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Hybrimune Hybridoma Production System
Publication 015-1010191 Rev 4.0 • www.btxonline.com
Cell Preparation Before
Electrofusion
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 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 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.
Producing Fusion Products Using Hybrimune
™
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
Pages 19–21
2. Cell Preparation on Electrofusion Day
Pages 21–23
3. Electrofusion
Page 23
4. Cell Handling After Electrofusion
Pages 24–25
The description in this section refers to Hybridoma production.
For immunotherapy refer to Trevor (2004).