[CANCER RESEARCH 38, 3560-3562,
0008-5472/78/0038-OOOOW2.00
October
1978]
Communication
A Greatly Simplified Method of Establishing B-Lymphoblastoid Cell
Lines1
Hiroko Tohda, Atsushi Oikawa, Toshio Kudo, and Takehiko Tachibana
Departments
Japan
ol Pharmacology
¡H.T.. A. O.¡and Immunology
¡T.K.. T. T.¡.Research
Institute
Detection
ABSTRACT
Ten lymphoblastoid cell lines were established by Epstein-Barr virus-induced transformation directly from 0.04
to 0.15 ml of peripheral whole blood of one patient with
xeroderma pigmentosum and four normal healthy adults.
All these lines expressed B-lymphocyte characteristics.
The advantages of this method are: (a) only a few drops
of blood are required for establishing a permanent line;
(b) damage and loss of cells in separation procedures are
minimal; and (c) the method is simple, reliable, and
applicable, if desired, to any patient, even babies.
INTRODUCTION
In a previous paper (13) we reported a convenient method
of establishing
permanent lymphoblastoid
cell lines from
xeroderma pigmentosum
patients, in which the leukocyte
fraction was separated from peripheral blood and trans
formed by EBV.2 In this study the method was further
simplified by omitting the separation procedure, and thus
only a few drops of whole blood were necessary to obtain
the cell line.
The present method is better than the previous method
(13), especially when only a limited amount of blood is
available.
This paper describes the procedure and evidence that the
established lines have the characteristics
of B-lymphocytes.
for Tuberculosis
of EBNA.
and Cancer. Tohoku University.
EBNA was detected
Sendai 980.
by the anti-
complement
immunofluorescence
test according
to the
method of Reedman and Klein (8). Daudi and MOLT-4F cells
were used as EBNA-positive and EBNA-negative controls,
respectively.
Detection of Immunoglobulin. Surface immunogobulin
and cytoplasmic immunoglobulins
were assayed with fluorescein
isothiocyanate-conjugated
monospecific
rabbit
anti-human
IgM, IgG, and IgA (Behringwerke,
MarburgLahn, West Germany) as described by Jondal and Klein (2)
and Hinuma and Grace (1), respectively.
Detection of Membrane Receptors. Membrane receptors
were examined by measuring rosette formation according
to the method of Tachibana and Ishikawa (11). Indicator
cells used were as follows: (a) EAC" [sheep erythrocytes
sensitized with the IgM fraction of rabbit anti-sheep erythrocyte serum and incubated with fresh human serum (6)]
for the detection of C3b receptor (9). (fa) EAC"1 [sheep
erythrocytes (1 x 109/ml) sensitized with the IgM fraction of
rabbit anti-sheep erythrocyte
serum and incubated with
fresh Co-deficient mouse serum (1:10) at 37°for 120 min]
for the detection of C3d receptor (9); (c) EN (neuraminidasetreated sheep erythrocytes)
for the detection
of sheep
erythrocyte receptor on T-cells.
Peroxidase Stain. The cytochemical peroxidase reaction
was performed as described by Kaplow (3).
RESULTS
MATERIALS
AND METHODS
Blood Donors. Peripheral blood samples were obtained
from a xeroderma pigmentosum
patient (XP18SE) and 4
normal healthy adults (N2, N5, N6, and N7). In one case
(NL2-WE) the sample was obtained from an ear lobe.
Virus. The culture filtrate from a B95-8 marmoset cell
culture (5) was used as a source of virus, as described in
the previous paper (13).
Culture Medium. Roswell Park Memorial Institute Me
dium 1640 (Nissui Seiyaku Co., Tokyo, Japan) supple
mented with 20% heat-inactivated
fetal bovine serum
(Grand Island Biological Co., Grand Island, N. Y.), 2 mw Lglutamine,
and 50 u.g kanamycin
per ml was used.
1This work was partly supported by Grants-in-Aid for Cancer Research
from the Ministry of Health and Welfare and the Ministry of Education,
Science, and Culture. Japan, and a grant from the Princess Takamatsu
Cancer Research Fund.
2The abbreviations used are: EBV, Epstein-Barr virus; EBNA, EpsteinBarr virus-associated nuclear antigen.
Received June 19, 1978; accepted August 11, 1978.
3560
Establishment of Lymphoblastoid Cell Lines. From 1 to
3 drops of heparinized peripheral whole blood were diluted
with 2.5 ml of culture medium, mixed with 0.2 or 0.4 ml of
the filtrate containing EBV in Falcon plastic culture flasks
(50 ml), and incubated at 37°.Every 3 or 4 days, 2 to 3 ml of
fresh culture medium were added to the cultures. Within 1
to 3 weeks after EBV infection, transformed
cells were
recognized microscopically
as proliferating foci embedded
in a thick layer of erythrocytes
(Fig. 1), and eventually
lymphoblastoid
cell lines were established. In this way all
10 trials were successful, and the resulting cell lines with
their characteristics
are listed in Table 1.
Characteristics of Established Cell Lines. Morphologi
cally, the established cell lines were indistinguishable
from
each other and also from cell lines obtained from a leuko
cyte-enriched
fraction as described in the previous paper
(13).
All the cell lines carried EBNA and cytoplasmic
IgM.
Some of them had cytoplasmic
IgG or IgA in addition to
IgM. Six of 9 cell lines examined had surface IgM, although
CANCER
RESEARCH
VOL. 38
Simplified Method of Establishing B-Lymphoblastoid
Lines
Table 1
Establishment of lymphoblastoid cell lines and their characteristics
formation
Cytoplasmictransforma
before
b
withSurfaceIgM
ofblood
line"XPL18-W1XPL18-W2XPL18-W3NL2-W2NL2-WENL5-W3NL6-W3NL7-W1NL7-W2NL7-W3Amount
Cell
EAC"NTC
(ml)0.040.080.120.100.040.120.120.050.100.15Time
tion (days)
IgA5
IgM
IgG
+5
+5
+14
+19
+12
+14
+11
+11
+11
979592+
+
+
+
+
+
+
+
+
9699+
97+
94+
99+
98+
+Rosette
95EACm86797590938890989895EN0.52.02.01.00.61.02.01.64
" Symbols preceding hyphens, donors; W and numbers after hyphens, "whole blood" and
number of drops used; E, from ear lobe.
* Cells were examined for receptors for EACh,EACm,and EN.Numbers represent percentages of
cells forming rosettes with the indicator cells. We examined 300 cells of each cell line.
c NT, not tested.
' ' ': .&>
!»v?<
&"°:
,%'-. /
-*/
' ff-e
Fig. 1. A proliferating focus embedded in the erythrocyte layer. NL7-W1
cells 12 days after EBV infection, x 250.
staining was very weak and could not be detected in 3 lines.
Cells of all lines formed many rosettes with EAC'1and EAC'"
but few rosettes with EN.The cells gave a negative reaction
for peroxidase. These results indicate that all the cell lines
originated from B-lymphocytes.
DISCUSSION
In the method described in this paper, lymphoblastoid
cell lines were established from 1 drop of peripheral whole
blood by EBV-induced transformation, without previous
purification of the lymphocytes. According to the report of
Katsuki ef a/. (4), 1 drop (40 /il) of the blood should contain
more than 500 surface IgM-bearing B-lymphocytes, the
target cells for EBV. Thus the volume of whole blood used
in our method could be reduced still further if necessary.
Consistent with EBV-induced transformation used, all the
established cell lines were confirmed to originate from Blymphocytes by demonstrating that they contained cytoplasmic immunoglobulins and the surface receptors for
C3b and C3d fragments of the third component of comple
ment and that they did not have sheep erythrocyte receptor,
which is a characteristic of T-lymphocytes (2). From the
OCTOBER
1978
negative results on peroxidase staining, it is unlikely that
they originated from monocytes or granulocytes (14). The
presence of cytoplasmic IgM in all the cells is consistent
with the claim that surface IgM-bearing cells are the major
target among B-lymphocytes for transformation by EBV (4),
if surface IgM is easily lost after transformation.
Transformation of B-cells by EBV/n vitro is influenced by
cellular components in the blood; namely, macrophages
have an enhancing effect (7, 10), while T-lymphocytes,
especially those from adult donors, have an inhibitory effect
(12). In the present method the number of target cells in
transformation culture was much less than that in the
conventional method, which uses a lymphocyte-enriched
fraction, but the reliability and the time required for estab
lishing cell lines were almost the same as in the conven
tional method. Possible reasons for this are: (a) damage to
target cells due to separation of cells was avoided; (b) blood
was diluted 20- to 70-fold with culture medium in the
transformation culture, so that the inhibitory action of Tlymphocytes would be lowered; and (c) loss of macro
phages due to their adhesion to the glass wall during cell
separation was minimal, favoring transformation. It is not
known whether the presence of a great number of erythrocytes is favorable or not.
Since a very small amount of blood is required in the
present method, B-lymphoblastoid cell lines can be estab
lished from blood samples of practically any clinical case,
such as those discussed in the previous paper (13).
ACKNOWLEDGMENTS
We are grateful to Dr. M. Seiji of Tohoku University School of Medicine
for the blood sample from a xeroderma pigmentosum patient.
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3561
H. Tohda et al.
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CANCER
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VOL. 38