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Hybrimune

™

Hybridoma

Production System

USER’S MANUAL

Publication 015-100191 REV 4.0

Содержание BTX Hybrimune

Страница 1: ...Hybrimune Hybridoma Production System USER S MANUAL Publication 015 100191 REV 4 0 ...

Страница 2: ...rt team constantly tracks published literature for any reference to electroporation and electrofusion We extract the pertinent experimental conditions and yields from these papers to help us in our efforts to help you In addition to tracking publications we are available to you for support at any time for advice in experimental design product recommendations troubleshooting and any other relevant ...

Страница 3: ...est 12 13 Elementary Verification Tests 14 The Waveform Generator Interface Software 14 The Tool Bar 15 Tools Area 15 Status Area 15 16 Last Protocol Log Window 16 Control Panel Area 16 17 Running a Protocol 17 File Management 17 Programming Waveform Protocols 18 Programming the Protocol 18 Producing Fusion Products Using Hybrimune 19 26 Cell Preparation Before Electrofusion Day 19 Myeloma Cells 1...

Страница 4: ...tion Experiment to Illustrate Turbulence 26 Optimization 26 Chamber Cleaning 26 Hybrimune Electrofusion Protocol Summary 27 28 Cell Electrofusion Tutorial 29 43 Applications 29 Hybridoma Production 29 Immunotherapy 30 Nuclear Transfer 30 Cell Fusion Methods 31 Electrofusion 32 Cell Alignment Dielectrophoresis 32 37 Waveforms for Electrofusion 37 43 Cell Alignment and Compression 38 39 AC Sine Wave...

Страница 5: ...ncer Center Fusion Example 30 Figure 9 Electrofusion Example 30 Figure 10 Nuclear Transfer Example 30 Figure 11 Uniform Field and Non Uniform Fields 32 Figure 12 Cell Alignment 32 Figure 13 Clausius Mossotti Function 34 Figure 14 Chamber Definitions 35 Figure 15 Cell Alignment in Optimization Chamber 36 Figure 16 Force on a Cell 37 Figure 17 Cell Alignment Sequence 38 Figure 18 Sine Wave Definitio...

Страница 6: ...ithin the warranty period promptly contact BTX 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 support hbiosci 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 charg...

Страница 7: ...in accordance with local regulations and practices This product should not be used in the presence of a flammable atmosphere such as an anesthetic mixture with air oxygen or nitrous oxide Please read the following safety precautions to ensure proper use of your generator If the equipment is used in a manner not specified the protection provided by the equipment may be impaired To Prevent Hazard or...

Страница 8: ... BTX Cytofusion Medium C is a required component of the Hybrimune system Other ionic medium should not be used it will generate significant heating and kill cells 6 The chamber must be cleaned after each use to prevent ionic build up which will cause heating kill cells and reduce efficiency Please refer to chamber cleaning procedure page 27 The Hybrimune Voltage Waveform Generator The waveform gen...

Страница 9: ...ber 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 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 or...

Страница 10: ...ic 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 Fi...

Страница 11: ...ve 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 70 Vpk 1 MHz sine wave is delivered to a 47 0020 large volume fusion chamber Chamber filled with 9 ml of a 100 µS cm Conductivity Standard at 25 C Waveform Generator Interface Software Installation The Hybrimune waveform generator interface s...

Страница 12: ...replacement of the fuse and the correct fuse type 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 What should happen The screen should appear like that as shown in Figure 5 The CommLink window should show OK This display indicates that the compute...

Страница 13: ...ow Figure 6 The Last Protocol Log for the TEST pro Run Protocol Information Filename if applicable Date MM DD YY DoW Time 24 Hour Clock System Mode Delivered Waveform Parameters Subdivided by Group Number Where GRP Pulse Group Dur AC duration sec Freq AC Frequency MHz BegV Start AC Amplitude EndV Stop AC Amplitude volts NUM Pulse in Group WIDTH Pulse Duration msec INTVL Time between pulses sec Set...

Страница 14: ... from several places on the screen The default installation directory is C Program Files BTX Hybrimune It will be different if you have selected another directory during installation 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 System Status area right see Figure 5 W...

Страница 15: ...equently 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 The Toolbar Across the top left of the main window is a list of standard Windows pull down menus File Pull down Menu Open Opens a previously saved protocol Save Saves the current protoco...

Страница 16: ...he 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 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 Control Panel Area The Electroporation Mode Control Panel Area in the center of the scree...

Страница 17: ...ser 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 Start or Alt S Clicking Start begins delivery of the protocol to the test sample The pulse groups are exec...

Страница 18: ...protocol will be demonstrated 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 To program Enter the values for Group 1 in the user interface Click Add Enter the values for Group 2 in the user interface Click Add Save as BTX This can also b...

Страница 19: ...age pathway Thus the chemicals thymidine and hypoxanthine are provided as source 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 from the B cells and imm...

Страница 20: ...ws Immunize mice every 3 to 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 determine if they are producing polyclonal antibody to the antigen of interest Three days before harvesting spleen cells a final boost of antig...

Страница 21: ...pleen cells in complete culture medium Re suspend cells at 1 million cells ml in complete growth medium plus 2 4 µg ml PHA L Place cells in T 75 flask and incubate at 37 C 5 CO2 for 3 days Cell Preparation on Electrofusion Day 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 develop...

Страница 22: ...lls 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 It is also advisable to do the washes immediately prior to electrofusion Electrofusion medium is non toxic but fusi...

Страница 23: ... in the 9 ml Batch Production Chamber The causes of turbulence if it is observed are listed below High current due to high ion content Dirty electrode Contaminated or high conductivity medium Inadequate cell wash Excessive force applied to cells AC amplitude too high AC amplitude too long On the front of the Hybrimune voltage waveform generator there is a bar graph labeled Relative Load Indicator ...

Страница 24: ...cal tube and fill tube to 40 ml mark with complete culture medium Allow cells to rest an additional 25 minutes Dilute the cells to an appropriate volume for plating The cells can be immediately plated and cultured without washing Materials Needed for Post Electrofusion Culture Cloning Medium 1 DMEM 20 Fetal Bovine Serum 10 Origen Supplement Note this supplement is no longer commercially available ...

Страница 25: ... for antibody screening Re feed with 150 ml complete medium with HAT medium 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 Methods of Analysis The most common method of analysis is to determine the num...

Страница 26: ...e 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 10 µs Unless the post pulse sine wave is causing excessive movement of the cells changing ...

Страница 27: ...ncy Computer Preparation 1 Turn on the computer 2 Turn on the BTX Hybrimune system 3 Load the Hybrimune PulseAgile program from the Start button in Windows or shortcut from the desktop 4 Click the electrofusion button 5 Load the optimized protocol for this set of conditions Found under the file menu Overall Fusion Summary 1 Sterilize the hood 2 Clean and sterilize the electrode 3 Turn on computer ...

Страница 28: ... dry a couple of minutes 5 Dunk it into fresh 95 ethanol 5 seconds 6 Allow to air dry 7 Place the slide in the Wright s Giemsa for 1 minute 8 Dunk the slide up and down in the first tube of DI water to remove most of the stain 9 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 10 Knock the side of the slide on p...

Страница 29: ...n 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 ...

Страница 30: ... Katrina Trevor Arizona Cancer Center Figure 9 was produced in the laboratory by fusing an immune system cell and a tumor cell The image was taken 45 minutes after electrofusion Figure 8 Arizona Cancer Center Fusion Example 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 ...

Страница 31: ...h these steps The following table summarizes some of these 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 ce...

Страница 32: ...l 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 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...

Страница 33: ...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 Th...

Страница 34: ...une 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 µS cm After adding properly washed cells the suspension will generally have a conductivity of 100 to 200 µS cm Clausius Mossotti Function cell radius 7 um Frequency MHz 1 000 0 800 0 600 0 400 0 200 0 000 0 200 0 400 0 600 0 800 1 000 0 001 0 01...

Страница 35: ...on the cell is always in one direction the force does not reverse direction when a sine wave voltage waveform goes from plus to minus If the electric field is uniform then E2 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 a...

Страница 36: ...try 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 Parameter Optimization Chamber Batch Production Chamber Volume 2 ml 9 ml Outer Inside Radius b 22 86 mm 22 86 mm Inner Outside Radius a 19 05 mm 19 05 mm Gap 3 81 mm 3 81 mm Well Height 5 mm 18 mm a b 0 8333 0 8333 Figure 15 Cell Alignment in Optimi...

Страница 37: ... for example see the Trevor et al 2004 2 There are many types of AC waveforms rectangular square trapezoid triangle and sine wave Hybrimune technology uses sine wave AC waveforms due their smooth amplitude transition AC Waveform Amplitude The AC waveform amplitude and duration are the only variables under user control The user can control force by changing amplitude In addition the duration of the...

Страница 38: ...e 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 decrease...

Страница 39: ...kely 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 6 Care has to be taken with the amount of time the AC sine wave 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 wh...

Страница 40: ...nt system LINEAR RAMP AMPLITUDE SET BY RAMP K 1 This function appeared on the original Zimmerman system a number of years ago 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 Hybrimune Interface Software Figure ...

Страница 41: ...gure 20 for a Start Amplitude of 20 volts a Stop Amplitude of 50 volts and duration of 10 seconds Ramp k 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 AC Amplitude t AC Duration ...

Страница 42: ...in 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 Post Fusion Pulse AC Sine Wave 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 sine wave causes...

Страница 43: ...s 0 8 MHz 75 volts peak 30 seconds 75 volts peak 10 seconds Linear Step Step Pulse Amplitude 800 volts 800 volts 800 volts Pulse Width 40 µs 40 µs 40 µs Pulse Number 1 1 4 Pulse Interval NA NA 0 125 seconds AC Post Fusion 60 to 5 volts peak 30 seconds 1 4 MHz 60 volt peak 30 seconds Freq unknown 45 volts peak 55 seconds 0 8 MHz AC Post Fusion Linear Constant Constant Waveform Parameter Value AC Be...

Страница 44: ... 1 to 10 groups Maximum Separation Pre Pulse AC End to Pre Pulse AC start 1 ms Maximum Separation Post Pulse AC End to Post Pulse AC Start 1 ms Maximum Separation Pre Pulse AC End to Pulse Start 10 ms Maximum Separation Pulse End to Post Pulse AC Start 20 ms Electrical and Mechanical Current Ratings 2A Input Voltage Frequency Ratings 100 to 240 VAC 50 60 Hz Dimensions HxWxD 6 5 x 12 x 16 in 16 5 x...

Страница 45: ... 0 8333 0 8333 Overall Dimensions 13 5 cm x 7 7 cm x 2 2 cm 11 cm x 10 cm x 5 cm Weight 210 g 7 oz 270 g 9 oz Operating temperature 20 to 30 C Storage temperature 0 to 50 C Cytofusion Medium Formula C Parameter Tolerance Sterility Sterile filtered no growth on random samples using four bacterial test media pH 7 2 0 2 Conductivity 80 µS cm 5 µS cm Endotoxin 0 2 IU ml Osmolarity 270 to 290 milliOsmo...

Страница 46: ...g into other parts of the cabinet Put a small amount of cleaner on the towel and gently rub the screen or enclosure Avoid hard rubbing abrasives or harsh solvents like alcohol or ammonia Fuse Replacement 1 Make sure the power cord is disconnected from the main supply before servicing the fuse 2 Replace fuse with 250V 2A slo blo 5 x 20 mm Turn Off power and remove power cord from power module Use a...

Страница 47: ...fusion Generator Only 1 Ea Chambers 47 0020 9 ml Coaxial Chamber for Production 1 Ea 47 0030 2 ml Coaxial Chamber for Optimization 1 Ea Item No Description Qty Accessories 47 0001 BTXpress Cytofusion Medium C 500 ml Volume 1 Ea 47 0302 Hybrimune Chamber Cable 1 Ea 47 0301 Hybrimune User Interface Application Software 1 Ea Service Offerings 45 9996 Hybrimune 1 Year Extended Warranty 1 Ea Ordering I...

Страница 48: ...oclonal antibodies J Immunol Methods 2008 Jul 336 2 142 151 Niedbala WG Scott DI A comparison of three methods for production of human hybridomas secreting autoantibodies Hybridoma 1998 17 299 304 Neil GA Zimmerman U Electrofusion Methods Enzymol 1993 220 174 196 O Shannessy DJ et al Novel antibody probes for the characterization of endosialin TEM 1 Oncotarget 2016 Oct 7 43 69420 69435 Schmidt et ...

Страница 49: ... 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 to 100 million cells t...

Страница 50: ...Experiment Ag E fusion PEG Fusion t g k Ag g g k Ag g 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 1100 400 83 23 6 TT 582 151 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 5 323 0 47 0 12 Ag 5 246 0 36 0 Appendix B Medarex Data Appendices ...

Страница 51: ...ement Relief Meets the 200 hour requirement Resistivity Decreased 25 Magnetic Characteristics Permeability reduced 20 Adhesion to Base Metal No separation occurred Biological Compatibility USP Class VI Certification Acute Systemic Toxicity No systemic toxicity Intracutaneous Toxicity No localized tissue reaction Surgical Muscle Implantation No irritation to human tissue ISO Tripatite Testing Cytot...

Страница 52: ...US Canadian Customers phone 508 893 8999 fax 508 429 5732 e mail support hbiosci com website www btxonline com International Customers To locate a distributor near you please visit our website at www btxonline com ...

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