EFFECTS OF OSMOTIC PRESSURE ON NORMAL AND
MALIGNANT FIBROBLASTS
AUSTIN M. BRUES
AND
CLAIRE McTIERNAN MASTERS
(From the Medical Laboratories of the Collis P. Huntington Memorial Hospital
of Harvard University)
In view of the importance of osmotic pressure as a factor in the internal
environment of the organism, the investigation here described was made for
the purpose of noting the reactions of surviving and growing cells in vitro to
changes in osmotic conditions. Little previous work has touched on this problem. Hogue in 1919 (1) grew tissue cultures of chick embryo tissues and
established the limits of hypertonicity and hypotonicity in which they will survive and grow. She found that they grow (or at least migrate) more rapidly
in slightly hypotonic solutions and grow well in a medium consisting of NaCI
diluted to 0.5 per cent, and made a number of interesting morphological observations on cells grown under these conditions. In the early days of tissue
culture, Lewis (2) had observed that tissue cells would migrate under a wide
range of inorganic salt concentrations with various degrees of tonicity.
Fischer (3) discusses the permeability of macrophages and fibroblasts briefly,
with particular reference to the phenomenon of phagocytosis.
Shear and Fogg, in two recent papers (4, 5), have made observations on
the peripheral cells of excised tissues placed in various solutions, and noted in
the case of malignant tissues and some normal mouse tissues that cells show
globular swelling around their free margins in hypertonic and hypotonic solutions alike, beginning within the first few minutes of immersion and continuing
for hours. This phenomenon can be retarded by increasing the protein content of the solution and is minimal in a solution of 10 or 15 per cent horse
serum protein. Swellings which had already appeared in saline solutions were
seen to disappear on increasing the protein content of the solutions.
METHOD
It seemed important to devise some method of estimating the volume of
cells in tissue culture, and in this some difficulty was encountered. Fibroblasts grown in a fluid medium are found attached to the coverslip on which
the explant has been placed, and such cells have an unknown thickness, so that
it did not seem advisable to use them. Attempts to measure cells growing in a
meshwork of glass wool and on the fibrous net found in silkworm cocoons, and
on similar frameworks, failed because of the optical conditions and because
cells were asymmetrical wherever they touched the matrix.
In cultures of fibroblasts maintained in clotted plasma, the cells are found
growing through the plasma at various levels. Many of these cells are irregular in outline and have ameboid processes at the progressing end. Others
appear stretched by attachments at various points; some of these are attached
314
EFFECTS OF OSMOTIC PRESSURE ON FIBROBLASTS
315
at two points only and a spindle-shaped cell lies between. The length of
these cells is of the order of magnitude of 100 to 150 [J.; malignant fibroblasts
appearing from a freshly explanted sarcoma are in general broader than other
fibroblasts.
If we can assume that such bipolar cells are radially symmetrical around
the long axis, it should be possible to compute the volume on a basis of the
shape and apparent area. This seems to be a reasonable assumption, since
(1) all such typical spindle-shaped cells have reasonably regular borders as
far as can be seen from the point of observation, and (2) because such cells
as do not show marked ameboid processes have probably a sufficiently great
surface tension along the surface of contact with the medium to give a symmetrical shape at equilibrium. As a matter of fact, similarly shaped cells and
organisms are frequently seen in nature and their borders are made up of the
type of surface known as the unduloid (6). That the shape of the cell is not
distorted under the influence of gravity is obvious as a result of several observations made on cells viewed on the side instead of from above. A series
of explants were made by the usual coverslip technic and were kept on side in
the incubator for several hours and days. A number of cells were observed in
the same position as that in which they had been grown, and there was no
tendency of these cells to sag from the horizontal. Such sagging would not
be expected, since much larger cells show little or no tendency to sag or lose
symmetry under gravitational force (7).
It has, therefore, been assumed in these experiments that we are dealing
with radially symmetrical spindle-shaped cells, and such cells have been chosen
for measurement as appeared to approach this ideal shape as nearly as possible.
The material for the experiments consisted of explants in plasma of chick
and rat embryo heart, mouse sarcoma 180, and Walker rat tumor 256. In
some instances cells migrating from freshly explanted tissue were used; and in
others cultures were employed which had been grown under constant conditions for a greater or less length of time. The cultures were grown on mica
coverslips and suspended in a hanging plasma drop over a cylindrical well in
the culture slide. The tissues were grown with the customary precautions
used in tissue culture work, and special care was taken to seal the edges of the
coverslip as rapidly as possible with paraffin, in order to cut down evaporation. A drop of Tyrode solution, brought to pH 7.6 to 7.8 by passing CO~
through it, was placed in the well of the culture slide immediately before applying the coverslip in order to prevent further evaporation and to retard loss
of CO~ from the culture medium, in view of the relatively large size of the well.
The chambers in the slides had an average diameter of 1.4 em. and an average
depth of 0.38 cm., so that the average volume of the chamber would be about
0.58 c.c. As might be expected from this, the average weight of water which
these chambers held under experimental conditions was 600 mg. When desired, the fluid under the culture could be changed by filling the well through
a hypodermic needle, or by using a special chamber which gave a constant
flow of fluid under the coverslip.
The primary explants used were bits of tissue not over 1 mm. in diameter,
freshly excised from embryos or tumors. These were placed in a drop of fluid
316
AUSTIN M. BRUES AND CLAIRE MCTIERNAN MASTERS
consisting of 25 per cent heparinized chicken plasma in Tyrode solution with
a little chick embryo extract. The average diameter of these drops was 6 to
7 mm., and the average thickness at the deepest point was 0.2 to 0.3 mm.
The usual weight of these plasma cultures was 4 to 12 mg.
In estimating cell volume, such cultures were chosen for observation as
contained a symmetrical bipolar fibroblast which could be seen clearly and
was entirely in focus at one time. When a cell was chosen for study, it was
observed under a 90 X oil-immersion objective and drawn by means of an
Abbe camera lucida, so calibrated that the total magnification was 1400 diameters. Hence, a distance of 1 micron in the object examined would be
represented by a line 1.40 mm. long in the drawing.
After a series of drawings had been made, they were measured in the following way. A series of equidistant parallel lines were drawn across the celldiagram, perpendicular to its long axis, with a celluloid guide made for the
purpose. The area of the cell-diagram subtended by each adjacent pair of
lines then appeared as a trapezoid representing a section of the cell which, assuming radial symmetry, would have approximately the shape of a cylinder or
of a truncated cone. The lines were drawn 5 mm. apart and the sides cutting
across the cell were measured. Each segment was then calculated as a cylinder 5 rnm. high and with a radius equal to the mean of the two measured radii;
and by means of a table, the volume and lateral surface of each corresponding
segment of the original cell were calculated, taking into account the amount
of magnification. These quantities were then summed to give the volume
and surface of the cell, in cubic and square micra. It was soon discovered
that no significantly greater accuracy could be gained by dividing the celldiagram into a larger number of segments, or by calculating them as truncated
cones instead of as cylinders.
In order to check the accuracy of the camera lucida drawings, a few experiments were recorded photomicrographically, appropriately enlarged, and
measured. Neither method appears particularly superior to the other in respect to accuracy.
RESULTS
All healthy appearing cells which were placed in a hypotonic solution could
be observed to swell. This swelling took place rapidly at first, after a short
latent period in which, in all probability, the salts in the plasma medium began
to diffuse out. Then the rate of swelling became slower as the cell approached
an equilibrium volume. In another report (p. 324) we have attempted to determine under the same conditions the permeability of these cells to water as a
function of time.
Cells which have been in the same culture medium until they have ceased
to show activity will not swell under the influence of hypotonic solutions. If
the solution is sufficiently hypotonic (below 0.2 per cent N aCI), however, the
visible granules within the cell can frequently be observed to go into Brownian
movement after a few minutes. The most likely interpretation of this is that
these dead or dying cells have lost their differential permeability, so that water
and solutes alike diffuse freely across the cell surface. As is well known, this
loss of semi-permeability is characteristic of cell death. No normal or sar-
EFFECTS OF OSMOTIC PRESSURE ON FIBROBLASTS
317
comatous cells were found in transitional stages between the normal condition
and this complete loss of semi-permeability.
A series of measurements were made on fibroblasts in cultures which, after
being in equilibrium with 0.9 per cent NaCl, were surrounded by 0.3 per cent
NaCl. It is well known that most cells, under such circumstances, do not increase to a volume exactly in inverse proportion to the osmotic pressure. A
correction must be made in the ideal pressure.volume equation for a quantity
FIG.
1.
CHICK EMBRYO FIBROBLAST:
(l) IN EQUILIBRIUM WITH 0.9 PER CENT NACL;
(2) AFTER EIGHT MINUTES IN 0.3 PER CENT NACL
which has been called the" osmotically inactive" portion of the cell, although
it may indicate that the cell is in some other way not a perfect osmometer (8).
If this quantity is called b, the corrected equation for pressure :volume relationships may be written as follows:
P(V-b)
= P,,(Vu-b),
where P is the osmotic pressure at the final concentration and Po that at the
initial concentration, and V and Vo are the corresponding cell volumes at
equilibrium. The quantity b has been ascertained in the studies of the
318
AUSTIN M. BRUES AND CLAIRE MCTIERNAN MASTERS
Arbacia egg and the erythrocyte, and we have attempted to estimate it in the
cells of our study. The measurements and calculations are summarized in
Table I.
TAil!.!' I:
Calculation of "Osmotically lnactive " Portion uf Cell
Chick fibroblasts
1
2
3
4
5
h
v.,
\'".3
IJ
2002
1013
742
843
447
5038
2443
2056
2239
1149
484
298
85
195
96
24
29
12
23
926
1457
663
5084
690
2134
3603
1526
14620
1763
321
379
231
632
154
35
26
35
12
22
V I-J.
"
~,
,(I
23
Mean value
Sarcoma fibroblasts
1
2
3
4
5
Mean value
26.0',;,
----------~
Thus the" osmotically inactive" correction amounts to about one-fourth
of the total cell volume at equilibrium with 0.9 per cent salt solution, or probably a little more than the dry weight; it seems to be slightly greater in sarcomatous cells, and is subject, perhaps partly due to inaccuracies in the method
of measurement, to great variations from one cell to another.
A few macrophages were included in the observations, but since they are
either highly irregular or nearly spherical, it was impossible to obtain satisfactory measurements. Such spherical cells as were measured, in both normal and sarcomatous cultures, showed less swelling at final equilibrium than
did the fibroblasts. In no case was the equilibrium volume in 0.3 per cent
NaCl more than twice the volume in 0.9 per cent NaCl. It is possible, of
course, that the more active, irregular cells may behave differently.
In view of Shear's observations (4, 5) of swelling of cells in fragments of
sarcoma removed from their normal environment to salt solutions of various
concentrations, a few volume observations were made on fibroblasts in their
normal plasma medium, and then after six or more minutes in 0.9 per cent
NaCl. These are recorded in Table II, which includes only cases in which a
sufficient number of measurements was made in both media (four or more)
to make the results significant.
It seems from these figures that fibroblasts in general swell when removed
from plasma to 0.9 per cent sodium chloride solution, but that occasional cells
shrink a little. The swelling has been over 20 per cent of the original volume
only in cells which were very small to begin with. Of course, due to the conditions of making the cultures, there is reason to suspect that they may not
have been in media of the same osmotic pressure, and that most cultures would
tend to be on the side of hypertonicity. It seems clear that there is no sys-
319
EFFECTS OF OSMOTIC PRESSURE ON FIBROBLASTS
ternatic difference in the behavior of normal and malignant fibroblasts growing in a plasma medium upon removal to a salt solution.
Occasional cells, however, have been seen to develop colorless blister-like
TABLE
Embryo cells
I
2
3
4
5
6
7
8
9
II: Volume Observations
In Plasma
In 0.9 NaCI
1318
520
320
1087
2759
1228
1662
3739
627
1347
742
447
1258
2792
1447
1735
3326
724
Change
+
1.5
+42.6
+39.6
+15.7
+1.2
+20.1
4.2
-12.2
+10.6
+
+13.7
Mean change
Sarcoma 180
I
2
3
4
Mean change
1982
835
2503
978
1904
926
2530
1068
- 4.1
+10.9
+1.1
+
+
9.3
4.3
structures on their lateral surfaces during the course of changing from one
medium to another. These structures appeared rapidly and did not include
any of the intracellular structures. They appeared outside the still visible cell
surface and had a faintly visible surface of their own. They were seen in perhaps 10 per cent of both normal and sarcomatous cells, and were dome-shaped,
with a volume at most not over one-fifth that of the cell to which they were
attached. Clearly, they were outside an apparently undamaged cell and were
filled with a clear fluid of about the same refractive index as the external saline
medium. These swellings are in all respects similar to those which Shear has
observed (9) and have in each case been seen in cells which were apparently
undamaged, and which were capable of swelling internally in response to a
hypotonic salt solution.
All fibroblasts which were placed in a hypertonic medium up to 2.0 per
cent NaCI could be observed to shrink. In so doing, the cell surfaces were
seen to buckle and become irregular, as if decrease in volume took place more
rapidly than contraction of the surface could. After a few minutes, however,
the cells were again smoothly spindle-shaped. A concentration of 4 per cent
NaCI or over caused the cell surface to become much more refractile than
before, and such cells then showed no further osmotic phenomena.
Upon placing cells in a hypotonic medium, swelling took place to an equilibrium value if the medium was not below 0.3 per cent N aCl. Such cells, unless left in the medium for an hour or more, could be shrunk in 0.9 per cent
NaCI to approximately their original volume, which we feel justifies the idea
that the cells in these experiments were not irreversibly damaged. Cells which
320
AUSTIN M. BRUES AND CLAIRE MCTIERNAN MASTERS
were left in 0.2 per cent NaCI for four or five minutes could usually be shrunk
back to their original volume, but if left longer than that were so altered that
no more hypertonic solution would shrink them. Such cells, and likewise cells
left in 0.1 per cent NaCI and distilled water for a short time, showed Brownian
movement of the visible particles within the cell soon after swelling ceased,
indicating that probably, as in the case of dead cells, their semi-permeability
was lost. This happened usually when the volume reached about three times
the original volume. This limit of tolerance at between 0.2 and 0.3 per cent
NaCI was the same for both normal and sarcomatous fibroblasts.
During the course of swelling by endosmosis, it was noted that the cluster
of granules ordinarily seen at the poles of the nucleus in fibroblasts usually
remained together, and in the center of the cell, while the surface expanded
away from them. Occasionally a single granule or two would wander away
and remain near the surface. Aside from these larger particles, however, the
consistency of the protoplasm looked much the same throughout the experiment, and in all parts of the cell. Large highly refractile globules, which as
seen in embryonic fibroblasts frequently filled almost the whole width of the
cell at one place, could not be seen to increase during the course of increase
in the total cell volume. The nuclear volume in no case showed an increase.
This was difficult to observe directly, but cells stained with neutral red at the
conclusion of an experiment showed a relatively small nucleus, which would
fit well within the original cell.
The cell surface almost always showed no change in appearance during the
course of swelling. In no case did a fibroblast under observation rupture suddenly, but the surface always maintained its continuity, except in the case of
the blister-like structures mentioned previously, which appeared as often in
isotonic saline as in hypotonic; and in the case of a few cells in which, after
considerable expansion, the surface slowly became less visible. An occasional
cell appeared to lose its attachment at one end, so that that end retracted and
became hemispherical during the course of swelling. Some fibroblasts swelled
throughout their length, so that their surface increased greatly, while others
swelled largely in the middle, so that in some cases the diameter even decreased at the ends and the surface area was very little changed. These cells
all retained an approximately unduloidal surface, however. These various
types of swelling were seen in both normal and malignant cells.
A few spherical macrophages in both normal and malignant cultures, which
contained many highly refractile granules, were seen to break up below 0.3
per cent NaCI, discharging the granules.
The writers have performed a few experiments on the effect of osmotic
pressure on the migration of cells from explants of chick embryo heart and
sarcoma 180. These tissues were grown in D-4 Carrel flasks, a total of
twenty drops of medium being used for the solid phase; one of concentrated
embryo extract, three of chicken plasma, and the rest of Tyrode solution with
suitable additions of 4 per cent N aCl or distilled water. The liquid phase consisted of Tyrode solution made up to the appropriate osmotic pressure in the
same way. The average radius of the original fragment was measured by
camera lucida, and the average radius of the fringe of migrating cells was
measured after two days' incubation. The ratio of the latter radius to the
321
EFFECTS OF OSMOTIC PRESSURE ON FIBROBLASTS
former was taken as an index of the degree of inhibition to migration of the
medium in question. Table III shows the results: four cultures were made
of each tissue at each concentration. It would seem that sarcoma 180 is more
susceptible to anisotonicity in both directions than chick embryo tissue. The
migration radius of the latter is very little affected till the lethal limit is
reached.
T ABLE II I: Effect of Osmotic Pressure on Fibroblast Migration
Salt concentration of the medium
NaCI
0.20
0.30
0.50
0.70
0.86
1.20
1.45
2.00
2.50
Ratio of growth-radius to radius of explant
Embryonic
tissue
milliequivalents
per liter
o
34
51
85
120
150
205
(few cells)
0.85
0.58
0.86
0.67
0.64
(few cells)
248
342
427
o
Sarcoma
tissue
o
o
0.41 (cells dead)
0.33
0.83
0.30
0.17
o
o
Additional cultures of both tissues, after growing for twenty-four hours
in Tyrode solution, were placed for twelve hours in 0.2 and 0.3 per cent salt
solution, as prepared above, after washing the clot with the new solution. In
all cases, no additional growth or activity was seen, even after returning these
cells to normal Tyrode solution.
DISCUSSION
Our observations on the behavior of various cells in media of different
osmotic pressures show nothing which would indicate that the elasticity or
surface tension of the cell surface differs between normal and malignant fibroblasts. The buckling of the surface in hypertonic solutions and the changes
in configuration of the cells in hypotonic solutions, as described, are similar in
both types of cell. This is in accord with the microdissection studies of
Chambers and Ludford (10). The occasional fragility of the spherical
macrophage in hypotonic media has not been observed in any fibroblasts.
It is noteworthy that the nucleus shows no apparent swelling during the
course of acute swelling experiments. A few cultures of rat testis were made,
and such migrating spermatozoa as were seen could not be made to swell
measurably in hypotonic solutions or water, although fibroblasts in the same
cultures followed the usual course. The nucleus may account for some of the
" osmotically inactive" portion of fibroblasts.
We have not observed any great difference in the content of normal and
malignant cells in " osmotically inactive" fractions. Roffo (11), as a result
of experiments in dehydrating fragments of embryonic and sarcomatous tissue,
and subsequently explanting them, has suggested that malignant cells contain
much more bound water than embryonic cells. He reports that embryonic
tissue will stand a much greater loss of water without losing its capacity to
322
AUSTIN M. BRUES AND CLAIRE MCTIERNAN MASTERS
grow than will sarcoma, although the water content of the two tissues is not
very dissimilar. Certainly, if other factors are equal, our experiments show
that the amount of cell water taking part in endosmosis in both types of cell is
nearly the same, and a large proportion of the total water content.
It is most interesting to consider the findings of Shear (5), whose results,
indicating a certain type of swelling of tissue cells of mice dependent wholly
upon the protein content of the medium, would at first sight seem incompatible
with those here reported. It must be recalled, however, that we have sometimes noted clear bulges, similar to those described by him, in fibroblasts in
isotonic saline media. These bulges, as we have seen, have occurred in occasional cells only, unlike the phenomenon of swelling of the whole cell homogeneously in hypotonic solutions, which has occurred in all the living cells we
have observed. His technic, unfortunately, is probably not capable of detecting the latter type of swelling, because of the irregularity of the cells which he
uses for observations.
It is possible that the cell environment in some of our experiments was
never depleted of the serum protein originally present, because of the probable
slow diffusion of protein from a plasma clot. It is necessary to remember,
however, that the classical method of growing tissues in Carrel flasks, in which
the plasma is washed with Tyrode solution and then covered with dilute
embryo extract or split proteins, may eventually lead to a considerable depletion of the protein content of the clot without cell swelling. It is possible that
the fibrin may in many instances exert an opposition to this type of swelling,
and that the pressure of surrounding tissues may do the same thing in vivo.
The tissues in these experiments were kept under conditions which would allow
them to grow as well as to live, until the acute experiment was started. It
sems fair to say that the swelling of the cell body dependent on the salt concentration of the medium, and the globular swelling phenomenon described by
Shear, are both characteristic of tissue cells. Whether the globular type dependent on colloid osmotic pressure is something which might occur under
physiological conditions, it is impossible to say.
SUMMARY
1. A method is described for estimating the volume and surface of fibroblasts growing under tissue culture conditions.
2. The volume of the entire fibroblast, whether embryonic or malignant,
depends largely upon the osmotic pressure of salts in the medium, as is the
case with the erythrocyte. Occasionally, clear blister-like swellings have also
been seen, and may be due to changes in colloid osmotic pressure.
3. Observations made on the osmotic behavior of these cells do not suggest
any difference in the elasticity or surface tension between embryonic and sarcomatous cells.
4. The correction for" osmotically inactive" material is about 22 per cent
in embryonic cells and 26 per cent in sarcomatous cells, with wide individual
variations.
5. The migration and growth of sarcoma 180 are more greatly affected by
EFFECTS OF OSMOTIC PRESSURE ON FIBROBLASTS
323
changes in tonicity of the medium in both directions than are the migration
and growth of cultures of chick embryo tissue.
NOTE: The writers are indebted to Dr. J. C. Aub for suggesting this problem, and to
Mrs. Beula B. Marble for assistance in many of the tedious details.
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