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Hybrimune Hybridoma Production System
Publication 015-1010191 Rev 4.0 • www.btxonline.com
Harvesting Spleen Cells
• Euthanize immunized mice.
• Immerse mice in alcohol.
• Sterilely collect spleen and place into 10 ml cold DMEM.
• Pour spleens onto Cellector screen.
• With a 25-27 gauge needle, inject 0.5 ml sterile PBS
into spleen.
• Tease spleen apart using two syringes with 21 gauge
needles. Use the needle tips to cut the spleen into
small parts.
• Express spleen cells through 100 mesh sterile screen
using Cellector pestle or rubber end of a syringe plunger.
• Drop-wise, flush screen with 7 ml PBS.
• Pipette cells into 15 ml conical tube.
• Let settle for 5 minutes to remove large pieces.
• Collect cells in supernatan.
• Centrifuge 300 Xg for 7 minutes.
• Re-suspend pellet in 5 ml red blood cell lysis buffer
(pre-warmed to 37°C).
• Mix well and incubate 3 minutes.
• Add 5 ml PBS.
• Centrifuge cells and wash cells once in complete medium.
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:
• Wash spleen 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% CO
2
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 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 13,
page 34) 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 persists, the temperature of the medium will rise to
critical levels and kill the cells.
Producing Fusion Products Using Hybrimune
™