune Hybridoma Production System toll free 800 272 2775 508 893 8999 e www btxonline com 44 Hybrimune Electrofusion Protocol Summary Computer Preparation 1 2 3 Turn on the computer Turn on the BTX Hybrimune system Load the Hybrimune PulseAgile program from the Start button in Windows or shortcut from the desktop Click the electrofusion button Load the optimized protocol for this set of conditions Found under the file menu Overall Fusion Summary 5 a eS eS Sterilize the hood Clean and sterilize the electrode Turn on computer and load fusion protocol Wash and count the cell
58. or are receiving adequate power It should be illuminated at all times when the waveform generator is turned on Ready green LED Indicates that the waveform generator Is ready to deliver a electrofusion waveform to an attached Chamber It will only be illuminated when the internal microprocessor detects no problems and when the Chamber Cable is properly connected Internal Fault red LED Indicates that a problem occurred inside of the unit during a protocol run If illuminated the system requires a Reset If the problem persists BTX Customer Service should be contacted before proceeding Under normal operating conditions this LED will not be illuminated User s Manual 26 4 6 2 Process Status Group The function of this group is to inform the user of state of an electrofusion protocol run This group consists of three LED indicators and a multi color bar graph e Chamber Connected Green LED Indicates that the Chamber Cable is properly connected If a Chamber is plugged into the cable then it is ready to receive a waveform from the waveform generator e Charging Power Supply On Green LED Indicates that the internal reservoir capacitor is charging or is fully charged When not illuminated the reservoir capacitor is at zero volts It is only illuminated after the user clicks Turn HV On and remains illuminated during the entire process run e Process Running Green LED I
59. oved from a patient to produce a personal immunotherapeutic vaccine against the tumor A significant number of experiments were conducted at the Arizona Cancer Center The results of this research were published in Trevor et al 2004 Cancer Immunol Immunother 53 8 705 714 Figure 3 2 Arizona Cancer Center Fusion Example Figure 3 2 shows an electrofusion of dendritic cells with A549 human lung carcinoma cells Fusion efficiencies of 10 were obtained Cells were fused at 8 million cells ml With that concentration in a full 9 ml electrofusion chamber up to 7 2 million therapeutic hybrid cells can be produced Data provided courtesy of Dr Katrina Trevor Arizona Cancer Center Figure 3 3 was produced in the laboratory by fusing an immune system cell and a tumor cell The image was taken 45 minutes after electrofusion Figure 3 3 Electrofusion Example Hybrimune Hybridoma Production System toll free 800 272 2775 508 893 8999 www btxonline com 10 gt a division of Harvard Bioscience Inc 3 1 3 Nuclear Transfer Nuclear transfer is the transfer of a nucleus from one cell to the cytoplasm of another cell The donor cell can be either from an embryo or from somatic cells The recipient cell is usually an enucleated oocyte The process uses electrofusion in the final steps of nuclear transfer First a recipient oocyte is enucleated Second a nucleus from another cell is inserted in the recipient egg und
60. ow voltage megahertz frequency sinusoidal alternating voltage that results in an alternating electric field that aligns and compresses the cells in a process called dielectrophoresis e Fusion Pulse a high voltage fusion pulse that forms pathways in the cell membranes that results in fusion e Post Fusion Pulse AC Wave a second sinusoidal alternating electric field that holds the fused cells together while they mature A detailed description of the waveforms Is presented in Section 3 3 2 The parameter values available are listed in Section 3 3 2 5 The User Interface software provides the user with the ability to fine tune each of the three parts of the waveform The instructions on how to program the waveforms may be found in Section 4 11 User s Manual le Figure 4 2 Large Fusion Chambers 4 3 The Large Volume Chambers The Hybrimune Waveform Generator connects to a coaxial large volume fusion chamber The chamber produces the various electric fields required in the hybridoma production process These chambers were very carefully designed to provide optimum alignment forces and intense electric fields necessary for efficient electrofusion processes When the alternating sinusoidal voltage is applied to the inner and outer electrode rings of the chamber a highly non uniform electric field is established that causes the cells to align into pearl chain formations Two types of chambers are provided The first
61. parameters are set edited and reviewed The following is a description of each of the sections and input display boxes found in the Control Panel Hybrimune Hybridoma Production System toll free 800 272 2775 508 893 8999 e www btxonline com 32 Group ID Displays the group number whose characteristics are shown in the rest of the Control Panel Area The Group ID is also shown highlighted in the Group List Pre SINE Beg Voltage Beginning voltage Duration Duration in seconds Frequency Frequency in Megahertz End Voltage End voltage Ramp k Non linear ramp constant Pulse Amplitude Voltage of the electroporation fusion pulse Duration Length of the pulse Pulses Number of pulses to deliver Interval Delay between the start of each pulse Post Sine Beg Voltage Beginning voltage Duration Duration in seconds Frequency Frequency in Megahertz End Voltage End voltage Ramp k Non linear ramp constant User s Manual E Hybrimune System Components amp Set Up 4 10 The Waveform Generator Interface Software cont d 4 10 5 Control Panel Area continued e Group List Displays the list of groups in the current protocol The groups will be executed in the order displayed There are three buttons to control the list Add or Alt A Used to add a group to the list Replace or Alt P Used to change data within a group Changes made to values within a group will not take effect until Replace has been selected If a
62. rcuit to the Wave Form Generator Therefore a significant amount of current will begin to flow In general frequencies above 1 MHz should not be used 4 10 The Waveform Generator Interface Software The Waveform Generator Interface conforms to standard Microsoft Windows conventions and this manual assumes that the user is familiar with Windows The software performs the following e Setting up Protocols e Running Protocols e File Management to save and recall protocols e Data Log Display printing and saving e Status Display The following sections describe operation based on the above list Some things to remember e Some commonly used functions have redundant control features i e they can be accessed from several places on the screen e The default installation directory is C Program Files BTX Hybrimune It will be different if you have selected another directory during installation e The screen is divided into four areas within the main window The Title Bar and pull down menus top the Tool buttons left the Control Panel for each operating mode center and the Status area right see Figure 4 5 e When the electrofusion chamber cable is not attached to the system the Chamber window will display OPEN As a safety feature the high voltage power supply cannot be enabled and protocols cannot be run while the system is in this state However protocols can be programmed and saved in this state Hybrimune
63. rnal high voltage power supply is now enabled and the reservoir capacitor is being charged After approximately seven seconds the START button is enabled The Power Supply Voltmeter window will display 500 5 volts Then e Click Start What should happen The waveform generator runs the protocol The green Process Running LED will illuminate cycling through its display phases The Relative Load Indicator display will not illuminate since there is no conductive medium in the chamber A protocol log as shown in Figure 4 6 on the next page should appear in the Last Protocol Log window elivered Waveform Parameters Protocol File HYBRIMUNE TEST PRO Sin ee DY Group Numve Date 9 03 2011 4 7 52 24 GRP Pulse Group System Serial Number 08 9937HY Dur AC duration sec Firmware Version 5 81 Freq AC Frequency MHz Mode Electrofusion BegV Start AC Amplitude Delivered Waveform Parameters EndV Stop AC Amplitude volts gt Pre Sine NUM _ Pulse in Group gt GRP Dur Freq Begv EndV WIDTH Pulse Duration msec 5 0 1 4 50 50 INTVL Time between pulses sec gt Pulsing SEV Programmed PS vonage gt GRP NUM WIDTH INTVL SetV MonV MonV Measured PS Voltage gt 1 0 100 0 50 500 500 2 0 100 0 50 500 500 gt 1 3 0 100 0 50 500 500 Protocol Information gt 4 0 100 050 500 500 EHENEmE UF apeieae 1 5 0 100 0 50 500 500 Date MM DD YY DoW Time 24 Hour Clock gt Post Sine System Mode gt GRP Dur Freq BegV EndV gt
64. s Plug the machine into the electrode Carefully load the resuspended cells into the chamber and place the plastic cover on the chamber Note The following step should be performed within 30 seconds e g as soon as possible following loading the cells into the chamber or cells may settle to the bottom and give poor alignment If allowed to sit more than 30 seconds resuspend the cells before proceeding 7 8 9 Click the Start button The fusion process will start and the computer will run the pre sine pulse and post sine steps Allow the cells to sit undisturbed for 5 minutes Gently re suspend the cells It is important to get settled cells back into suspension 10 Transfer the cells to a 50 ml conical tube with a 11 disposable transfer pipette Gently rinse the chamber with electrofusion media to remove all the cells and transfer these cells to the 50 ml conical tube User s Manual Hybrimune Electrofusion Protocol Summary Overall Fusion Summary continued 12 13 Add selection media to fill the tube Incubate 10 to 20 minutes more minutes to allow fused cells to stabilize 14 Do not wash or centrifuge the cells prior to plating 15 Dilute the fused cells to the appropriate volume a division of Harvard Bioscience Inc for plating 16 Plate the fused cells and incubate at 37 C BTX Harvard Apparatus a i 84 October Hill Road Staining optional QC step Holliston MA 01746 USA 1 Dilute
65. s the suspension will generally have a conductivity of 100 to 200 uS cm T Hybrimune Hybridoma Production System User s Manual ae toll free 800 272 2775 508 893 8999 e www btxonline com 14 3 3 1 Cell alignment Dielectrophoresis continued F is the characteristic of the chamber used The character is the mathematical operator to differentiate with respect to spatial volume It is important to note that the E is the electric field vector and the electric field intensity is squared Thus the force on the cell is always in one direction the force does not reverse direction when a sinewave voltage waveform goes from plus to minus If the electric field Is uniform then E 0 Thus with uniform electric fields such as those produced in a cuvette parallel plate the force is zero There have been publications claiming to have fused cells in a cuvette The geometry of the chamber and the cell radius are the two major elements that determine the force on the cells Non uniform fields can be produced in a number of ways by two parallel wires by a wire and a plate etc The optimum chamber is two coaxial cylinders called a coaxial chamber The mathematical model of a coaxial chamber is given by the following see Pohl Chapter 7 Chamber dimension definitions are given in Figure 3 8 V V In Ir bl V varies with rg the radial E Ae position from a to b in the In la b chamber gap V o E 2 E field varies with rg
66. s undisturbed e Harvest cells from the chamber using a sterile disposable transfer pipette e Flush chamber with culture medium to remove cells that have settled to bottom e Add cells to 50 ml conical tube and fill tube to 40 ml mark with complete culture medium e Allow cells to rest an additional 25 minutes e Dilute the cells to an appropriate volume for plating e The cells can be immediately plated and cultured without washing 5 4 2 Materials Needed for Post Electrofusion Culture 5 4 2 1 Cloning Medium 1 DMEM 20 Fetal Bovine Serum 10 Origen Supplement Note this supplement is no longer commercially available A filtered supernatant from J774A 1 cells can substitute 1 OPI Supplement Sigma 05003 Penicillin Streptomycin 1 X Sodium pyruvate 110 mg L Glutamax 50 uM 2 mercaptoethanol HAT diluted to 1X 5 4 2 2 Cloning Medium 2 RPMI 1640 HFCS 50X diluted to 1X Roche 11 363 735 001 HAT diluted to 1X 24 uM 2 mercaptoethanol Penicillin Streptomycin 1 X Sodium pyruvate 110 mg L Glutamax 5 4 2 3 Complete Growth Medium DMEM or RPMI 1640 10 Fetal Bovine Serum Penicillin Streptomycin 1 X 50 UM 2 mercaptoethanol Glutamax Sodium pyruvate 110 mg L 5 4 2 4 HAT Stock 50X Hypoxanthine 5 mM Aminopterin 2 mM Thymidine 0 8 mM 5 4 2 5 HT Stock 50X Hybrimune Hybridoma Production System Toll free 800 272 2775 508 893 8999 e www btxonline com 40 Hypoxanthine 5 mM Thymidine 0 8 mM
67. sh For instance myeloma cells are larger than B cells and may centrifuge differently Another advantage of combining the cells after the last wash is that the two cell populations may be stained with fluorescent dyes to differentiate the two cells after fusion and to identify fused cells Stained cells must be washed in separate washes to prevent bleeding of the dye from one population to the other during the washes While most Labs mix the cells in the last step others obtain better yields by mixing the cells before the first wash If the cells used in the electrofusion are immortalized tissue culture cells the myeloma cells the process is most efficient when cells in log growth phase are used Primary cells may not divide in tissue culture and therefore may not be obtained in log phase Hybrimune Hybridoma Production System toll free 800 272 2775 508 893 8999 www btxonline com 38 It is also advisable to do the washes immediately prior to electrofusion Electrofusion medium is non toxic but fusion efficiencies have been known to decrease if the total time in the Cytofusion medium pre and post fusion exceeds 30 minutes However 30 minutes is a sufficient amount of time to complete the process Generally cells should be mixed in a 1 1 ratio Any decrease in the myeloma cells to B cell ratio may result in decreased efficiency If one cell type is particularly rare such as antigen specific B cells then more myeloma cells
68. state E Hybrimune Hybridoma Production System User s Manual Ak toll free 800 272 2775 508 893 8999 e www btxonline com 2 Table of Contents SUBJECT 1 Introduction 2 Items of Particular Interest 3 Cell Electrofusion Tutorial 3 1 Applications 3 1 1 Hybridoma Production 3 1 2 Immunotherapy 3 1 3 Nuclear Transfer 3 2 Cell Fusion Methods 3 3 Electrofusion 3 3 1 Cell Alignment Dielectrophoresis 3 3 2 Waveforms for Electrofusion 3 3 2 1 Cell Alignment and Compression 3 3 2 2 AC Sinewave Waveforms 3 3 2 3 Electrofusion Pulse 3 3 2 4 Post Fusion Pulse Sinewave 3 3 2 5 Parameters Range of HybrimuneTM Waveform Generator 3 3 2 6 Published Waveform Protocols 4 Hybrimune System Components and Set up 4 1 Introduction24 4 2 The Hybrimune Waveform Generator 4 3 The Large Volume Chambers 4 4 Cytofusion Medium 4 5 Setting up the Hybrimune System 4 6 Waveform Generator Front Panel Display 4 6 1 System Status Group 4 6 2 Process Status Group 4 7 Waveform Generator Interface Software Installation 4 7 1 Set up using a USB to Serial Converter 4 8 System Test 4 9 Elementary Verification Tests 4 10 The Waveform Generator Interface Software 4 10 1 The Tool Bar 4 10 2 Tools Area 4 10 3 Status Area 4 10 4 Last Protocol Log Window 4 10 5 Control Panel Area 4 10 6 Running a Protocol 4 10 7 File Management 4 11 Programming Waveform Protocols 4 11 1 Programming the Protocol 2417 1718 18 23 18 19 19 22 22 22 22 23 24 24 2
69. stem for a given cell radius are the chamber configuration and the voltage applied to the chamber to produce the electric field In the last section the chamber parameters were discussed In this section the voltage waveform parameters will be discussed There are two types of waveforms used alternating current AC in the form of a sinewave and pulsed direct current DC The functions of these waveforms in the electrofusion process are Function Waveform Type Typical Cell alignment AC sinewave Non linear and compression increasing amplitude Fusion DC Pulse One short pulse Linear decreasing amplitude Fusion partner AC sinewave stabilization Notes 1 There have been optimized protocols that use waveforms other than those shown as typical for example see the Trevor et al 2004 2 There are many types of AC waveforms rectangular square trapezoid triangle and sinewave Hybrimune technology uses sinewave AC waveforms due their smooth amplitude transition User s Manual Cell Electrofusion Tutorial 3 3 2 Waveforms for Electrofusion continued 3 3 2 1 Cell Alignment and Compression Section 3 3 1 described the method of aligning cells using dielectrophoresis There were two important parameters the chamber configuration and the voltage amplitude The chamber configuration has been optimized and is fixed However the voltage parameters can be set by the user The objecti
70. t indicate which cell lines are better suited as fusion partners for the species indicated It is meant to provide a starting point for finding a good fusion partner 5 1 2 1 Human e U 226AR e GM1500GTGALZ2 Coriell Institute GMO4672 e HuNS1 ATCC CRL8644 e SHM D33 ATCC CRL 1668 e KR 12 ATCC CRL8658 Derived from GM1500 5 1 2 2 Mouse IgG secreting mouse myeloma cells e MOPC 21 e P3X63AG8 ATCC TIB9 Non antibody mouse secreting cells some produce light chains e SP2 0 Ag14 ATCC CRL 1581 e NS 1 ATCC TIB18 e P3 X63AG8 653 ATCC CRL 1580 e FO ATCC CRL1646 e 194 5 XXO BU 1 ATCC CRL 1580 TIB20 e FOX NY ATCC CRL 1732 5 1 2 3 Rat e 210 RCY3 AgI 2 3 5 1 2 4 Rabbit e 240E1 1 Spieker Polet 1995 Hybrimune Hybridoma Production System toll free 800 272 2775 508 893 8999 e www btxonline com 36 5 1 3 B Cells Preparation of B cells is more complicated The general steps are in vivo or in vitro immunization collection and purification of B cells and optional activation of B cells There are many ways to do this series of procedures and all of them will not be covered 5 1 4 Immunization One immunization procedure follows Immunize mice every 3 4 weeks The first Immunization can use complete Freund s adjuvant Subsequent immunizations should employ other adjuvants such as incomplete Freund s adjuvant Other more modern adjuvants can be used Bleed the mice periodically to det
71. t when the start bar becomes active e Observe orderly alignment of cells e Next add 50 ml PBS to the cells in the chamber e Click turn on HV Pulse then Start when the start bar becomes active e Observe turbulence and that the current indicator shows red bars lit 5 7 Optimization The protocol presented on page 3 17 is a good starting point for producing hybridomas by electrofusion using the Hybrimune System The cells from each lab probably have a different optimum The factors that most dramatically affect fusion efficiency are e Pre pulse sine wave amplitude and duration e Pulse voltage and pulse width The pre pulse sine wave second step amplitude is 75 volts peak If there is excessive turbulence it may be reduced slightly Decrease the duration until the turbulence cannot be visually detected Another function such as Ramp k may also improve performance Next try decreasing pulse voltages and then increase pulse width in steps of 10us Unless the post pulse sine wave Is causing excessive movement of the cells changing that parameter will have minimal effect on the results EY Producing Fusion Products Using Hybrimune 5 8 Chamber Cleaning Chamber cleaning Is necessary to remove biological contaminants and ions The electrofusion process is sensitive to the presence of ions Clean chambers are essential to prevent excess ion contamination Cleaning Process The following cleaning process is recommend
72. ters are widely avallable however they are not available from BTX See section 4 7 1 for more information BTX HARVARD APPARATUS DOES NOT GUARANTEE THAT A USB TO SERIAL CONVERTER IS 100 APPLICABLE THE USE OF AN RS 232 SERIAL INTERFACE IS RECOMMENDED Line Mains Power Cord Plug this cord into the power input receptacle labeled Line Mains In Plug the other end into an outlet supplying suitable electric power Chamber Connection Cable There are two connections made with this cable assembly Using the figures shown in the Chamber Cable Connections area of the back panel connect the SHV type coaxial plug to the jack labeled Waveform Out Then connect the RCA type phono plug to the jack labeled Chamber Enable Generally this cable only needs to be connected at the time that a chamber is to be used 4 6 Waveform Generator Front Panel Display There are eight status indicators on the front panel divided into two groups Before powering up the system the user should become familiar with them 4 6 1 System Status Group The function of this group is to inform the user of the overall state of the waveform generator when it is powered up This group consists of four LED light emitting diode indicators Hybrimune Hybridoma Production System Toll free 800 272 2775 508 893 8999 e www btxonline com Power green LED Indicates that the internal power supplies and the system microprocess
73. the 9 ml Batch Production Chamber The causes of turbulence if it is observed are listed below e High current due to high ion content Dirty electrode Contaminated or high conductivity medium Inadequate cell wash e Excessive force applied to cells AC amplitude too high AC amplitude too long EY Producing Fusion Products Using Hybrimune Hybrimune Hybridoma Production System toll free 800 272 2775 508 893 8999 e www btxonline com 39 On the front of the Hybrimune voltage waveform generator there is a bar graph labeled Relative Load Indicator It will illuminate bars during waveform delivery depending on a number of factors See Sections 4 6 2 and 4 9 Generally the Load Indicator should have green and possibly yellow lights illuminated during sine wave delivery If the indicator shows red bars illuminated then the current is too high and aqueous heating and turbulence can occur 5 3 2 Fusion Pulse The next step after aligning and compressing the cells is to apply one or more high voltage pulses to induce pores in adjacent cell membranes followed by fusion of the same adjacent cell membranes The principles of pore formation for electrofusion are the same as those for pore formation for electroporation The difference is that in electrofusion the pores are forming in intimately touching cell membranes This is a short but essential step in the electrofusion process An example of an electrofusion pulse used for
74. to 100 million cells to allow direct comparison of differed fusion GB Appendix B Medarex Data Hybrimune Hybridoma Production System toll free 800 272 2775 508 893 8999 www btxonline com 49 Symbols used Y The immunoglobulin heavy chain for IgG antibody includes IgG 1 3 k One of two immunoglobulin light chains Y k The number of wells that contained IgG antibody of any specificity the number of wells with hybridoma clones secreting IgG antibody Ag Y The number of wells with hybridoma clones secreting IgG antibody with specificity for the antigen of interest Ag antigen the specific antigen used in the screening TT Tetanus toxoid The table shows the results of 12 experiments For electrofusion the average number of wells in the 12 experiments that produced IgG secreting hybridomas was 542 Of those 91 bound to the antigen of interest For the PEG fusions the average number of wells in the 12 experiments that produced IgG secreting hybridomas was 58 and 12 of those were antigen specific This means that on the average electrofusion produced approximately 9 times as many IgG producing hybridomas and 8 times as many antigen specific hybridomas However there were many cases where E fusion produced results and PEG did not User s Manual GB Appendix B Medarex Data Experimental Results Experiment Ag E fusion PEG Fusion k Ag
75. variables under user control The user can control force by changing amplitude In addition the duration of the AC waveform applies the force for a given period of time which is also under user control Frequency is available but Figure 3 7 says It has little effect Waveforms are described in Section 3 3 2 A graph is presented in Figure 3 10 which presents the approximate force applied to a cell as a function of cell radius of 5 10 and 20 um as a function of AC peak voltage The AC peak voltage range is that provided by the Hybrimune waveform generator The effect of the cell radius cubed is quite dramatic This force Is on a cell in isolation Figure 3 10 Force on a Cell Cell Electrofusion Tutorial Hybrimune Hybridoma Production System toll free 800 272 2775 508 893 8999 e www btxonline com 17 In summary the design of the Hybrimune System has been optimized for electrofusion and many of the variables are fixed by system design such as the chamber configuration and the conductivity of the medium The one powerful tool available to the user is the selection of the waveform protocol The waveform protocol sets the force on the cells for alignment and compression the pulse to fuse the cells and the waveform to stabilize the fused cells after the pulse The next section covers the available waveforms 3 3 2 Waveforms for Electrofusion The two most important metrics in an electrofusion sy
76. ve of this step in electrofusion is to align the cells in an optimum manner and then apply the fusion pulse There are a number of important considerations 1 A perfect alignment process would alternate the two cell types to be fused In general that does not occur in any known process The way the cells align is a statistical event If the cells all have the same radius and equal numbers of each are mixed then see Figure 3 11 XY 25 YX 25 XX 25 YY 25 XY YX 50 Figure 3 11 Cell Alignment Sequence In addition not all cells fuse in pairs some products have 3 4 or more nuclei This can be controlled but not eliminated by optimization of waveform amplitude A practical result of the stochastic nature of cell alignment is that the maximum yield is obtained when the percent of each cell population is 50 shown above However Hybrimune Hybridoma Production System toll free 800 272 2775 508 893 8999 www btxonline com 18 A practical result of the stochastic nature of cell alignment is that the maximum yield is obtained when the percent of each cell population is 50 shown above However when 3 cells of X are present for every one cell of Y 75 25 then the following yield is obtained YX 19 XY 19 XX 56 YY 6 XY YX 38 Unequal ratios of cells are sometimes used to conserve a rare cell population However when this is done the total yield decreases As show
77. whether or not the chamber cable is connected and ready to receive pulses OK The chamber cable is connected the safety interlock is satisfied Open The chamber cable is not connected the safety interlock is not satisfied High Voltage Displays the state of the internal high voltage power supply HVPS On The power supply is on and the reservoir capacitor is charging Off The power supply is off Status This box shows the current functional status It displays Ready The system Is ready to accept instructions to begin a protocol Download The computer is loading protocol to the internal microprocessor Charging The system is charging the reservoir capacitor to the set voltage Pulsing The protocol is being delivered to the chamber Monitors Power Supply Voltmeter Displays the voltage of the reservoir capacitor not the pulse amplitude This display is operational at all times except when pulses are being delivered During charging Displays the voltage updated every second or so After charging Displays the final voltage on the reservoir capacitor W Generator at reset Displays the system rest voltage 4 10 4 Last Protocol Log Window The window at the bottom center of the screen is the Last Protocol Log window The window displays the last protocol executed 4 10 5 Control Panel Area The Electroporation Mode Control Panel Area in the center of the screen is where the pulse protocol
78. ys EY Producing Fusion Products Using Hybrimune Hybrimune Hybridoma Production System toll free 800 272 2775 508 893 8999 www btxonline com 37 5 2 Cell Preparation on Electrofusion Day 5 2 1 Medium BTX Cytofusion Medium must be used for the fusion Establishing the conductivity and purity of this medium was part of the system design process The medium was developed over a two year period and has been used in commercial applications since 2003 The medium is produced in the BTX facility under strict quality system including mixing bottling and labeling Each batch is full tested for pH endotoxin conductivity sterility and other quality control parameters One example of a poor medium would be one containing excessive ions due to the addition of inorganic acids or bases in order to adjust the pH The presence of the ions will increase conductivity enough to result in excessive heat that will kill the cells Cells need to be in a high resistance low conductivity medium for several reasons One is that the cell alignment step dielectrophoresis is dependent upon there being a difference between the conductivity of the cell cytoplasm and the conductivity of the medium outside a cell See Figure 3 7 page 3 6 Second the high current generated in a highly ionic media will cause excessive heating The heating will at first cause convection currents and disrupt the cell alignment process If the heating pers

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