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Effects of concentration of media and elevated temperature on cell division in tissue cultures of chick tissue.

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EFFECTS O F CONCENTRATION O F MEDIA AND
ELEVATED TEMPERATURE ON CELL
DIVISION I N TISSUE CULTURES
O F CHICK TISSUE1
E. FRANCES STILWELL
Department of Anatomy, Woman’s Medical College
of Pennsylvania, Philadelphia
FIVE FIGURES
Many diverse chemical and physical agents have been used
experimentally in the study of cells in relation to normal
and neoplastic growth phenomena. The range of visible
responses of cells to injurious agents appears to be limited
for the most part to mitotic aberrations which reveal themselves microscopically more readily than insults to the interphase cell. Ludford (’53) reviews some of the early, as well
as more recent experimental work in this field and points
out that “the property of disturbing cell division in some
way or other is one which can be exhibited by almost any
substance under appropriate conditions, particularly in tissue
cultures, though its only action may be to stop mitosis completely.” Although quite unrelated agents have been found
to evoke similar mitotic derangements, it does not follow
that the same cell constituent or fundamental metabolic
process is disturbed by the various agents employed.
It has been shown in a series of experiments that mitotic
aberrations, especially multipolar division figures, increase in
number in primary cultures of embryonic chick heart tissue
exposed to elevated temperatures (42 rt O C.) for brief periods
This investigation has been supported in part by grants from the American
Philosophical Society. The photomicrographs were made by Mr. Paul J. Hollway,
Woman ’s Medical College of Pennsylvania.
15
16
E. FRANCES STILWELL
of time (4hour and three hours) as well as for longer periods
of time (8 2 days) (Stilwell, '44,'47, '52a,b). At the same
time attention was directed to the possible role a nutrient
medium, rich in nucleoprotein fractions of embryonic extract,
might play in these experiments. It is the purpose of this
paper to present the results of a series of experiments designed
to test the effect of low and high concentration of embryonic
extract in the culture media upon the production of multipolar
mitoses in tissue cultures of embryonic chick heart tissue
incubated at normal and elevated temperatures.
MATERIAL A N D METHODS
The technique of culturing involved no unusual features.
The double cover glass method of Maximow was employed in
three series of cultures of 8 day embryonic chick heart tissue
which were incubated for 48 hours at three temperatures,
namely: 38" C.; 40" C.; and 42 -L. C.
The culture media consisted of reconstituted fowl plasma
(Difco Company) and freshly prepared embryonic extract of
8 day old chick embryos. The following procedure was carried
out in the preparation of embryonic extract. Upon removal
from the eggs, the embryos were washed in saline solution
before transfer to a mincing apparatus. The undiluted embryonic brei was allowed to stand in the centrifuge tube at
room temperature for 20 minutes before centrifugation at high
speed. The highly viscid supernatant fluid was withdrawn
and diluted as follows:
( a ) 1part embryonic juice to 6 or 7 parts TC balanced
saline solution (Difco Company) constituted dilute
embryonic juice.
(b) 6 parts embryonic juice to 1 part TC balanced
saline solution (Difco Company) constituted concelztrated embryonic extract.
The final culture medium consisted of one drop of plasma
to which was added an equal amount of either dilute or
concentrated embryonic extract, yielding a medium of approximately 7% and 42% embryonic extract respectively.
MULTIPOLAR MITOSES
17
Following the period of incubation all cultures were fixed
according to the method of Champy as in toto mounts and
stained by Heidenhain's iron hematoxylin method as modified
by Patten and Wigoder ( '29).
The microscopic study and mitotic counts of cultures were
made with the aid of a 2 mm apochromatic oil immersion lens
(N.A. 1.4) and 12.5 )( compensating oculars except in a
specified instance (see table 1). The method employed in
previous work (Stilwell, '47,52a) was adopted for the counting
of mitotic figures. A statistical treatment of the results
is not warranted since multipolarity in itself is an all or
none process and the mitotic counts were checked in only a
few instances. In all cases the location of each multipolar
division figure and certain other atypical configurations encountered were recorded as readings from the vernier scales
accompanied by a free hand sketch of the cell involved.
CYTOLOGICAL STUDY O F CULTURES
Series 1. Temperature of incubatiorz 38' C.;
48 hours i.rz vitro
Of the 8 cultures comprising this series, 4 received dilute
embryonic extract and 4 received concentrated embryonic
extract.
Microscopic examination of the living cultures following 24
hours incubation revealed a marginal growth of comparable
width and density in all eight cultures. There was, however,
a slight increase in the number of fat droplets within the cells
of the marginal growth of the cultures which received the
stronger extract.
The microscopic study and mitotic counts (table 1) of the
fixed and stained cultures likewise fail to reveal significant
differences in the two groups of cultures, attributable to the
concentrations of the embryonic extracts employed. The
marginal growth appears to be typical for primary cultures
of 48 hours cultivation. The zone of cell growth and migration
immediately surrounding the circumference of the original
tissue fragment consists of a dense felt work of more or less
Cone.
Dilute
4
4
Cone.
Dilute
4
4
Cone.
4
Dilute
42
-+ "C.
42 h "C
40°C
40°C.
38°C.
38°C
TEMPERATURE O F
INCUBATION
Counted with high dry objective.
Series 3
Series 2
Series 1
4
C0N CE N.
NUMBER
TRATION
OF
OF
CULTURES EMBRYONIC
EXTRACT
-+
512 &
299 &
2082
1848 2
1433 &
2025 &
TOTAL
NUMBER
OF
MITOSES
52
3
3
+ 38
+18
6+1?
5+3?
7
MIPOSES
5,":;-
NUMBER
OF
TOTAL
Mitotic counts
TABLE 1
:
:96
:694
:308
1:287
:289
RATIO O F
MULTIPOLAR
MITOSES TO
TOTAL NUMBER
OW MITOSES
DISTRIBUTION
Culture 1 3 - 2
Culture 1 4 - 1
Culture 15 - 1
Culture 32 - 2
Culture 27 - 1 4
Culture 30 - 18
Culture 12 - 0
Culture 1 3 - 1P
Culture 24 - 12
Culture 25 - 8
Culture 15 - 2
Culture 16 - 1
Culture 26 - 3
Culture 27 - 0
+ 17
+ 2B
+18
+ I?
Culture 23 0
Culture 24 - 0
-
Culture 11 2
Culture 13 - 1
-
C u l t u r e 2 3 - 1 + I ? Culture27-3
Culture 24 - 11
Culture 28 - 1
Culture 11- 4
Culture 12 - 0
O F MULTIPOLAR MITOSES
F
G
ld
1?I?
cn
5
M
MULTIPOLAR MITOSES
19
stellate cells outside of which there is an area of equal width
consisting of closely associated, radially oriented, spindle
shaped fibroblasts.
Reference to table 1 shows that the distribution of the
12 (+351) multipolar division figures present in this series
of cultures gives no evidence of any influence of the dilute
versus the concentrated embryonic extract upon the occurrence of this type of aberrant mitoses; the ratio of one multipolar figure to approximately 300 bipolar mitoses holds for
both concentrations of extract.
Figure 1shows one of 6 similar reconstruction figures which
are present in this group of cultures. The two or three incipient
daughter cells, inte!rpreted as arising from a tetrapolar
mitosis, are connected by a tenuous cytoplasmic strand in
the midst of which a midbody is located. It is noteworthy that
this midbody and those of similar cells is conspicuously larger
in size than those present in bipolar division figures. Another
feature of interest is the presence of chromosome vesicles
of variable size in the vicinity of the reconstructed nuclei.
Such vesicles may result from lagging chromosomes or from
chromosomes which fail to become attached properly to fibers
of the spindle. I n either instance the chromosomes may be
reorganized during late telophase as chromosome vesicles
which fail to be incorporated in the daughter nuclei. Further
evidence of disturbance in the orderly processes characteristic
of normal bipolar mitosis is seen in the uneven cytoplasmic
cleavage which in this instance could have led to two binucleated cells or one binucleated and two-uninucleated cells
rather than 4 uninucleated daughter cells. There seems to be
little doubt that an unequal distribution of the number of
chromosomes among the daughter cells may result in either
event.
Series 2. Temperature of incubation 40' C.;
48 hours
vih-0
Of the 8 cultures comprising this series 4 received dilute
embryonic extract and 4 received concentrated embryonic
extract.
20
E. FRANCES STILWELL
The microscopic study and mitotic counts (table 1) of the
fixed and stained cultures fail to reveal consistent differences
in the two groups of cultures attributable to the concentration
of embryonic extract employed. While the marginal zone of
cell growth and migration is quite comparable in width and
density to that observed in cultures of Series 1, the cells in
the peripheral area appear to be less regular in their radial
orientation. Furthermore, a considerable number of cells in
this area tend to be irregular rather than spindle shaped.
Variations in the size of cells are also observable. Early and
late prophases of extraordinary size (polyploid) are illustrated in figures 2 and 3.
Reference to table 1shows that the total number of mitoses
as well as the total number and distribution of multipolar
division figures is within the same order of magnitude as that
found in Series 1. It will be noted that the number of multipolar mitoses (6 1?) present in the 4 cultures which received
dilute extract is twice that found in the group of cultures
which received concentrated extract. This difference may be
more apparent than real since culture 23 of the latter group
did not permit study with oil immersion and it is quite
possible that atypical division figures present among the
316
mitoses in this culture failed to be recognized.
+
Series 3. Temperature of incubation 42
48 hours in vitro
c.;
Of the 8 cultures comprising this series 4 received dilute
embryonic extract and 4 received concentrated embryonic
extract. During the period of incubation the temperature of
the incubator fell to 41 O C. and rose to 43" C. within a period
of three hours.
Microscopic examination of the fixed and the stained cultures
of this series reveals striking differences between it and the
preceding series. The pattern of growth is markedly inferior
to that found in cultures of Series 1 and Series 2. The
marginal zone of cell growth and migration is not only narrow
MULTIPOLAR MITOSES
21
and irregular but there are numerous signs of cell degeneration, such as cell fragments and cellular debris; vacuolated
cytoplasm and indistinct boundaries of irregularly shaped
interphase cells. The differences observable between the two
groups of cultures comprising Series 3 are quantitative rather
than qualitative ; cultures which received concentrated embryonic extract show fewer cells exhibiting signs of injury
than those which received dilute embryonic extract. Among
the 52 (+34) multipolar figures present in the group of
cultures which received concentrated extract all stages of the
mitotic cycle (except prophase) are represented. The vast
majority of these figures are tripolar rather than tetrapolar.
Many variations involving the size and number ( 2 or 3) of
spindles as well as distribution and number of chromosomes
are presented by the multipolar division figures. A tripolar
mitosis with two spindles over which the chromosomes are
widely scattered is shown in figure 4. Similar disturbances in
the distribution of chromosomes upon the spindle are also
encountered in bipolar mitotic figures. Contiguous cells each
exhibiting a tripolar anaphase are illustrated in figure 5.
Mitotic aberrations other than multipolar division figures
are present in these cultures but only those of rare occurrence
and of particular significance require comment. Cultures 24
and 27 (Series 3) each exhibit a single cell undergoing endomitosis. While binucleated interphase cells occur not infrequently only two examples of the synchronous division
(anaphases) of the nuclei of such cells are present (culture 30,
Series 3; culture 13, Series 2).
Reference to mitotic counts (table 1) shows that the total
number of mitoses as well as the total number of multipolar
division figures of Series 3 is of a different order of magnitude
compared to the number obtained f o r Series 1 and Series 2.
I n both groups of cultures comprising Series 3 the total
mitotic counts are much lower than those of Series 1 and
Series 2 but the ratio of multipolar mitoses to total mitoses is
much higher than in any group of Series 1 and Series 2.
22
E. FRANCES STILWELL
I n the group of cultures (Series 3) which received dilute
embryonic extract the multipolar mitoses are distributed as
follows : 2 in culture 32 ; 1each in cultures 13 and 15 ; none in
culture 12. A striking increase in the number of multipolar
division figures per culture occurs in the group of cultures
which received concentrated embryonic extract. I n these
cultures the distribution is the following: 18 11 in culture
30; 14 2 ? in culture 27; 12 in culture 24; 8 in culture 25.
It is noteworthy that the lowest number (8) per culture in
this group is double the highest number present in any culture
(Culture 11, Series 1)of Series 1and Series 2.
+
+
DIBCUSSION
Cytological study and mitotic counts have been carried out
on primary tissue cultures of embryonic chick heart tissue
grown under experimental conditions in an attempt to disclose
the influence of elevated temperature versus concentration of
growth promoting substances (nucleoprotein fractions) of the
media upon the occurrence of multipolarity in dividing cells.
The variables introduced in the experimental procedure were
accordingly, temperature of incubation (38" C., 40" C., and
42 O C.) and concentration of embryonic extract (7% versus
42%) in the culture media.
Cytology. While the cytological study of the cultures
reveals a wide range of variations in the configurations of
multipolar mitoses, only a few are illustrated in figures 1-5.
There is no indication of any specificity of action in the experimental variables, but it should be noted that the colchicine
effect, i.e. inhibition of spindle formation, is absent.
It seems pointless to speculate regarding the potentialities
and ultimate fate of the depicted daughter cells of aberrant
mitoses since it was not possible to obtain cinematographic
records of the living cultures. Instead attention will be
focused upon certain minutiae of the dividing cells as they
appear in fixed and stained preparations.
The late reconstruction phase (fig. 1) resulting from a
tetrapolar mitosis is representative of silmilar figures present
MULTIPOLAR MITOSES
23
in the cultures. I n all cases a conspicuous midbody is located
within the slender cytoplasmic strand connecting the daughter
cells. The midbodies of these cells appear to be appreciably
larger than those of bipolar division figures. Although there
is little agreement upon the nature and significance of the
midbody (Schrader, '53) it is well known that in animal cells
it is generally less well developed than in cells of higher
plants (Wilson, '25). Recent studies (Jacobson and Webb,
'52; Ris and Kleinfeld, '52) on the shedding of ribonucleoproteins by chromosomes during anaphase suggests the possibility that the midbody may be the residuum of such superfluous material, in which case, the size of the midbody would
be a reflection of the number of chromosomes involved in a
multipolar mitosis of a specific type of cell.
Figure 4 shows a tripolar mitosis in which the numerous
chromosomes are widely scattered over the two spindles
presumably due to a disturbed synchronization during anaphase in the poleward movement of chromosomes. The phenomenon is not confined to multipolar mitosis but occurs in
bipolar figures as well. It has been observed in cells treated
with carcinogenic hydrocarbons and arsenicals (Ludford, '53).
Two modifications of cell division, namely endomitosis and
synchronous mitoses in a binucleate cell are rare phenomena.
However, they, as well as multipolar mitosis, may lead to a
condition of polyploidy and/or heteroploidy in daughter cells
(Beams and King, '42; Biesle, Poyner and Painter, '42;
Geitler, '39 ; Jacoby, '40 ; Stilwell, '47, '52a, '52b ; Beatty,
'54). The extraordinary size of the prophases in figures 2
and 3 $may be attributed to a condition of polyploidy, the
origin of which was undoubtedly some type of aberrant mitotic division. The assumption of Hsu ('52 and '53) that
endomitosis is the most probable abnormality in nuclear
phenomena leading to variation in chromosome number seems
to call for substantiation.
Mitotic counts. Attention may be drawn to certain features inherent in the experimental material before an evaluation of the results of mitotic counts is presented. It is well
24
E. FRANCES STILWELL
known that conditions of life in vitro “per se” may affect
cells adversely, evoking changes in cell behavior. This fact
has been expressed succinctly by Ludford (’53) who states
“With tissue cultures the problem is rather how to attain
the right conditions in order to sustain the normal growth of
cells. Almost any devitaion from the proper culture technique
will influence mitosis adversely.” Furthermore, it is recognized that cells of primary cultures are in a less stable state
of equilibrium with the culture medium than those of cultures
which have undergone a series of transfers in vitro. It is,
then, not surprising but rather to be anticipated, that a cell
by cell study reveals atypical cells including mitotic aberrations, such as multipolar division figures, in primary cultures
grown under “non-experimental conditions. ” The problem is
complicated further by the fact that multipolarity in cell
division is an all or none phenomenon which occurs sporadically in so-called normal tissue.
With these facts in mind only a marked change in the ratio
of multipolar mitoses to total number of mitoses has been
interpreted as an effect attributable to the experimental procedure. Table 1 shows that only in Series 3, incubated at
42 +- C., is there a marked deviation from the results
obtained in Series 1 and Series 2. I n both groups of cultures
of Series 3 the total mitotic counts are much lower than those
of Series 1and Series 2. On the other hand the ratio of multipolar mitoses to total mitoses in both groups of Series 3 is
much higher than in any group of Series 1and Series 2. Under
the conditions of the experiments, it appears that the rate of
cell division decreased but the ratio of multipolar mitoses to
total mitoses increased with the elevation of temperature of
incubation to 42 “C.
On the basis of mitotic counts no effect attributable to the
concentration of embryonic extract (776 versus 42%) in the
culture medium is evident in the groups of cultures of Series
1and Series 2. However, the mitotic counts reveal conspicuous
differences between the two groups of cultures comprising
Series 3 (table 1). Comparison of the group of cultures which
O
MULTIPOLAR MITOSES
25
received dilute medium (7% embryonic extract) with the
group which received concentrated medium (42% embryonic
extract) shows that the total number of mitoses is in the ratio
of 3 : 5 whereas multipolar mitoses are in the ratio of 1:10.
Until data from a more extensive series of experiments are
available no definite conclusions will be drawn from the present study. The results do lend support to the writer's earlier
suspicions (Stilwell, '44,'47 and '52a) that a culture medium
of high nucleoprotein content may be instrumental in evoking
a particular type of mitotic aberration, namely, multipolar cell
division, when cultures are incubated at elevated temperature
(42 O C.). The experiments yield no information as to
what mitotic mechanism o r metabolic process is disturbed in
the production of multipolarity. The utilization of extracellular ribonucleic acid has,been demonstrated by Montalenti et
al. ('50) in the initiation of meiosis during the spermatogenic
cycle of a crustacean, Asellus aquaticus. On the basis of cytological studies Darlington ( '47) and Koller ( '43 and '47) postulate quantitative changes in the nucleic acid metabolism of
cells as the origin of mitotic disturbances in both normal
(experimental) and malignant tissues. Convincing data on
the effect of the constitution of culture media upon the synthesis of nucleic acids in short term, primary cultures of chick
heart tissue under conditions of inadequate and adequate
nutrition have been presented by Kirk and his co-workers
('50). Their analyses, however, do not include data from
cultures grown in a medium of unusually high concentration of
embryonic extract.
SUMMARY
A cytological study and mitotic counts have been carried
out on primary cultures of embryonic heart tissue grown
under experimental conditions in an attempt to disclose the
effect of elevated temperatures versus concentration of embryonic extract of the media upon the occurrence of multipolarity in dividing cells. Three series of cultures, each comprised of a group of cultures which received dilute extract
26
E. FRANCES STILWELL
(7% embryonic extract) and a group which received concentrated extract (42% embryonic extract) were incubated
at 38 O C. (Series 1); 40 C. (Series 2) ; and 42
C.
(Series 3).
A marked change in the ratio of number of multipolar mitoses to total number of mitoses present in the groups of
cultures is interpreted as an effect attributable to the experimental procedure. The results of the mitotic counts and microscopic study lend support to the writer’s earlier suspicions
that a culture medinm of high nucleoprotein content may be
instrumental in evoking multipolarity in dividing cells in
cultures incubated at elevated temperature (42 f O C.). The
experiments are discussed briefly in relation to current reports
on the role of nucleic acids in cell division.
O
O
LITERATURE CITED
BEAMS, H. W., AND R. L. KING 1942 The origin of binucleate and large
mononucleate cells in the liver of the rat. Anat. Rec., 89: 281.
BEATTY,R. A. 1954 How many chromosomes in mammalian somatic cells?
Internal. Rev. Cytology, 3: 177.
BIESELE,J. J., H. POYNER
AND T. S. PAINTER
1942 Nuclear phenomena in
mouse cancers. The University of Texas Publication, No. 4243.
DARLINQTON,
C. D. 1947 Nucleic acid and the chromosomes. Symp. SOC.exp.
Biol., 1: 252.
GEITLER,L. 1939 Die Entstehung der polyploiden Somakerne der Heteropteren
durch Chromosomenteilung ohne Kernteilung. Chromosoma, 1 : 1.
HSU, T. C. 1952 Tissue culture studies on human skin. 111. Some cytological
features of the outgrowth of epithelial cells. Texas Reports on Biol.
and Med., 10: 336.
Hsu, T. C., AND C. M. POMERAT
1953 Mammalian chromosomes in Vitro. 111.
On somatic ‘meuploidy. J. Morph., 93: 301.
HULL,W., AND P. L. KIRK 1950 Tissue culture studies. 11. The relationship
of nucleic acid increase t o growth of cells in vitro. J. Gen. Physiol.,
33: 327.
JACOBSON,
W., AND M. WEBB 1952 The two types of nucleoproteins during
mitosis. Exp. Cell Research, 9: 163.
JACOBY,
F. 1940 Synchronous mitosis in binucleate macrophage in vitro. J.
Physiol., 98: 6P.
KOLLER,P. C. 1943 Origin of malignant tumor cells. Nature, 152: 244.
1947 The experimental modification of nucleic acid systems in the
cell. Symp. SOC.exp. Biol., 1: 270.
MULTIPOLAR MITOSES
27
LUDFORD,
R. J. 1953 I. Chemically induced derangements of cell division. J.
Roy. Micr. Soc., 73: 1.
MONTALENTI,
G., G. VITAQLIANO AND M. DE NICOLA1950 The supply of ribonucleic acid to the male germ cells during meiosis in Asellus aquaticus.
Heredity, 4 : 75.
PATTON,
R. E. P., AND S. B. WIGODER1929 The cytological changes observable
in irradiated bean root tips, Quart. J. Micr. Sci., 73: 633.
RIS, H., AND R. RLEINFELD
1952 Cytochemical studies on the chromatin elimination in Solenobia (Lepidoptera). Chromosoma, 5: 363.
SCHRADER,
F. 1953 Nitosis. Second Ed. Columbia University Press, New York.
STILWELL,
E. F. 1944 The production of multipolar mitoses in normal somatic
cells of embryonic chick tissue grown in vitro. Anat. Rec., 90: 115.
1947 The influence of temperature variation upon the occurrence
of multipolar mitoses in embryonic cells grown in vitro. Anat. Rec.,
99: 227.
1952a The effect of elevated temperature upon the occurrence of
multipolar mitoses in embryonic cells grown in vitro. Anat. h.,
12.2: 195.
1952b Observation of endomitosis in embryonic chick cells grown
in vitro. Anat. Rec., 114: 9.
WILSON,E. B. 1925 The Cell i n Development and Heredity. Third Ed. The
Macmillan Co., New York.
PLATE 1
EXPLANATION OF FIGURES
Photomicrographs and outline tracings (facing). Reduction one-tenth.
1 Tetrapolar late reconstruction phase exhibiting 4 nuclei and chromosome
vesicles; conspicuous midbody of unusual size. Culture 11, series 1 (38"C., dil.
extract, 48 hrs. i n vitro). X 1350.
2 Early prophase (polyploid) exhibiting intact nuclear membrane and 4
conspicuous nucleoli. Culture 11, series 2 (4OoC., dil. extract, 48 hrs. in vitro).
X 1350.
3 Late prophase (polyploid).
hrs. in vitro). X 1350.
Culture 13, series 2 (40"C., dil. extract, 48
4 Tripolar anaphase exhibiting chromosomes scattered over 2 spindles. Culture 30, series 3 (42 & "C., conc. extract, 48 hrs. in vitro). X 1350.
5 Tripolar anaphases of contiguous cells only one of which is in the optical
plane of the photomicrograph. Culture 30, series 3 (42 2 OC., cone. extract, 48
hrs. in vitro). X 1350.
MULTIPOLAR MITOSES
E. BRANCES STILWELL
Fig 2
\
28
PLATE I
29
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