Harvard Bioscience BTX Hybrimune User manual
Hybrimune™
Hybridoma
Production System
USER’S MANUAL
Publicaon 015-100191 REV 4.0
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Hybrimune Hybridoma Producon System
Publicaon 015-1010191 Rev 4.0 • www.btxonline.com
Since its founding in 1983, the main focus of BTX has been in the area of applying
controlled electric elds for genec engineering applicaons. Because of this,
we quickly established a reputaon as the technological leader in the elds of
electroporaon and electrofusion. Our systems have been installed in many
presgious instutes around the globe where they are used successfully for high
eciency transfecon, transformaon and cell fusion applicaons. We oer a
variety of waveforms, electrodes and chamber opons to provide you with the
best tools to achieve your goals.
We are vested in your success. To that end, the BTX technical support team
constantly tracks published literature for any reference to electroporaon and
electrofusion. We extract the pernent experimental condions and yields
from these papers to help us in our eorts to help you. In addion to tracking
publicaons, we are available to you for support at any me for advice in
experimental design, product recommendaons, troubleshoong, and any other
relevant technical advice.
We thank you again for your investment and we look forward to assisng you in
any way we can.
Finally, please read this manual carefully before aempng to operate the
electroporaon system. If you have any quesons about the unit or about
parcular applicaons, please contact us:
BTX
84 October Hill Road
Holliston, MA 01746 USA
Toll Free: 1-800-272-2775
Internaonal Callers: 508-893-8999
Fax: 508-429-5732
Web: www.btxonline.com
Email: [email protected]
For any customers outside the US or Canada, please call your local BTX dealer or
call us directly.
A Message from BTX
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Hybrimune Hybridoma Producon System
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SUBJECT PAGE #
6
7
8–18
Introducon 8
The Hybrimune™ Waveform Generator 8
The Large-Volume Fusion Chamber 9
Cytofusion® Medium 9
Seng up the Hybrimune™ System 10
Waveform Generator Front Panel Display 10
System Status Group 10
Process Status Group 11
Waveform Generator Interface Soware Installaon 11
Set-up using a USB-to-Serial Converter 11
System Test 12–13
Elementary Vericaon Tests 14
The Waveform Generator Interface Soware 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
19–26
Cell Preparaon Before Electrofusion Day 19
Myeloma Cells 19
Available Myelomas or Heteromyelomas 20
Human 20
Mouse 20
Rat 20
Rabbit 20
B-Cells 20
Immunizaon 20
Screening 20
Harvesng Spleen Cells 21
In Vitro Acvaon 21
Cell Preparaon on Electrofusion Day 21
Medium 21–22
Chamber Cleaning 22
Washes and Other Preparatory Needs 22
Cell Preparaon Process 22
Table of Contents
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Hybrimune Hybridoma Producon System
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SUBJECT PAGE #
Electrofusion 23
Pre-Fusion Pulse AC 23
Fusion Pulse 23
Post-Fusion Pulse AC 23
Cell Handling Aer Electrofusion 24
Immediate Post-Fusion Care of Cells 24
Materials Needed for Post Electrofusion Culture 24
Cloning Medium 1 24
Cloning Medium 2 24
Complete Growth Medium 24
HAT Stock (50X) 24
HT Stock (50X) 24
Method, Culture of Cell and Clone Selecon 25
Culture of Cells 25
Feeding Cells 25
Cloning Cells 25
Weaning Clones 25
Method of Analysis 25
Demonstraon Experiment to Illustrate Turbulence 26
Opmizaon 26
Chamber Cleaning 26
Hybrimune Electrofusion Protocol Summary 27–28
29–43
Applicaons 29
Hybridoma Producon 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 Waveforms 39–41
Electrofusion Pulse 42
Post Fusion Pulse Sine Wave 42
Parameters Range of Hybrimune Waveform Generator 42
Published Waveform Protocols 43
44–45
46
Fuse Replacement 46
47
48
49–51
Appendix A: Medarex Data 49–50
Appendix B: MEDCOAT 200 Summary Test Results 51
Table of Contents
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Table of Contents
PAGE #
8
9
9
10
12
13
29
30
30
30
32
32
34
35
36
37
38
39
40
41
42
46
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Hybrimune Hybridoma Producon System
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Warranty Information
BTX
84 October Hill Rd
Holliston, MA 01746, USA
Phone: 1-508-893-8999
Fax: 1-800-429-5732
Web: www.btxonline.com
BTX warranes Hybrimune systems for a period of two years
from the date of purchase. At its opon, BTX will repair or replace
the product if it is found to be defecve as to workmanship or
materials. This warranty does not extend to any device which
has been (a) subjected to misuse, neglect, accident or abuse,
(b) repaired or altered by anyone other than BTX without BTX
express and prior approval, (c) used in violaon of instrucons
furnished by BTX. This warranty extends only to the original
customer purchaser.
. Some states do
not allow exclusion or limitaon of incidental or consequenal
damages so the above limitaon or exclusion may not apply to
you.
Some states do not allow this limitaon on an implied warranty,
so the above limitaon may not apply to you. Without liming the
generality of the foregoing, BTX 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 BTX Hybrimune system is changed in any
way from its original factory design or if repairs are aempted
without wrien authorizaon by BTX. Warranty is void if parts,
connecons or electrodes not manufactured by BTX are used
with the BTX Hybrimune instrument. If a defect arises within the
warranty period, promptly contact BTX, 84 October Hill Road,
Building 7, Holliston, Massachuses, USA 01746-1388 using our toll
free number 1-800-272-2775 (US Only) or 508-893-8999 (E-mail:
[email protected]). Goods will not be accepted for return
unless an RMA (Returned Materials Authorizaon) number has
been issued by our customer service department. The customer
is responsible for shipping charges. Please allow a reasonable
period of me for compleon of repairs, replacement and return.
If the unit is replaced, the replacement unit is covered only for the
remainder of the original warranty period dang from the purchase
of the original device. This warranty gives you specic rights, and
you may also have other rights, which vary from state to state.
Proceed exactly as for Warranty Service above. If our service
department can assist you by phone or other correspondence, we
will be glad to help at no charge.
Repair service will be billed on the basis of labor and materials.
A complete statement of me spent and materials used will be
supplied. Shipment to BTX should be prepaid. Your bill will include
return shipment freight charges.
Disassembly by the user is prohibited. Service should only be
carried out by experienced BTX technicians.
BTX stocks replacement and repair parts. When ordering, please
describe parts as completely as possible, preferably using our part
numbers. If praccal, enclose a sample photo or drawing.
The BTX Hybrimune systems are intended for laboratory use
only and can be used in research and development applicaons.
These systems have been designed to meet the standards for
electromagnec compability (EMC) and safety intended for
laboratory equipment applicaons.
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Hybrimune Hybridoma Producon System
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Caution Risk
of Electric Shock
Caution Protective
Ground Terminal
These instruments contain a high voltage power supply adjustable
to 1,000 V. High voltage power supplies present a serious risk of
personal injury if not used in accordance with design and/or use
specicaons, if used in applicaons on products for which they
are not intended or designed, or if they are used by untrained or
unqualied personnel.
• The user must read this manual carefully before the
instruments are placed into operaon.
• Removing the cover will 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 o and
unplug line (mains) cord.
• Do not touch the electrode p while the waveforms are
being applied.
• Do not under any circumstances turn the power switch o
while the system is pulsing; if an emergency occurs push the
emergency buon on the front panel
If there are any quesons about the operaon of this instrument,
call BTX Customer service at 1-800-272-2775, or 1-508-893-8999.
The BTX Hybrimune Systems are intended for laboratory use
only and can be used in research and development applicaons.
These systems have been designed to meet the standards for
electromagnec compability (EMC) intended for laboratory
equipment applicaons as well as the applicable safety
requirements for electrical equipment for measurement, control,
and laboratory use. The unit itself does not generate waste, but
may be used to treat samples that are hazardous. Please use
appropriate PPE and ensure disposal in accordance with local
regulaons and pracces.
Please read the following safety precauons to ensure proper
use of your generator. If the equipment is used in a manner not
specied, the protecon provided by the equipment may be
impaired.
If a problem occurs during a run, push the STOP/RESET buon on
the front panel.
Use only the specied line cord for this product and make sure line
cord is cered for country of use. The operang voltage range for
the Hybrimune is 100 to 240 VAC, 50/60 Hz.
This product is grounded through the grounding conductor of the
power cord. To avoid electric shock, the grounding conductor must
be connected to earth ground. Before making any connecons
to the input or output terminals of the product, ensure that the
product is properly grounded.
Make sure all connecons are made properly and securely. Any
signal wire connecons to the unit must be no longer than three
meters.
Review the operang manual to learn the rangs on all
connecons.
Use only specied fuses with product.
Do not touch any electronic circuitry inside of the product.
If damage is suspected on or to the product do not operate the
product. Contact qualied service personnel to perform inspecon.
Do not orient the equipment so that it is dicult to operate the
disconnecon device.
Review the operang manual for guidelines for proper operang
environments.
Read all labels on product to ensure proper usage.
Safety Information
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Hybrimune Hybridoma Producon System
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The Hybrimune™ Hybridoma Producon System was developed
for large volume commercial applicaons using cell electrofusion.
This includes hybridoma producon and tumor/dendric cell
immunotherapy. The system has not been approved for clinical or
in vitro diagnosc use. The system can fuse up to 180 million cells
in one process run. The Hybrimune is designed to be simple to set-
up and to operate. The system is shown in Figure 1.
The Hybrimune System is comprised of four components: the
Hybrimune Waveform Generator, the user-interface soware,
two large volume coaxial chambers (2 ml opmizaon chamber
and 9 ml producon chamber), and the low conducvity BTXpress
Cytofusion® Medium C in which the electrofusion takes place.
These components were all designed to operate as a system. Using
other components may damage the system and may result in
degraded performance. A computer is required for the applicaon
soware but is not included in the system.
The Hybrimune Hybridoma Producon System is the most
sophiscated system available for fusing dissimilar cells. The
system is available only under license directly from Cellecs.
Please note the following important informaon about the
system:
1. The System must remain in the possession of the licensee.
3. The System shall only be used as described in this User
Manual.
4. Certain concepts and processes are covered by BTX patents
and patents pending.
5. BTX Cytofusion Medium C is a required component of the
Hybrimune system. Other ionic medium should not be used;
it will generate signicant heang and kill cells.
6. The chamber must be cleaned aer each use to prevent
ionic build up which will cause heang, kill cells, and reduce
eciency. Please refer to chamber cleaning procedure,
page 27.
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 applicaon
soware. When the Start buon is clicked, the soware 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 eld. Depending
on the user-input instrucon, this microprocessor-controlled
waveform generator will generate:
• a low voltage,
megahertz frequency sinusoidal alternang
voltage that results in an alternang electric
eld that aligns and compresses the cells in a
process called dielectrophoresis
• a high voltage fusion pulse that
forms pathways in the cell membranes that
results in fusion
• a second
sinusoidal alternang electric eld that holds
the fused cells together while they mature
A detailed descripon of the waveforms is presented in page 37.
The parameter values available are listed on page 42. The User-
Interface soware provides the user with the ability to ne-tune
each of the three parts of the waveform. The instrucons on how
to program the waveforms may be found on page 18.
Figure 1: Hybrimune™ System
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The Hybrimune Waveform Generator connects to a coaxial
large-volume fusion chamber. The chamber produces the various
electric elds required in the hybridoma producon process.
These chambers were very carefully designed to provide opmum
alignment forces and intense electric elds necessary for ecient
electrofusion processes. When the alternang sinusoidal voltage
is applied to the inner and outer electrode rings of the chamber, a
highly non-uniform electric eld is established that causes the cells
to align into ”pearl chain” formaons.
Two types of chambers are provided. The rst is the 2 ml chamber
(le image in Figure 2) that holds a maximum volume of 2 ml. This
chamber permits light to enter in from the boom so that the
cell fusion process can be observed with a microscope. The 2 ml
chamber is used for process opmizaon. The second chamber is
the 9 ml chamber (right image in Figure 2) that holds a maximum
volume of 9 ml. This chamber is used for large batch producon.
The electrical characteriscs of the two chambers are equivalent.
The electrodes are coated with MEDCOAT 2000™, a chromium
coang by Electrolyzing Corporaon of Ohio.
BTX has developed a proprietary formula cell fusion medium
over several years that has under gone extensive tesng. The
conducvity of this medium is low in order to minimize electrical
current ow during the applicaon of the intense alternang
electric elds generated by the Waveform Generator. By liming
current ow, aqueous heang is minimized which improves fusion
eciency and viability. The medium is shown in Figure 3.
The Hybrimune Waveform Generator and the large-volume
fusion chamber were designed to work with low conducvity cell
suspensions. Do not use highly ionic buers in the chambers. If
ionic buers are used, two catastrophic problems will occur. First,
the cell suspension will rapidly heat causing cell death. Second,
signicant convecon currents will be generated in the suspension
that will inhibit cell alignment.
Figure 2: Large-Volume Fusion Chamber Figure 3: Cytofusion Medium C
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All of the cables necessary to install the equipment are included in
the Hybrimune System. They are:
Connect the serial cable between the
D-Subminiature 9-posion (DB9) connector labeled “Computer
Control” on the back of the unit and a serial port on the computer.
This is the communicaon link between the microprocessor within
the waveform generator and the computer. It is a standard RS-
232 serial communicaon. If only USB ports are available on the
computer, then a USB-to-Serial converter will be necessary. These
converters are widely available; however they are not available
from BTX. See next page for more informaon.
: Plug this cord into the power input
receptacle labeled “Line/Mains In”. Plug the other end into an
outlet supplying suitable electric power.
: There are two connecons made with
this cable assembly. Using the gures shown in the “Chamber Cable
Connecons” 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 me that a chamber is
to be used.
Figure 4: Cable Connections
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.
The funcon 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 eming diode) indicators.
• “Power”, green LED: Indicates that the internal
power supplies and the system microprocessor
are receiving adequate power. It should be
illuminated at all mes when the waveform
generator is turned on.
• “Ready”, green LED: Indicates that the
waveform generator is ready to deliver a
electrofusion waveform to an aached 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 operang condions, this LED
will not be illuminated.
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The funcon of this group is to inform the user of state of an
electrofusion protocol run. This group consists of three LED
indicators, and a mul-color bar graph.
• 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.
•
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 aer the
user clicks Turn HV On and remains illuminated during
the enre process run.
• Indicates that the
waveform generator is delivering an electrofusion
waveform to an aached Chamber. As the process runs,
the LED will alternate between a steady illuminaon and
a brief ashing 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 sequenally.
•
This indicator is a series of 10 LED
segments that provides an indicaon of the amount of
electrical current ow being delivered to the coaxial
chamber during waveform delivery. The number of
segments that illuminate is dependent on a number of
factors: buer conducvity, cell concentraon, cell
soluon temperature, the volume of cell soluon in the
chamber, the sine wave voltage and frequency being
applied, and the length of me the wave form is
applied. If many segments illuminate, then signicant
heat and turbulence is being generated in the coaxial
chamber, which will have a negave eect on the fusion
process. During the factory calibraon 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 lled with
9 ml of a 100 µS/cm Conducvity Standard at 25°C.
The Hybrimune waveform generator interface soware must be
installed on a local computer with these minimum specicaons:
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 rst. If the
soware 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 “{CDRomDriveDesignaon}:\setup”, then
click “OK”.
5. The setup program will begin. The default le
locaons are for the applicaon
and for protocol and log les.
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 compable with the system. The
following procedure was developed using the Dynex USB PDA/
Serial Adapter Cable (Model: DX-UBDB9) not currently provided
by BTX.
Use the manufacturer’s instrucons to install the converter’s
soware on the computer. Aer installaon, disconnect the
converter from the computer’s USB port. Connect the serial cable
between the D-Subminiature 9-posion (DB9) connector labeled
“Computer Control” on the back of the waveform generator
and the DB9 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 connecon.
Hybrimune™System Components & Set-Up
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The following is a basic test of the waveform generator system
using the preprogrammed protocol located
in the default \ protocol folder. For this inial test, the Chamber
Cable and Chamber should be fully connected, but it is not
necessary to ll the chamber.
• Plug in the Line/Mains power cord for the
waveform generator.
• Turn the Line/Mains Power rocker switch at the lower
le front of the front panel to the ON (I) posion
The rocker switch, “Power”, “Ready”, and
“Chamber Connected” LED’s should illuminate.
If the rocker switch fails to illuminate, then return it to
the OFF posion (O), and:
• Verify that the Line/Mains cord is properly
plugged into the receptacle on the back panel
and into the source of electric power.
• It may be necessary to check the fuse. See Maintenance
secon for instrucons, proper 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.
Start the Waveform Generator Interface soware.
The screen should appear like that as shown in
Figure 5. The CommLink window should show .
This display indicates that the computer is
communicang with the internal microprocessor of the
waveform generator. Addionally, the Electrofusion
Chamber window should display , the High Voltage
window should display, and the Status window
should say . The Power Supply Voltmeter window
will display 0 volts.
If ERROR appears in the CommLink window, then turn
o the Waveform Generator and check the serial cable
connecon to the waveform generator and computer
(or USB-to-Serial adapter, if applicable). Once
communicaon is established, the test may proceed.
• Click on the le folder icon on the le side of the screen
• Open the protocol le called
• Click Turn HV
Figure 5: User Interface Application Opening Screen
Hybrimune™System Components & Set-Up
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Hybrimune Hybridoma Producon System
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The protocol sengs 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 internal high voltage power
supply is now enabled and the reservoir capacitor is being charged. Aer approximately seven seconds the START
buon is enabled. The Power Supply Voltmeter window will display 500 ± 5 volts.
• Click Start
The waveform generator runs the protocol. The green “Process Running” LED will illuminate, cycling through
its display phases. The “Relave Load Indicator” display will not illuminate since there is no conducve
medium in the chamber. A protocol log as shown in Figure 6 on the next page should appear in the Last
Protocol Log window.
Figure 6: The Last Protocol Log for the TEST.pro Run
Protocol File: HYBRIMUNE TEST.PRO
Date: 1-03-2019-4 7:52:24
System Serial Number: 08-9937HY
Firmware Version: 5.81
Mode: Electrofusion
Delivered Waveform Parameters
>Pre-Sine
>GRP Dur Freq BegV EndV
>1 5.0 1.4 50 50
>Pulsing
>GRP NUM WIDTH INTVL SetV MonV
>1 1 0.100 0.50 500 500
>1 2 0.100 0.50 500 500
>1 3 0.100 0.50 500 500
>1 4 0.100 0.50 500 500
>1 5 0.100 0.50 500 500
>Post-Sine
>GRP Dur Freq BegV EndV
>1 5.0 1.4 50 50
>Normal Compleon
$
Log: C: \My Documents\Harvard Apparatus\Hybrimune\ log\
PulseAgile 2006-06-27 15_52_44.txt
>CPS System v5.81
Hybrimune™System Components & Set-Up
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Hybrimune Hybridoma Producon System
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Set the AC waveform to start and stop 70 volts,
the duraon 10 seconds and the frequency to
2 MHz. All bars should be illuminated. This test
demonstrates the eects of frequency.
Although the conducvity of the medium in the
chamber is sll the same as Test 1, since the
chamber is also a capacitor, at 2 MHz it begins
to look like a short circuit to the Wave Form
Generator. Therefore a signicant amount of
current will begin to ow. In general,
frequencies above 1 MHz should not be used.
The Waveform Generator Interface conforms to standard
Microso Windows® convenons and this manual assumes that
the user is familiar with Windows®. The soware performs the
following:
The following secons describe operaon based on the above list.
Some things to remember:
• Some commonly used funcons have
redundant control features, i.e. they can be
accessed from several places on the screen.
• The default installaon directory is
.
It will be dierent if you have selected another
directory during installaon.
• The screen is divided into four areas within the
main window. The Title Bar and pull down
menus (top), the Tool buons (le), the
Control Panel for each operang mode (center),
and the System Status area (right), see Figure 5.
• When the electrofusion chamber cable is not
aached 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.
There are two elementary tests that can be run to determine if the
AC waveforms are operang properly:
Fill the 47-0020 large-volume fusion 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 duraon to 10 seconds and the frequency
to 1 MHz. The LED bar segment display should
show between one and four bars. This test shows:
• The cable between waveform generator
and chamber is OK.
• The conducvity in the chamber is OK.
• The AC waveform is working properly.
T EST
TES T
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Tools Area
The Tools area is found on the le side of the screen and contains
a set of frequently used funcon buons. These funcons can also
be accessed from the Toolbar as described above.
Opens a protocol.
Protocol “Save As…” Saves a
protocol under a user specied lename.
Prints the Last Protocol Log or
the Log History (user selectable) to a le or printer.
Opens the
Electric Field Converter applicaon.
The Microso Windows®
Calculator.
Saves the protocol Log
under a user specied lename.
Exits the program.
This area at the upper right corner of the screen displays up-to-date
informaon about the system condions.
This feature shows that there is proper internal connecon of the
system when the box labeled Electrofusion is checked.
This secon shows the current system status. These condions
frequently change during operaon.
This box displays the status of the
RS-232 serial communicaons link.
Communicaons is established and funconal.
Error: There is a problem with the
communicaons link.
Across the top-le of the main window is a list of standard
Windows® pull-down menus.
Opens a previously saved protocol.
Saves the current protocol, overwring
the le if it had been previously saved.
Saves the current protocol.
The user is prompted for a lename.
Saves the protocol Log. The user
is prompted for a lename.
Prints the Last Protocol Log.
Exits the program.
Applicaon to help
convert between absolute voltages and
electric eld.
The Microso Windows®
calculator.
Runs the open protocol without
addional user input. Pressing F12 achieves the
same result. The soware acvates Turn HV ON
and START. May be used to run the same
protocol repeatedly. A protocol must be open
for this to work.
General opons to set sound mode,
auto save, provide communicaons check, etc.
Shows the BTX logo, soware version,
and phone numbers.
Hybrimune™System Components & Set-Up
16
Hybrimune Hybridoma Producon System
Publicaon 015-1010191 Rev 4.0 • www.btxonline.com
This box displays whether or not the
chamber cable is connected and ready to
receive pulses.
The chamber cable is connected; the
safety interlock is sased.
The chamber cable is not connected;
the safety interlock is not sased.
Displays the state of the internal
high voltage power supply (HVPS).
The power supply is on and the reservoir
capacitor is charging.
The power supply is o.
This box shows the current funconal status.
It displays:
The system is ready to accept
instrucons to begin a protocol.
The computer is loading
protocol to the internal microprocessor.
The system is charging the
reservoir capacitor to the set voltage.
The protocol is being delivered to
the chamber.
Displays the voltage
of the reservoir capacitor, not the pulse
amplitude. This display is operaonal at all
mes except when pulses are being delivered.
Displays the voltage,
updated every second or so.
Displays the nal voltage on
the reservoir capacitor.
Displays the system
rest voltage.
The window at the boom center of the screen is the Last Protocol
Log window. The window displays the last protocol executed.
The Electroporaon Mode Control Panel Area in the center of the
screen is where the pulse-protocol parameters are set, edited, and
reviewed. The following is a descripon of each of the secons,
and input/display boxes found in the Control Panel.
Displays the group number whose
characteriscs are shown in the rest of the
Control Panel Area. The Group ID is also shown
highlighted in the Group List.
Beginning voltage
Duraon in seconds
Frequency in Megahertz
End voltage
Non-linear ramp constant
Voltage of the electroporaon fusion pulse
Length of the pulse
Number of pulses to deliver
Delay between the start of each pulse
Beginning voltage
Duraon in seconds
Frequency in Megahertz
End voltage
Non-linear ramp constant
Hybrimune™System Components & Set-Up
17
Hybrimune Hybridoma Producon System
Publicaon 015-1010191 Rev 4.0 • www.btxonline.com
• Displays the list of groups in the
current protocol. The groups will be executed in
the order displayed. There are three buons to
control the list:
A Used to add a group to the list.
Re Used to change data within a
group. Changes made to values within a group will
not take eect unl Replace has been selected. If a
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 rst.
Re Deletes a group. If a group is
deleted, the group numbers below the deleted group
(if any) are renumbered accordingly.
Non-linear step protocols containing mulple constant-amplitude
sine waves are programmed by the concatenaon of mulple
groups. Typically, the rst 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 seng the duraon to zero
seconds. Likewise, a pulse parameter set can be skipped by seng
the number of pulses to zero.
The following funcon buons are used to run the current
protocol:
Aer all of the electroporaon
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. Aer seven seconds, the Start buon is
highlighted and the system is ready to deliver the
pulse protocol.
Clicking Start begins delivery of the
protocol to the test sample. The pulse groups are
executed in sequenal order. A double beep signals the
end of protocol execuon, if sounds were enabled. If the
Start buon is not clicked within 80 seconds aer turning
on the HVPS, the system will automacally reset itself.
Clicking Reset stops the capacitor
charging and/or the delivery of pulses, and can be used
at any me.
Pushing F12 on the keyboard is the same as
clicking Turn HV ON followed by Start. Please note that
the reservoir capacitor takes me 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.
File management conforms to standard Windows® convenons.
The protocols are saved to and retrieved from a folder created
at the me of soware installaon or to any user created folder.
The le extension used for a protocol le is .pro. The soware
automacally applies this extension when a protocol is saved. Log
les are saved as plain text and are given the .txt extension.
508.893.8999
www.btxonline.com
Hybrimune™System Components & Set-Up
18
Hybrimune Hybridoma Producon System
Publicaon 015-1010191 Rev 4.0 • www.btxonline.com
This secon describes various methods to program waveform
protocols. As stated previously there generally are three waveform
components:
•
The waveform generator user interface uses the concept of Groups.
A group is dened as a set of AC + Pulse +AC waveforms in which
the parameters of the rst AC, the pulse and the last AC do not
change. The one excepon 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.
•
The soware automacally concatenates all of the Groups in the
proper order to be executed in series.
The programming of the recommended protocol will be
demonstrated.
This protocol requires two groups because the pre fusion pulse AC
has two parts:
• 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
• AC1
• AC2
• Pulse
• AC
Start 40 volts peak
Stop 40 volts peak
Funcon Linear (Ramp k =1)
Duraon 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
Start set at Group 2 value
Stop set at Group 2 value
Funcon set at Group 2 value
Duraon 0
Frequency set at Group 2 value
Start 75 volts peak
Stop 75 volts peak
Funcon Linear (Ramp k =1)
Duraon 20 seconds
Frequency 0.8 Mhz
Pulse Amplitude 800 volts
Width 40 µs (entered as 0.040
milliseconds)
Interval 0.125 seconds
Number 1
Start 60 volts peak
Stop 5 volts peak
Funcon Linear (Ramp k=1)
Duraon 30 seconds
Frequency 0.8 MHz
Hybrimune™System Components & Set-Up
19
Hybrimune Hybridoma Producon System
Publicaon 015-1010191 Rev 4.0 • www.btxonline.com
Myeloma cells are B cell leukemia cells that have been selected
for or engineered to have certain properes. One is that the
myeloma should not secrete anbody 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 suscepble 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 selecon agent. This leaves
B cells to die on their own and myeloma cells to die because of
the selecon agent.
A common selecon medium is hypoxanthine-aminopterin-
thymidine (HAT) containing medium. Aminopterin blocks the de
novo biosynthesis of purines, resulng in inhibion of cell growth.
Specically, the folic acid antagonist aminopterin interferes with
the donaon 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 producon of the
necessary nucleodes using purine bases and thymidine (a salvage
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 potenal 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 immortality from the myeloma cells.
Early preparaon of myeloma cells is relavely 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 suscepbility to HAT medium selecon. Culture of
the myeloma cells in either 8-azaquanine or 6-thioquanine will
eliminate cells that are not sensive to HAT.
Producing Fusion Products Using Hybrimune™
Pages 19–21
Pages 21–23
Page 23
Pages 24–25
The descripon in this secon refers to Hybridoma producon.
For immunotherapy refer to Trevor (2004).
20
Hybrimune Hybridoma Producon System
Publicaon 015-1010191 Rev 4.0 • www.btxonline.com
Note: This list is not exhausve. It was made from literature
searches and a search of the ATCC and Coriell Instute’s web sites.
Carefully research intellectual property issues relang to these cells
before using them since some of these cell lines may be patented.
It does not indicate which cell lines are beer suited as fusion
partners for the species indicated. It is meant to provide a starng
point for nding a good fusion partner.
• U-226AR
• GM1500GTGAL2 (Coriell Instute, GMO4672)
• HuNS1 (ATCC CRL8644)
• SHM-D33 (ATCC CRL-1668)
• KR-12 (ATCC CRL8658): Derived from GM1500
Mouse
IgG secreng mouse myeloma cells:
• MOPC 21
• P3X63AG8 (ATCC #TIB9)
Non-anbody mouse secreng cells (some produce light chains):
• SP2/0-Ag14 (ATCC #CRL 1581)
• NS-1 (ATCC #TIB18)
• P3.X63AG8.653 (ATCC #CRL 1580)
• F0 (ATCC CRL1646)
• S194/5.XXO.BU.1 (ATCC #CRL 1580, TIB20)
• FOX-NY (ATCC #CRL 1732).
• 210. RCY3.Agl.2.3
• 240E1-1 (Spieker-Polet 1995)
B Cells
Preparaon of B cells is more complicated. The general steps are in
vivo or in vitro immunizaon, collecon and puricaon of B cells,
and oponal acvaon of B cells. There are many ways to do this
series of procedures and all of them will not be covered.
One immunizaon procedure follows. Immunize mice every 3
to 4 weeks. The rst immunizaon can use complete Freund’s
adjuvant. Subsequent immunizaons 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 anbody to the angen of interest. Three days before
harvesng spleen cells, a nal boost of angen without adjuvant
can be given intravenously to acvate B cells and localize B cells to
the spleen.
An alternate immunizaon procedure is to use DNA vaccines
delivered using intradermal inoculaon of plasmid plus
electroporaon. Mice are immunized every 3 to 4 weeks. The
immunizaon can be either a series of DNA vaccines or a DNA
prime with protein boost. A nal boost is given 3 to 7 days prior
to harvest. For details, consult BTX regarding the AgilePulse™ DNA
vaccine delivery system.
Test Bleeds
• Idenfy mice with ear tags or ear clips.
• Apply grease to tail (an area of 3/4 inch, from base
toward p).
• 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.
• Place blood into mouse clot tubes.
• Apply pressure to tail wound to stop the bleeding.
• Spin down the blood at max speed for 5 minutes in the
microfuge.
• Draw o and store sera at -20oC unl you can test on
a blot or ELISA at 1:100 or at 1:50.
Use an ELISA or Western Blot to evaluate
anbody producon in mice.
Producing Fusion Products Using Hybrimune™
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