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The cultivation of adult rabbit testicle in roller tubes.

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Syphilis Division, Department of Yetdicine, Johns Hopkins University Medical
School, and Department of Embryology, Cnrnegie Institution of
Washington, Baltimore, Maryland
The growth of tissue in vitro has been made possible by
the use of a variety of methods, many of which possess advantages f o r a particular type of reasearch. Recently Gey
('33), Gey and Gey ('36) and Lewis ( '34, '35) have described
the roller tube method, in which an ordinary 150 X 16 mm.
test tube is employed. This method offers a convenient
technic for the study of the effects of drugs, toxins, viruses
and hormones on the growth and viability of tissue cells.
Relatively large amounts of tissue can be cultivated for long
periods of time, with the production of unusually good
growths of cells. The technic is comparatively simple and
involves the use of nutrient media that are readily available
in most laboratories.
The testicle of the adult rabbit provides an excellent source
of living tissue cells in roller-tube cultures. Under sterile
precautions, the testicle is removed and cut with small curved
scissors into 2 mm. cubes in 0.85% salt solution. The test
tubes are coated on the inside with 4 drops of heparinized
chicken plasma, evenly disposed with a glass rod. The fragments of testicle are taken up in a small pipette and distributed along the inside of the tube. Usually two rows of
From the Syphilis Division of the Department of Medicine, Johns Hopkins
University Medical School, and the Department of Embryology, Clarnegie Institution of Washington, Baltimore, Maryland.
explants of six each are used, but the number of rows may be
increased to four. Within 10 minutes the chicken plasma
clots and forms a smooth lining which holds the explants
firmly in place. One cubic centimeter of nutrient medium is
then added and the tube is tightly closed with a sterile
rubber stopper. The tubes a r e placed in a slightly inclined
drum which rotates at 8 revolutions per hour inside an incubator at 37.5"C. The nutrient medium consists of 0.5 cc.
of rabbit serum and 0.5 cc. of balanced salt solution (Gey,
'33). Locke solution can be substituted f o r the balanced salt
solution, and will give a satisfactory growth of cells, but
better results are obtained with the balanced salt solution.
The growth and migration of cells begins early, progresses
rapidly and reaches its maximum in 7 days. Within 24 hours
a few isolated sprouts can be observed extending from the
free edge of the explant. At this time the convoluted tubules,
embedded in the thickness of the testicle explants, are barely
visible. Within 48 hours, slender spindle-shaped cells begin
to migrate out and form a radial growth around the entire
explant. From this time on there is a progressive and rapid
increase in growth and migration of cells, characterized by
numerous mitotic divisions. For the most part, these cells
are the fibroblasts and endothelial cells derived from the
supporting tissue cells. As the stroma cells migrate away,
the convoluted tubules become more prominent and their
constituent epithelial cells begin to multiply. After 5 days
the tubules are clearly outlined within the thickness of the
explant. A tubule which earlier presented a free edge, is
now continuous with a broad sheet of flattened and rhomboid
polymorphous shaped cells. These are the germinal epithelium. A number of large cells with large nuclei were observed intermingled with the germinal epithelium ; they appear to be similar to the cells described in fixed and stained
sections as Sertoli cells. Occasionally, spermatozoa were
seen in these cultures, they retained their motility for at
least 48 hours and degeneration forms were observed as
long as 1 2 days after growth in roller tubes. I n the stained
preparations these are represented by tiny rods. The tails
were not stained by hematoxylin. The spermatozoa may have
been introduced as adult forms within the testicle explants;
however, it is highly probable that maturation from spermatocytes takes place within the roller tubes.
After 7 days small areas of liquefaction form near the
explant. It is necessary to patch these areas by relining the
tubes with chicken plasma, which it clotted in the liquefied
areas by means of a drop of chick embryo extract. Fresh
supernatant fluid is then added. The cells continue to grow
out in abundance (fig. 1). Cultures used in this investigation were maintained 21 days and patched, and the supernatant fluid was changed every 7 days. They can be maintained for longer periods if desired.
Permanent preparations of these cultures can be made
by fixing and staining. The supernatant fluid is drained off
as completely as possible and the tubes are washed with
three changes of physiologic salt solution. Zenker ’s solution, containing 5% formalin, is used as the routine method
of fixation, but any of the usual fixing solutions may be
substituted. After 15 minutes the Zenker’s solution is
poured off, and the tubes are washed in three changes of
water. The chicken plasma lining is then scraped away
from the inside of the tube with a slender wooden applicator.
It is desirable to leave an ample margin of chicken plasma
clot surrounding the rows of explants. The preparations
are subjected to the usual changes of alcohol, stained with
hematoxylin, dehydrated and cleared in xylol. Because of
the presence of the plasma clot, differential staining seldom
proves to be satisfactory. About 3 cc. of balsam is placed in
the tube and a Wasserman tube, filled with cedar oil and
closed with a cork stopper, is then allowed to slide into the
culture tube. The balsam rises and fills the space between
the two tubes just as it does between a slide and a cover glass.
The excess of balsam continues to rise about the level of the
Wasserman tube and, when dry, forms a permanent seal.
The large growth of cells obtained in roller tube cultures
of the testicle from the adult rabbit has been used to test
the toxicity of a few arsenical drugs (unpublished results).
The actively growing explants of the testicle have also
served as a culture medium in an attempt to grow the
Spirochaeta pallida (Eagle and Mendelsolin, in press). This
method is especially suited for the study of the viability of
living cells during exposure t o certain hormones, toxins and
viruses. It may be possible t o recover from the supernatant
fluid of these tissue cultures certain products of metabolism,
such as hormones.
GEY, G. 0. 1933 A n improved technic f o r massive tissue cultures. Am. J.
Cancer, vol. 17, p. 752.
GEY, G. O., AND M. X. GEY 1936 Maintenance of cells in continuous culture.
Ibid., vol. 27, p. 45.
LEWIS,W. H. 1932 Roller tube cultures. Anat. Rec., vol. 58 (Suppl.), p. 55.
1935 Roller tube cultures. Contrib. t o Embryology, no. 150,
Carnegie Inst. of Washington.
1 Living explant of testicle showing extensive migration and growth of
cells. Twenty-one-day culture. X 60.
2 A sheet of germinal epithelium with a group of spermatozoa shown as
small dark rods. Twelve-day culture fixed and stained. x 150.
3 Germinal epithelium and large Scrtoli cells. Twelve-day culture fixed and
stained. X 150.
TI11 A N A T O M I C A I . RECORD, VOT.. 69, NO. 3
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adults, testicle, rabbits, cultivation, roller, tube
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