Karyologic studies on cells from rabbit
cornea and other tissues
grown in vitro
P. Sarkar,* P. K. Basu, and Irene Miller
Individual layers of the cornea as well as lung tissues of rabbit were cultured separately in
vitro. The endothelial and stromal cells of the cornea maintained their distinctive tissue culture
morphology in serial subcultures for a period of over 20 months, and in the case of one endothelial line, even in passages after thatuing from storage at -79° C. rThese cell lines proved to
be very suitable material for chromosome study. Normal karyotype of rabbit has been studied,
from some of these lines derived from the corneal and. lung tissues. One cell line derived from
the corneal endothelium loas stored at -79° C. for over 3 months and subsequently thawed,
and subsequent passages after thawing, showed heteroploid. transformation of chromosomes,
and subsequent passages, after thawing shoiced heteroploid transformation of chromosomes.
The possible application of karyologic studies of corneal cells in ophthalmic research is discussed.
R
Karyologic studies of the ocular tissues
have been very limited. Mitotic figures
have been studied in histologic preparations of the corneal and lens tissues.:l"r'
Radioisotopes have been used recently to
study the desoxyribose nucleic acid (DNA)
synthesis in the lens epithelium and corneal
cells.0' 7 The sex chromatin, which is a
heterochromatic mass found in the female
nucleus, has been identified in ocular tissues.8 Basu and co-workers9 have studied
the fate of corneal grafts in cats with the
sex chromatin as a biologic cell marker.
However, no detailed account of the
chromosomes in ocular tissues, particularly
of the cornea, could be found in the literature. So far as we are aware, no attempt
has been made to study the chromosomes
in the stromal and endothelial cells of the
adult mammalian cornea.
The present investigation was undertaken to study the karyology of normal
corneal tissues of mammalian species, and
also to establish cell lines in vitro from the
'ecent advances in the field of mammalian cytology, particularly the use of
tissue culture techniques for the study of
chromosomes, may be profitably applied in
ophthalmologic research. The karyology of
the corneal cells would be of considerable
interest if the number and morphology of
the chromosomes could be utilized as a
tool for the study of the fate of corneal
grafts as well as the pathogenesis of abnormal cellular growths resulting from certain corneal lesions. Furthermore, routine
chromosomal studies would be essential in
maintaining cell lines in vitro for virologic
and other investigations on the corneal
tissue.
From the Department of Ophthalmology, Faculty
of Medicine, University of Toronto, Toronto,
Ontario.
The studies were carried out under the National
Health Research Grant 605-7-151.
•Department of Botany, University of Toronto.
33
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34 Sarkar, Basu, and Miller
different layers of the cornea. A number
of species have been used in our laboratory
for the supply of corneal tissues, namely,
rabbit, mouse, Chinese hamster, and cat.
However, the present report is confined to
the karyologic studies of cells from the
adult rabbit cornea cultivated in vitro. The
main observations relate to: (a) the
morphology of cells grown in vitro from
the different layers of the cornea, (b) the
normal karyotype of rabbit as studied in
cells from short-term cultures of the corneal
and lung tissues, and (c) the heteroploid
transformation in one cell line derived
from the corneal endothelium.
Materials and methods
For the tissue culture and subsequent cytologic examination of corneal cells, the eyes of
male and female adult albino rabbits were used.
The animals were sacrificed and the eyes removed
immediately and submerged for 10 minutes in
Hanks' buffered saline solution which contained
500 units of penicillin and 250 units of streptomycin per milliliter. For the comparison of the
karyotype, tissues from the lung of the same animal were also cultured.
The procedure employed for the separation of
the individual layers of the cornea for culturing
in vitro was essentially that of Stocker and coworkers.10
The technique used for growing a tissue explant
and the subsequent subculturing of the cells from
the primary outgrowth was as follows: Tissue
fragments were planted in a Carrel flask containing a smear of chicken plasma and clotted with
chick embryo extract. Two milliliters of Eagle's
basic medium with 10 per cent sheep serum, and
100 units each of penicillin and streptomycin per
milliliter were used for culturing the cells.
When there was adequate cellular outgrowth,
tissue explants were removed from the flask and
the cellular outgrowths exposed to a 0.25 per cent
trypsin in phosphate buffered saline solution (pH
7.0) for 10 to 15 minutes at room temperature.
The cell suspension thus obtained was centrifuged at 1,100 r.p.m. for 3 minutes and the supernatant removed. The cells were then resuspended
in growth medium and transferred to new culture
containers. The cultures were maintained in an
incubator at 37° C. with passages at weekly
intervals.
For the study of chromosomes, following
trypsinization of a subculture, the cells were transferred to a 100 mm. Petri dish which contained
two 75 by 25 mm. slides in 15 ml. of tissue
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Investigative Ophthalmology
February 1962
culture medium. The Petri dish was incubated
in a humidified atmosphere containing 5 per cent
CCX. In about a week there was sufficient cellular
growth on the slides. Ten micrograms of colchicine
per milliliter of medium were added to each
Petri dish 10 to 16 hours before the slides were
removed. The cells attached to the slides were
then fixed and stained by the air-drying method
of Rothfels and Siminovitch.11
Samples from one of the cell lines of endothelial
derivation were frozen for storage after the
seventh passage. For this purpose the cells were
trypsinized and suspended in a culture medium
containing 15 per cent glycerin. Two milliliters
of this cell suspension were introduced in a 10
ml. ampule and "shell-frozen" in a mixture of dry
ice and acetone,12 and stored at -79° C. After
storage for 2>Vz months, one such sample was
thawed rapidly in a water bath at 37° C. The
thawed cells were found to be viable and successful cultures could be initiated from them. They
were subcultured in 8 ounce bottles by trypsinization at weekly intervals for a period of over 1
year. This is the line referred to later as the
established endothelial line. Chromosome studies
were made on cells of this line at the fourteenth
and later passages after thawing.
Results
The morphologic patterns of the cellular
outgrowths from the epithelium, stroma,
and endothelium of the rabbit cornea were
recognizably distinct from one another
(Figs. 1-3).
The primary epithelial outgrowth could
be preserved for approximately 10 days.
Our attempts to subculture the epithelial
cells of the rabbit cornea have so far been
unsuccessful.
The stromal and endothelial cells maintained their respective in vitro morphology
after many passages (Figs. 4 and 5), and
in the established endothelial line, even
after thawing and subsequent subculturing
(Fig. 6).
The normal karyotypes* of male and
female rabbits were determined from cells
in short-term cultures (at passage 2) derived from the stroma and endothelium
of the cornea as well as from fibroblasts
of the lung (Figs. 7-10). The karyotype
studied from the cells of the cornea and
"Karyotype, the characteristic chromosome complement
of any individual or group of related organisms.
Volume 1
Number 1
Karyologic studies on corneal cells 35
Figs. 1-3. Primary cellular outgrowths from corneal tissues of rabbit cultured in vitro: Fig. 1,
epithelial; Fig. 2, stromal; Fig. 3, endothelial. (x75.)
Figs. 4-6. Endothelial cells from rabbit cornea cultured in vitro: Fig. 4, at passage 2; Fig. 5,
at passage 22; Fig. 6, at passage 7 after thawing from storage at -79° C. (xl95.)
of tlie lung from the same animal was
identical. In our experience, the corneal
tissues were very suitable material for
chromosome studies.
The number of chromosomes in the
rabbit is 44, including the X and Y in the
male and the two X's in the female. Fig.
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10 shows paired chromosomes from a
typical nucleus of a male. It can be seen
that although every chromosome cannot be
unequivocally identified the chromosomes
fall into distinct groups as regards total
length and arm-ratio. The Y chromosome
in the male is similar to the two smallest
InoeUigatioe Ophthalmology
February 1962
36 Sarkar, Basu, and Miller
Figs. 7-9. Diploid chromosome complement of rabbit in cells cultivated in vitro: Fig. 7, from
stromal cells of cornea (male); Fig. 8, from lung cells (male); Fig. 9, from stromal cells of
cornea (female). (xl,200.)
fiW Kit H H
0
«»«» 8
in
11
10
AA
18
12
13
v
Ml HA 00
••
14
**
19
20
21
Y
Fig. 10. Karyotype of male rabbit. Camera lucida drawing. (x2;600.
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15
OJlAO
16
17
Karyologic studies on corneal cells 37
48
?0
52 54
56
58 60
62
64 66
68
70 72
74
76 78
No. of chromosomes
Fig. 11. Distribution of chromosome numbers in cells of the altered line derived from corneal
endothelium of rabbit. (One cell with 124 chromosomes is not included in the histogram.)
autosomes. The X is a medium-sized
chromosome with arm-ratio of approximately 1 to 3. The chromosomes numbered
18 and 19 (Fig. 10) are distinct from the
rest in being almost telocentric.*
In the established endothelial cell line
accurate chromosome counts were made
after the fourteenth passage following
thawing. Thirty-nine well-spread colchicine
metaphases were studied. The cells showed
a range of chromosome numbers from 48
to 124, of which about 85 per cent were
at a hypertriploidf level with the mode
around 70 (Fig. 11). No cells with the
diploid number of chromosomes were
found. Apart from this change in chromosome number, these cells also showed
alterations in chromosome structure, the
most conspicuous being the presence of
dicentric| chromosomes. About 20 per cent
of the cells had dicentric chromosomes and
one cell had three of them (Fig. 12).
Furthermore, there was a reduction in the
number of telocentric chromosomes in
these aneuploid§ cells as compared to the
diploid karyotype. The two pairs of small,
telocentric chromosomes characteristic of
rabbit (Fig. 10) were either absent or
represented by only one or two such
chromosomes in the altered cells.
A second batch of slides was prepared
from the same endothelial line about 4
months after the first chromosome study
was made. These preparations showed
chromosome numbers grouped close to 70.
Cells with chromosome numbers in the
lower range of 40's and 50's, as well as the
occasional cells with veiy high chromosome
numbers (more than 100), seemed to have
been completely eliminated. Fig. 13 shows
a metaphase with 70 chromosomes in one
of the cells from the second set of preparations.
It is noteworthy that in spite of the
heteroploid* transformation of the chromosomes the morphology of these cells remained unaltered as compared with that
of the primary outgrowth and serial subcultures of the endothelium (Figs. 3-6).
Discussion
°Telocentric chromosome, a chromosome with a terminal
centromere (or kinetochore).
f Hypertriploid cell, a cell with a chromosome number
slightly greater than three times the haploid (gametic)
number.
tDicentric chromosome, a chromosome with two centromeres (or kinetochores).
JAneuploid cell, a cell, not containing an exact multiple
of the haploid (gametic) number of chromosomes, one or
more chromosomes being represented more or less times
than the rest.
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In the primary culture, the epithelial,
stromal, and endothelial cells of the cornea
were distinct from one another in their
morphologic characteristics.
Because of our failure to subculture
"Heteroploid cell, a cell with a chromosome number which
is not an exact multiple of the haploid (gametic) number.
3S Sarkar, Basu, and Miller
Investigative Ophthalmology
February 1962
Figs. 12 and 13. Chromosome complements of heteroploid cells in the altered line derived
from corneal endothelium of rabbit: Fig. 12, metaphase with 64 chromosomes showing 3
dicentrics (arrow); Fig. 13, metaphase with 70 chromosomes. (xl,100.)
epithelial cells, we were unable to compare
the morphology of the subcultured epithelial cells with that of the primary outgrowth from the explant. However, it was
possible to subculture the stromal and the
endothelial cells; they retained their distinctive morphology in serial passages.
Endothelial cells remained viable during
storage for 3V2 months at -79° C.
Our observations on the chromosomal
changes in the established endothelial cell
line are in agreement with the usual experience of tissue culture workers handling
mammalian cells. Almost all such cultures,
once they become established, show unbalanced numbers of chromosomes,13"10 a
phenomenon which has been termed
heteroploid transformation.
This particular endothelial cell line was
not checked for chromosomal abnormalities
during the early period of its growth.
Hence, it was impossible to determine the
number of passages after which the altered
cells began to appear and eventually replace cells with normal chromosome complement.
In spite of very careful handling, there
have been cases of contamination of one
cell line with another in various labora-
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tories.17' 1S In our case, two possible sources
of contamination, viz., live cells in chicken
embryo extract used for clotting the
original explant, and in sheep serum used
in the growth medium can safely be excluded because of the very different
chromosome morphology of these two
species. In our altered line, the karyotypes,
particularly in the nuclei with chromosome
numbers between 48 and 50 of which there
were a few in the first batch of slides, were
very similar to the normal karyotype of
rabbit. Furthermore, at the time of the
chromosome study the cells showed morphologic features characteristic of the
endothelial cells. Thus, it may be assumed
that the cell line we are carrying is
genuinely of rabbit derivation and did not
arise from contaminating cells of other
species.
An interesting aspect of heteroploid
transformation may be discussed here. Almost eveiyone studying established cell
lines derived from normal tissues encounters heteroploidy. It is now speculated
whether once a cell population becomes
heteroploid it acquires malignant properties since neoplastic cells are usually
heteroploid.
Volume 1
Number 1
Ability to produce tumors upon inoculation into suitable hosts is a widely used
method of estimating neoplastic properties.10"'- In recent experiments, Rothfels
and associates-'1 induced tumors by inoculating strains of heteroploid mouse cells
in C3H mice. We intend to undertake
transplantation experiments using transformed cell lines derived from corneal
tissues to test their tumor-forming potentialities in the eye and brain of experimental animals.
Unrestricted cellular growths may be
assumed to be due to a breakdown of the
balance between the growth potential of
a cell population and its environment. The
normal aqueous humor is known to have
an inhibitory action on cellular growth.24' 25
However, under certain circumstances,
corneal cells will grow in the anterior
chamber in the form of epithelial downgrowth or fibrotic membranes in cases of
abnormal corneal wound healing. Conceivably, this could be due to an adaptive
genetic transformation of these cells in a
new environment. If so, such pathogenesis
could be better understood if it were possible to study the cytology of these cells.
Recent researches tend to show that a
large proportion of the undamaged cell
population of a corneal graft is able to survive without being replaced by the host
cells.9-2C The chromosome constitution of
the surviving cells of the graft is not
known. It is not impossible that in the
process of establishing itself, the grafted
tissue would undergo genetic changes as a
means of adaptation to the new environment, analogous to the behavior of a cell
population in tissue culture. If any such
alteration can be recognized at the chromosomal level, a karyotypic study of the
grafted tissues would offer important clues
regarding the biologic individuality of the
transplants.
We are hopeful that, with karyologic
studies of the cells from grafted tissues, it
will be possible to determine the fate of
the auto-, homo-, and heterologous grafts
more definitely.
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Karyologic studies on corneal cells 39
Addendum
After sending this paper to the press we came
across a publication by Melander27 on the
chromosome complement of the rabbit. We also
received a personal communication (to P. Sarkar)
from Dr. Johanna Clausen of the University of
Minnesota together with her unpublished data
on the rabbit chromosomes. The karyotypes of
the rabbit as worked out by these investigators
are in close agreement and differ with that shown
in Fig. 10 in the present publication only in
having 3 pairs instead of 4 pairs of almost telocentric small chromosomes.
We wish to thank Dr. K. H. Rothfels for his
comments, Dr. R. C. Parker for providing tissue
culture facilities at certain stages of our experiments and Miss E. Kupelwieser and Mr. R. Dunbar for technical assistance.
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