| Abstract |
For study of immunoglobulin production in genetically identical cells, we
have developed a technique for cloning normal human lymphocytes in vitro
and modified the conventional methods for establishing lymphocytoid cell
lines. Our technique of cloning lymphocytes is the first method yet
described in the literature for obtaining lymphocyte clones, in a pure
sense, since this technique enables us to observe the fate of single,
isolated lymphocytes under the inverted microscope. The modified method
for the establishment of lymphocyte cell lines solved the most difficult
problem of conventional methods (i.e., the requirement of a large quantity
of blood) so that cell lines can be derived from virtually any individual,
including pediatric patients.
To clone lymphocytes, cell suspensions were diluted to a final
concentration of 1 cell per 10 microliters and plated out in microwells of
(Falcon) plastic microtest plates. These were then incubated at 37
Celsius, in a humidified 5% CO2 atmosphere, with sequential observation
under the inverted microscope. Initial experiments on phytohemagglutinin-
stimulated lymphocytes from the peripheral blood of normal persons
demonstrated that 9% of the single cells formed microcolonies of 30-100
cells after 10 days using medium in which HeLa cells have been grown.
However, when the peripheral blood lymphocytes were first established as
continuously growing cell lines, up to 50% of the single cells produced
clones of 100 or more cells, which were then selectively propagated as
sublines.
For establishment of the lymphocytoid cell lines, the buffy coat
cells of 10 ml of heparinized venous blood were cultured in RPMI medium
1640 with 20% fetal calf serum. At the initiation of the culture either
small amounts of intact cells or cell lysates from previously established
lymphocytoid cell lines were added as the "stimulating" agents. The cell
lines chosen for stimulation had sex chromosome complements which were
different from those of the donors.
When 13 cultures were set up from 5 different donors by using
intact cells from previously established cell lines, 3 new cell lines
were established from 2 donors. When cell lysates were used, however, in
36 cultures from 16 donors, 14 cell lines were developed (from 9 individuals) at the end of 10 weeks of culture. These included 2 cell lines
from a patient with the Lesch-Nyhan syndrome. These 2 cell lines are
deficient in hypoxanthine guanine phosphoribosyl transferase activity and
are the first reported, biochemically marked human lymphocytoid cell lines.
The modal chromosome number and the karyotype of each of the 17
new lymphocytoid cell lines were the same as those of the donor's cells.
Immunodiffusion and immunoelectrophoretic studies revealed the production
of immunoglobulins of various classes and types in 15 of these 17 new
cell lines.
Two cell lines, UM-1 and UM-5, which were derived from two healthy male
donors and established by our modified method, were cloned by our
technique to yield 8 and 7 clonal sublines, respectively. The parental
lines were found to produce consistently IgG and IgM of the kappa type,
and all of the cloned sublines of both lymphocytoid cell lines produced
the same classes and the same type of immunoglobulins as the parental cell
lines. This suggests that each lymphocytoid cell of these two cell lines
produces both IgG and IgM at the same time.
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