Tissue culture study of human retinoblastoma
Daniel M. Albert,* Alan S. Rabson, and Albert J. Dalton
Cells derived from four human retinoblastomas were maintained in culture for periods ranging
from 2 to 24 months. The tumors grew in vitro in a biphasic manner with an undifferentiated
stroma attached to the surface of the tissue culture flask and clusters of well-differentiated
retinoblastoma cells growing in suspension. The cells in suspension continued to produce
Flexner-Wintersteiner rosettes and on electron microscopy contained microtubules.
R,
nesium for 30 minutes at 37° C. The cells were
resuspended in fresh medium and were passed to
other flasks.
Suspension cultures were maintained in 50
ml. Falcon plastic flasks containing 20 ml. of
medium and suspended cells. Once a week, 10
ml. of medium and cells were removed and
replaced by fresh medium. The removed cells
and medium were then placed in a new bottle
and 10 ml. of fresh medium was added.
Light microscopy. The histopathologic study
of the cells in the culture was carried out in
several ways: (1) the cells were examined and
photographed as they grew in vitro, (2) smears
were made of floating cells, and (3) cells in
tissue culture were either scraped from the plastic, or, in the case of suspension cultures, poured
from the tissue culture flask and formed into a
pellet by centrifugation 748 g for five minutes.
Tissue culture material was fixed in Zenkerformol
solution and sections stained with hematoxylin
and eosin.
Electron microscopy. Specimens from the original tumors and cells from tissue culture were
examined. Tumor material was minced into fine
pieces. Cells in tissue culture were formed into
a pellet as described above. Fixation of tumors
and pellet was carried out in either Dalton's
chrome osmium fixative for one hour or 3 per
cent phosphate-buffered glutaraldehyde-sucrose for
one hour followed by chrome osmium fixation.
Tissue was dehydrated in graded ethyl alcohol
and embedded in Epon-Araldite mixture. Ultrathin sections were cut with an LKB microtome
and double stained in uranyl acetate followed
by lead citrate. Micrographs were taken with a
Siemens I electron microscope using an 80 kv.
accelerating voltage and a 50M objective aperture.
/etinoblastoma constitutes the most
common malignant intraocular tumor of
childhood and, after malignant melanoma,
the second most common primary intraocular malignancy of any type. The histogenesis and etiology of this tumor, however, remain topics of dispute. The present
report describes tissue culture studies of
retinoblastoma in which the light and electron microscopic appearance of the cells in
culture are compared to that of cells of
the original tumor.
Materials and methods
Culture methods. Retinoblastoma was obtained
from 4 enucleated eyes immediately following
surgery. Portions of the removed tumor were cut
into explants of approximately 0.5 mm.3 and
used to establish cultures in 30 ml. plastic tissue
culture flasks by methods previously described.1
All cultures were incubated at 37° C. in medium
number 1640 with 20 per cent fetal bovine serum.
The fluid was changed 3 times weekly. The
cells adhering to the surface of the culture flasks
were trypsinized with 0.25 per cent trypsin in
Dulbecco's Tris buffer free of calcium and mag-
From the Armed Forces Institute of Pathology,
Washington, D. C , and the National Cancer
Institute, Bethesda, Md.
°This work was done in part while in National
Institutes of Health Special Fellow in Ophthalmic Pathology at the Armed Forces Institute of
Pathology, Washington, D. C.
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Study of retinoblastoma 65
Fig. 1. Cells growing on plastic surface 12 weeks following establishment of the culture.
(Unstained; light-field microscopy; xl75.)
Results
From 36 to 96 hours after tissue was put
into culture, cells had begun to migrate
from the edges of some explants. These
were predominantly spindle shaped cells
and small epithelioid cells although polygonal cells and cells with long thin cytoplasmic processes with fan-shaped endings
were seen. As cell proliferation continued
and the cells became more confluent, spindle shaped cells predominated (Fig. 1).
Cells scraped from the plastic surface
during the first three weeks resembled cells
taken directly from the tumor. Microtubules were plentiful at this time. Thereafter, however, the cells gradually developed a large amount of cytoplasm, a highly
developed endoplasmic reticulum, cisternae filled with proteinaceous-appearing
material, and numerous cell processes (Fig.
2). Microtubules were rare in cells attached to the plastic surface after the third
week.
At periods ranging from 2 to 12 weeks,
clumps of cells were noted detaching from
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the surface of the flask and eventually
floating free in the medium (Fig. 3). In the
cultures from 2 of the 4 tumors, the floating
clumps of cells gradually increased in number and size and were subcultured independently of the cells adhering to the plastic. Light microscopic examination of the
cells in suspension culture revealed predominantly small, round, undifferentiated
cells with little cytoplasm, and darkly hyperchromatic nuclei (Fig. 4). In addition,
better differentiated cuboidal or low columnar cells were seen arranged radially
around a central lumen with processes projecting into the lumen and nuclei basally
located (Fig. 5). These had the appearance
of typical Flexner-Wintersteiner rosettes.
Numerous foci of necrotic-appearing cells
were also seen. The cellular components of
the suspension cultures were extremely
similar in appearance to the original tumor
material.
The electron microscopic appearance of
the suspension culture also closely resembled that of the original tumor (Figs. 6, 7,
66 Albert, Rabson, and Dalton
Investigative Ophthalmology
January 1970
Fig. 2. Electron micrograph of a cell from culture shown in Fig. 1. There is abundant cytoplasm and highly developed endoplasmic reticulum. (Glutaraldehyde fixation; approx.
xl3,000.)
and 8) with similarly appearing nuclei,
nucleoli, mitochondria, endoplasmic reticulum, and Golgi apparatus. Particularly
striking in both cells taken directly from
the tumors and cells in vitro, was the presence of large numbers of microtubules.
These structures were present both in glutaraldehyde and chrome osmium fixed specimens. The tubules had an outside diameter of 200 A and a wall thickness of 55 A.
The length varied in different cells but
continuous tubules of 1.2/A were observed.
On cross sections some appeared to con-
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tain a central rod. No vims particles or
secretory granules were observed in any
of the cells.
Clumps of retinoblastoma cells growing
in suspension culture were introduced into
flasks containing monolayers of WI 38 human embryonic fibroblasts. In these cultures the previously floating clumps adhered to the monolayer and appeared to
destroy the underlying fibroblasts with
clear zones developing around the retinoblastoma cells. On inverted microscope examination of the cultures, the tumor cells
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Fig. 3. Dark clusters of retinoblastoma cells developing from "stroma" on plastic surface
following three weeks in culture. (Unstained, light field microscopy; *85.)
Fig. 4. Low-power view of pellet formed from cells growing in suspension 8 months after
the establishment of the culture. (Hematoxylin and eosin; *40.)
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Albert, Rabson, and Dalton
Investigative Ophthalmology
January 1970
Fig. 5. Higher power view of portion of field shown in Fig. 4, with characteristic FlexnerWintersteiner rosettes (Hematoxylin and eosin; x800.)
appeared to grow in mounds, from which
were projecting columns with hollow centers (Fig. 9).
Discussion
In earlier studies of retinoblastoma
grown in tissue culture, free floating clumps
either did not develop or went unobserved.
Kersting2 described the characteristic cell
in his cultures as having a round nucleus
which was rich in chromatin, and a unipolar or bipolar shape. Yoneda and Van
Herick3 noted a variety of cell types in their
cultures and observed that the cells generally changed from polygonal shape to spindle shape and back again. In contrast to
these reports, in the present experiments
cells grew in clumps suspended in the
medium without attaching to the culture
flask surface. This manner of growth resembled that of cell lines established from
patients with Burkitt's tumor and leukemia
patients, and differed strikingly from the
usual growth of solid tumors in vitro. The
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appearance of the individual cells and the
production of Flexner-Wintersteiner rosettes make unlikely the possibility that the
clumps in suspension represented anything
other than cells derived from retinoblastoma.
The cells in suspension cultures showed
a marked tendency to undergo necrosis, a
prominent feature of retinoblastoma in
vivo. This degree of necrosis was not seen
in cultures of Burkitt's cells and leukemia
cells grown in a similar manner. It may
be that the rapid degeneration of retinoblastoma cells results from factors inherent
in the cells and is not the result of immunologic factors. This is consistent, moreover,
with the observation in certain patients
with bilateral retinoblastoma of necrosis
of the tumor and regression in one eye, and
exhuberant growth and extraocular extension in the fellow eye.
Micro tubules were observed both in the
original tumor and in cells in culture. These
structures are cytoplasmic inclusions of
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Study of retinoblastoma 69
Fig. 6. Parts of several typical retinoblastoma cells from original explants used for tissue culture. Microtubules are numerous. (Chrome osmium fixation; approx. xl7,500.)
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70 Albert, Rabson, and Dalton
Investigative Ophthalmology
January 1970
Fig. 7. Parts of several cells from a cluster growing in suspension in a culture which had
been maintained for 8 months. (Glutaraldehyde fixation; approx. xll,500.)
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Fig. 8. Microtubules present in cell growing in suspension 8 months after the culture was
initiated. (Glutaraldehyde fixation; approx. x50,000,)
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72 Albert, Rabson, and Dalton
Investigative Ophthalmology
January 1970
Fig. 9. A mound of retinoblastonia cells which were formerly growing in suspension adhering
to monolayer of WI 38 human embryonic fibroblasts. {Unstained, light-field microscopy;
x200.)
wide distribution which have an outside
diameter of 150 to 270 A, a wall thickness
of approximately 50 A, and are of indefinite length. Their presence in photoreceptor cells, bipolar cells, horizontal cells,
ganglion cells, Miiller cells, and other
glial cells, and in the nerve fiber layer has
been described by several investigators.4"8
Usually microtubules can be demonstrated
only after glutaraldehyde fixation. The
tubular structures observed by Fine,4 however, in the photoreceptor axon could be
demonstrated in chrome osmium fixed tissue as well as glutaraldehyde. In both
retinoblastoma cells taken directly from the
tumor and those observed in the suspension culture, microtubules are also well
fixed by both glutaraldehyde and chrome
osmium fixation. This finding is consistent
with recent evidence that retinoblastoma
cells in vivo have the potential to form
photoreceptor elements.9
REFERENCES
1. Albert, D. M., Rabson, A. S., and Dalton,
A. J.: In vitro neoplastic transformation of
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uveal and retinal tissue by oncogenic DNA
viruses, INVEST. OPHTHAL. 7: 357,
1968.
2. Kersting, C : Die Gewebszuchtung menschlicher Hirngeschwiiste, Heidelberg, 1961,
Springer-Verlag.
3. Yoneda, C., and Van Herick, W.: Tissue culture cell strain derived from retinoblastoma,
Amer. J. Ophthal. 55: 987, 1963.
4. Fine, B. S.: Observations on the axoplasm of
neural elements in the human retina, Third
European Regional Conference on Electron
Microscopy, 1964, p. 319.
5. Kuwabara, T.: Microtubules in the retina, in
Rohen, J. W., editor: Eye structure, II. Symposium, Stuttgart, 1965, Schattauer-Verlag, pp,
69-84.
6. Sheffield, J. B.: Microtubules in the outer nuclear layer of rabbit retina, J. Microscopie
5: 173, 1966.
7. Anderson, D, R., Hoyt, W. F., and Hogan,
M. J.: The fine structure of the astroglia in
the human optic nerve and optic nerve head,
Tr. Am. Ophthal. Soc. 05: 274, 1987.
8. Dowling, J. E., and Werblin, F. S.: Organization of retina of the mudpuppy, Necturus
muculosus. I. Synaptic structure, J. Neurophysiol. 32: 315, 1969.
9. Ts'o, M., Fine, B. S., Zimmerman, L. E., and
Vogel, M. H.: Photoreceptor elements in
retinoblastoma, Arch. Ophthal. 82: 57, 1969.