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The culture and karyotype of rat lymphocytes stimulated with phytohemagglutinin.

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The Culture and Karyotype of R a t Lymphocytes
Stimulated with Phyt ohemagglutinin '
WILLIAM 0. RIEKE AND M. ROY SCHWARZ
Department of Biological Stmcture, University of Washington School of
Medicine, Seattle, Washington
ABSTRACT
A simple method leading to growth and mitosis in over 95% of shortterm cultures of lymphocytes from the blood, lymph or thymus of the rat is described.
The method which is modified from Moorhead's original technique ('60) employs
standard tissue culture medium (Eagles MEM or TC no. 199), 20% fresh rat serum,
washed lymphocytes, penicillin, and phytohemagglutinin-P (0.01 cm3/cm3 of culture of
a 1:5 dilution of stock solution). Details of culture technique and factors contributing to growth failures are discussed.
The chromosomes of male and female Lewis rats were studied in metaphase
spreads of cultured cells. The karyotypes of these rats are presented and found to be
in agreement with those recently reported by Hungerford and Nowell ( ' 6 3 ) .
Since 1960, when Nowell reported that
phytohemagglutinin (PHA) initiates mitosis in cultures of human leucocytes, many
studies (Elves and Wilkinson, '62; Cooper,
Barkhan and Hale, '63; Galton and Holt,
'63; Humason and Sanders, '63; Aspegren
and Rorsman, '64) based on culture techniques described by Moorhead, Nowell,
Mellman, Battips and Hungerford ('60)
have confirmed his report and shown that
it is the lymphocytes which respond in
various laboratory animals. The value of
lymphocyte culture techniques makes it
important that they be suitable for lymphocytes from one of the commonest of all
laboratory animals, the rat, Rattus norvegicus. Unfortunately, attempts to culture rat
blood lymphocytes have often ended either
in failure or in only partial success. Schrek
and Rabinowitz ('63) noted that while
normal human lymphocytes stimulated
with PHA enlarged to form typical PHAblast cells, rat lymphocytes did not respond. Similarly, Ling and Husband ('64)
showed that while staphylococcal protein
induced a PHA-like transformation in
human blood lymphocytes, rat (and guinea
pig) lymphocytes were unreactive. In
contrast, Humason and Sanders ('63) reported that leucocytes from several species, including the rat, were successfully
grown with PHA, but gave no details specific to the culture of rat cells nor indicated the percentage of successful cultures. Most recently, Dowd, Dunn and
ANAT. REC., 150: 383-390.
Moloney ('64) have carefully described
the culture of lymphocytes from rat blood
in a medium requiring fresh glutamine
and newborn agamma calf serum. With
their method sufficient mitotic activity for
adequate chromosome preparations was
obtained in 70% of the cultures.
The present work describes modifications of Moorheads technique ('60) which
have allowed the development of a simple
and almost invariably successful method
for the culture of rat lymphocytes from
blood, thoracic duct or the thymus. Lymphocytes from both sexes in one randomly
bred and two inbred strains of rats have
been cultured, and the karyotype of one
of the inbred strains (Lewis) as obtained
from the cultured cells is presented.
MATERIALS AND METHODS
1. Animals. More than 60 rats and
300 cultures have thus far been studied.
The majority of the cultures were established with cells from inbred male Lewis
animals weighing 150-250 gm. However,
Lewis females, male and female Chocolate ( E N ) and Sprague-Dawley strain rats
ranging from 75 to 475 gm were also
used.
2. Obtaining cells for culture. Lymphocytes from the blood, thoracic duct lymph
or thymus were secured from rats anesthetized with pentobarbital. Blood leucocytes
1This work was supported by U.S.P.H.S. Grant
GM 0639.
383
384
WILLIAM 0. RIEKE AND M. ROY SCHWARZ
were obtained by cardiac puncture employing a syringe containing heparin (100
units/cm3 of blood) and phytohemagglutinin (0.01 cm3/cmyof blood of 1:5 dilution
of the stock PHA). After the blood was
mixed with the PHA and heparin, it was
allowed to stand in an ice bath for 20-30
minutes and then was centrifuged at 300500 rpm in a clinical centrifuge in a cold
room (4" C). The centrifuging was interrupted at 1-2 minute intervals in order
to determine the critical stage at which
there was separation of leucocyte-rich
plasma from erythrocytes before the formation of a buffy coat. The plasma with
its white cells was then removed, mixed
with an equal volume of Hanks balanced
salt solution and centrifuged at 600-800
rpm until all cells were sedimented. The
cell pellet was twice washed by resuspension and centrifugation from 5 cm3of Hanks
solution. Finally, the cells were suspended
in 1-2 cm3 of nutrient medium (Eagles
MEM or TC no. 199), and were counted in
a standard hemocytometer. The volume of
the nutrient medium was then adjusted so
that the final concentration of leucocytes
in culture would be 1-5 X loficells/cm'.
Lymph from the thoracic duct was obtained by cannulating the duct either in
the neck (Reinhardt and Li, '45) or the
abdomen (Bollman, Cain and Grindlay,
'48); and during the 1-14 hour collection
periods the lymph was heparinized and
kept in an ice bath. Lymphocytes were
separated by centrifugation; were washed
twice in Hanks solution as described
above; and finally were suspended in nutrient medium. The final concentrations
of cells in the cultures were varied from
0.4-15 X lofilymphocytes/cm3.
Lymphocytes were obtained from the
thymus by cutting thymic lobes into several pieces and gently stirring the pieces
in Hanks solution containing 10% serum.
In most cases the thymic pieces were further agitated by using a syringe to aspirate
and eject a portion of the solution in
which they were immersed. The pieces of
thymus were then removed and the cells
which had been released were washed as
described above. Thymus cells were cultured at final concentrations ranging from
5-20 X 10" cells/cm'.
3. Culture and nutrient substances.
The nutrient medium most frequently employed was Eagles Minimum Essential
Medium with Hanks balanced salt solution. However, tissue culture medium
no. 199 with NaHC03 also was successfully
used.
The contents of one bottle of Bacto
Phytohemagglutinin-P (Difco Laboratories, Inc., Detroit, Michigan) were mixed
with 5 cm3 of Bacto Phytohemagglutinin
buffer, pH 7.2-7.3 to form a stock solution. The stock solution was most often
used in a final concentration of 0.01 cm'/
cm3 of culture medium, but concentrations
of 0.002 cm3/cm3 and 0.02 cm3/cm3 were
also tested.
Sufficient Penicillin G to give a concentration of 100 units/cm3 of culture medium
was added to all cultures.
Rat serum in a final concentration of
20% was the only additive used to enrich
the nutrient medium. Serum was routinely used within 1-2 hours after being
prepared from cardiac blood, but serum
which had been refrigerated overnight or
deep-frozen (-20" C ) for 1-2 weeks was
also tested. Most commonly, isologous
serum was used. However, one experiment involved the sera and cells from rats
of three different strains in order to compare the growth-supporting characteristics
of homologous and isologous sera.
4. Culture vessels and conditions. Initially, sterile, polystyrene tubes or flasks
(Falcon Plastics, Los Angeles, California)
were used to culture the cells. These were
discontinued, however, in favor of standard 10, 25 or 50 cm3 Erlenmyer (Pyrex)
flasks which had been washed in Microsolv detergent (Microbiologcal Associates,
Bethesda, Maryland), thoroughly rinsed
(15 times) in tap and distilled water, and
oven dried. Tissue culture medium
amounting to 50-60% of the total volume
of the flasks was added and the flasks
were tightly stoppered. Room air remained
as the gas phase above the cultures which
were then incubated in an upright position at 37" C for 2-6 days. Cultures were
harvested either by hypotonic treatment
(Moorhead et al., '60) after the addition of
colchicine (0.1 ~gm/cm')),or by smearing
after washing the cells in Hanks solution
and resuspending them in 0.01-0.02 cm3
CULTURE O F RAT LYMPHOCYTES
385
when the cultures were sacrificed. In contrast, control cultures of blood, lymph or
thymus lymphocytes cultured without PHA
showed enlargement, division and labeling in only 0.1% of cells or less.
Cultures of blood, lymph or thymus
lymphocytes were similar with respect to
the rate of appearance and morphology of
enlarged cells. The sequence of development and morphology of these "blast"
cells have been clearly described for
human lymphocytes in culture ( Carstairs,
'62; Cooper, Barkhan and Hale, '63; Tanaka, Epstein, Brecher and Stohlman, '63)
and are very similar to the present findings. In contrast to cultured human
lymphocytes, the enlarged cells in cultures
of rat lymphocytes only rarely exhibit
clearly defined nucleoli in smears (fig. 1).
In rat cultures, as in human, however, the
number of enlarged cells and mitoses is
maximal at two and one-half-three days
and declines thereafter. The work of
others has established that the small
lymphocyte gives origin to the enlarged
cell in cultures of human blood cells
(MacKinney, Stohlman and Brecher, '62;
Marshall and Roberts, '63), and studies in
this laboratory indicate the same origin
for the enlarged cell in rat cultures
(Schwarz and Rieke, '64).
The total number of cells regularly declined during culture until the second day
and then (except for thymus cultures
[Schwarz and Rieke, '641) stabilized. When
RESULTS
the initial inoculum of cells was large
there were only 10-25%
Over 95% of the 300 cultures stimu- ( > 5 X 106/cm3)),
lated with PHA showed the following evi- as many found at the third day of culture.
dences of successful growth when sacri- When (with the exception of thymus
ficed at the second or third day of culture: lymphocytes) the initial number was small
( 1 ) significant numbers (up to 60% of (1-2 X 106/cm3), the percentage present
a11 cells) of large, primitive-appearing when the culture was sacrificed increased
"blast" cells (fig. 1 ) which had not been to 33-50%. While there was cell growth
present in the culture inoculum (fig. 2 ) ; in all ranges of cell concentrations tested,
and ( 2 ) mitotic figures (fig. 3 ) in suffi- the most consistent growth with highest
cient numbers to provide mitotic indexes mitotic indexes was found when cultures
of 1% or more per hour of colchicine were begun with leucocyte concentrations
treatment. In addition, those cultures in of 0.4-2 X lo6 cells/cm3 for cells from
which H3-thymidine was added terminally lymph; 1-2 X lo6 cells/cm3 from blood;
evidenced DNA synthesis in many nuclei and 10-20 X lo6 cells/cm3 from thymus.
Prior to the initiation of the above techand chromosome spreads (figs. 4, 5).
Sixty-five to 70% of the enlarged cells niques a number of cultures failed. The
were labeled by a 30-minute exposure to only factor which by itself led to failure
isotope, and these labeled cells amounted in every instance was an increase in pH
to as much as 30-40% of all cells present caused by a loss of carbon dioxide in those
of serum. Smears were air dried and fixed
four minutes in absolute methanol.
5. Labeling and radioautographic techniques. In order to determine DNA synthesis and cell growth, H3-thymidine
(Yz-1 vc/ml, specific activity 6.7 c/mM,
New England Nuclear Corp., Boston,
Massachusetts) was added to approximately one-fourth of the cultures during
the terminal one-half-three hours of incubation. In some cases colchicine was
added with the thymidine. The cultures
were well washed when sacrificed and
then used either for chromosome preparations (hypotonic treatment technique) or
for smears as described above. Smears
were made on slides previously subbed
with a gelatin base and chromosome
spreads were prepared on clean, unsubbed
slides. The smears were processed for
radioautography by painting on liquid
emulsion (Eastman Kodak NTBz or
NTB,) in a manner previously described
(Everett, Rieke, Reinhardt and Yoffey,
' 6 0 ) , and the unsubbed slides with the
chromosome spreads were coated by being
dipped in the emulsion. Film exposures
of 1-14 days were allowed. After developing the radioautographs, the dipped slides
were dried and the emulsion was wiped
from their back sides. They, together with
the smear preparations, were then stained
with MacNeal's tetrachrome (MacNeal,
'22).
386
WILLIAM 0. RIEKE AND M. ROY S C H W A R Z
cultures in which the stoppers on the
flasks had become loosened. Other factors
which in combination were sometimes determining included: ( 1 ) The serum. The
use of serum within 1-2 hours after it
was prepared was most important. Serum
refrigerated overnight supported only decreased or variable growth, and serum
deep frozen for 1-2 weeks often allowed
no growth. Serum from any age, sex or
strain rat was used successfully as long as
it was fresh. ( 2 ) The culture vessels.
While both round and flat bottom plastic
vessels allowed cell growth when all other
conditions were favorable, the number of
surviving cells and mitotic indexes were
usually less when plastic was used than
when glass vessels were employed. ( 3 )
Washing the cells. While at first it was believed that the use of washed cells was
critical in securing growth, later experiments showed that lymphocytes from the
blood at least could be grown, unwashed,
in the plasma in which they were isolated
(see also Dowd, Dunn and Moloney, '64).
The percentage of successful cultures and
mitotic indexes were higher, however,
when washed cells and serum were used.
( 4 ) Phytohemagglutinin. Although some
cells were stimulated with PHA in a concentration of 0.002 cm3/cm3 of culture
medium, a larger and more uniform response was observed in cultures with PHA
concentrations of 0.01 cm3/cm'. Twice the
latter amount of PHA did not produce a
greater number of enlarged cells and many
of those cells which responded were degenerated by the third day of culture.
Although PHA solutions which had been
stored in a refrigerator for a month or
more often retained their potency, stock
solutions were routinely changed every
two weeks.
At least ten metaphase plates from the
cultured lymphocytes of each of three
male and female Lewis animals were analyzed to prepare the karyotype shown in
figure 6. The chromosomes are arranged
in accord with the recent suggestion of
Hungerford and Nowell ('63) who have
obtained particularly clear preparations
from bone marrow and lymph node cells
treated with colchicine in vivo.
DISCUSSION
It is believed that the applicability of
the present culture technique to lymphocytes from various sites in the body as well
as its simplicity and reliability constitute
advantages over methods previously described. In essence, this method calls only
for the addition of washed lymphocytes to
culture medium containing 20% fresh
serum, penicillin, and an appropriate concentration of PHA (0.01 cm3/cm3 of culture of a l : 5 dilution of the stock solution).
No other additives are requisite. It seems
probable that the use of 20% fresh serum
obviated the need for the additives which
Dowd et al. ('64) found essential (fresh
glutamine) or helpful (agamma calf
serum and folic acid) in their cultures
which contained only 5% rat plasma.
The use of room air as the gas phase
over cultures not only contributes to the
convenience of the technique, but, as
shown by Nowell ('60), leads to as much
growth as may be obtained with many
combinations of oxygen and carbon dioxide. It is interesting, however, that
unlike the conditions Nowell described for
his standard cultures of human leucocytes,
it was not found possible to leave culture
bottles loosely capped without a loss of
carbon dioxide and a deleterious increase
in pH.
The problem of the percentage of small
lymphocytes which can be stimulated to
respond to PHA remains to be solved. The
percentage of responding cells cannot be
reliably estimated from the percentage of
enlarged cells found in culture for there
is too little known about the number and
kinds of cells which die, about the number, proliferative rates and progeny of cells
which divide, and about the number of
cells which persist but do not divide. It
may be that some small lymphocytes are
more responsive to PHA than others. This
possibility is strengthened by the evidence
from this laboratory (Caffrey, Rieke and
Everett, '62) and from the laboratory of
Fitzgerald ('64) that there are two populations of small lymphocytes with respect
to their rates of formation and circulating
life span.
The karyotype of the rat has been published several times in the last few years
(Fitzgerald, '61 ; Hungerford and Nowell,
CULTURE OF RAT LYMPHOCYTES
'63; Dowd et al., '64) but, unfortunately,
differences in the description of centromere location have led to different systems of classification. Most recently,
Hungerford and Nowell ('63), in a study
directed mainly at sex chromosome morphology, have classified the autosomes so
that the largest chromosomes and those
having subterminal centromeres are in
separate groups. Their system avoids some
of the previous difficulties and is followed
here (fig. 6). The present study confirms
their report that in Lewis rats X chromosomes have subterminal centromeres, Y
chromosomes are telocentric and larger
than pair no. 13, and that chromosome
pair no. 3 appears to be satellited. The
variability between a median and submedian location for the centromere in pair
no. 19 is also noted (fig. 6, cf. pair no. 19
in male and female).
LITERATURE CiTED
Aspegren, N., and H. Rcrsn?-:i 1361 Shortterm culture of lymphocj tes from gu..iea-pigs
allergic to tuberculin. Int. Arch. Mcrgy, 24:
119-123.
Bollman, J. L., 3 . C. Cain and J. H. Grindlay
1948 Techniques for the rollection of lymph
from the liver, small intestiiie and the thoracic
duct of the rat. J. Lab. Clm. Med., 33: 13491352.
Caffrey, R. W., W. 0. Rieke and N. B. Everett
1962 Radioautographic studies of small
lymphocytes i n the thoracic duct of the rat.
Acta Haemat., 28: 145-154.
Carstairs, C. 1962 The human small lymphocyte: Its possible pluripotential quality. The
Lancet, i: 829-832.
Cooper, E. H., P. Barkhan and A. J. Hale 1963
Observations on the proliferation of human
leucocytes cultured with phytohaemagglutinin.
Brit. J. Haemat., 9: 101-111.
Dowd, G., K. Dunn and W. C. Moloney 1964
Chromosome studies in normal and leukemic
rats. Blood, 23: 564-571.
Elves, M. W., and J. E. Wilkinson 1962 The
effects of phytohaemagglutinin on the morphology of culture leucocytes. Nature, 194:
1257-1259.
Everett, N. B., W. 0. Rieke, W. 0. Reinhardt and
J. M. Yoffey 1960 Radioisotopes in the study
of blood cell formation with special reference
387
to lymphocytopoiesis. Ciba Foundation Symposium on Haemopoiesis, J. & A. Churchill
Ltd., London, 43-66.
Fitzgerald, P. H. 1961 Cytological identification of sex in somatic cells of the rat, Rattus
norvegicus. Exp. Cell Res., 25: 191-193.
1964 The immunological role and long
life-span of small lymphocytes. J. Theoret.
Biol., 6: 13-25.
Galton, M., and S. F. Holt 1963 Culture of
peripheral blood leucocytes of the golden hamster. Proc. SOC. Exp. Biol. and Med., 114:
218-219.
Humason, G. L., and P. C. Sanders 1963 Culture and slide preparation of leukocytes from
peripheral blood. Stain Techn., 38: 338-340.
Hungerford, D. A., and P. C. Nowell 1963 Sex
chromosome polymorphism and the normal
karyotype in three strains of the laboratory
rat. J. Morph., 113: 275-285.
Ling, N. R., and E. M. Husband 1964 Specific
and non-specific stimulation of peripheral
lymphocytes. The Lancet, i: 363-365.
MacKinney, A. A,, Jr., F. Stohlman Jr. and G.
Brecher 1962 The kinetics of cell proliferation in cultures of human peripheral blood.
Blood, 19: 349-358.
MacNeal, W. J. 1922 Tetrachrome blood stain;
a n economical and satisfactory imitation of
Leischmann's stain. J. Amer. Med. Assn., 78:
1122-1 123.
Marshall, W. H., and K. B. Roberts 1963 The
growth and mitosis of human small lymphocytes after incubation with a phytohaemagglutinin. Quart. J. Exp. Physiol., 48: 146-155.
Moorhead, P. S., P. C. Nowell, W. J. Mellman,
D. M. Battips and D. A. Hungerford 1960
Chromosome preparations of leucocytes cultured f r t m human peripheral blood. Exp. Cell
Res., 20: 613-t'16.
Nowell, P. C. 1962 Phytohemagglutinin: An
initiator of mitosis in cultures of normal
human leukocytes. Cancer Res., 20: 462-466.
Reinhardt, W. O., and C. H. Li 1945 Cell
count, rate of flow, and protein content of
cervical lymph in the rat. Proc. SOC. Exp.
Biol., N. Y., 58: 321-323.
Schrek, R., and Y. Rabinowitz 1963 Effects of
phytohemagglutinin on rat and normal and
leukemic human blood cells. Proc. SOC.Exp.
Biol. and Med., 113: 191-194.
Schwarz, M. R., and W. 0. Rieke 1964 The
in vitro effect of phytohemagglutinin on cultures of rat thymus cells. In preparation.
Tanaka, Y., L. B. Epstein, G. Brecher and F.
Stohlman, Jr. 1963 Transformation of lymphocytes in cultures of human peripheral blood.
Blood, 22: 614-629.
PLATE 1
EXPLANATION OF FIGURES
1
Enlarged, “blast-like’’ cells shown in a smear preparation of rat
thoracic duct lymphocytes cultured two and one-half days with PHA.
x 2,000.
2
The appearance of the lymphocytes from the thoracic duct a t the
time of initiation of cultures leading to cells such as shown in
figure 1. Note that while an occasional large cell is present, it is
morphologically dissimilar from those large cells which develop with
PHA. X 2,000.
3 Enlarged and mitotic cells from a three-day PHA culture of rat blood
lymphocytes treated with colchicine and sacrificed in hypotonic
medium. x 1,600.
4 Radioautograph of rat thoracic duct lymphocytes cultured with PHA.
Following the addition oo H3-thymidine, labeled nuclei and labeled
and unlabeled mitotic figures are seen. x 2000.
5
388
Radioautograph of chromosomes from a rat thoracic duct lymphocyte
exposed to H3-thymidine during the terminal three hours of a PHA
culture. X 2,000.
CULTURE OF RAT LYMPHOCYTES
William 0. Rieke and M. Roy Schwarz
PLATE 1
389
CULTURE OF RAT LYMPHOCYTES
William 0. Rieke and M. Roy Schwarz
6
390
PLATE 2
The karyotype of male and female Lewis rats as obtained from thoracic duct lymphocytes
cultured with PHA.
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