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STUDIES ON THE GROWTH OF TISSUES
7Ar VITRO
II. AN ANALYSIS OF THE GROWTH OF CHICK HEART
FIBROBLASTS IN A HANGING DROP OF FLUID MEDIUM
BY E. N. WILLMER, M.A., M.Sc.
(Physiological Laboratory, Cambridge.)
{Received 4th February, 1933.)
(With Eight Text-figures.)
CONTENTS.
PAGE
Introduction
Experimental methods
Experimental results
Discussion
Summary
References
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325
328
338
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INTRODUCTION.
THE tissues of the animal body may increase in mass in four ways, by laying down
intra- or intercellular material, by increase in cell size or by increase in cell number,
and therefore presumably these methods of growth are available to tissues in vitro.
The question as to which of these processes shall predominate depends very largely
on the tissue and the manner in which it is cultured. Broadly speaking the methods
of tissue culture lead to two main types of growth, organised and unorganised. In
the former the tissue behaves much as it would do in the body and may grow by all
four methods: in unorganised growth, with which the experiments described here
are concerned, growth takes place almost entirely by cell division, and probably
sometimes to a small extent by increase in cell size. In unorganised growth inter- or
intracellular with the exception of fat material is rarely laid down.
Now when unorganised growth is occurring the apparent size of the culture
increases very rapidly, and since hitherto the most convenient method of measuring
the growth has been by measurement of the increase in the area of the culture it is
necessary to know exactly what processes are involved in this increase in area, to
what extent each is important and finally how far measurements of the areas of
cultures reflect the increase in mass. Under any given set of conditions the curves
showing the area of the cultures from day to day are regular and more or less
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accurately represent the rate of increase in size of the culture. For example, early in
the history of tissue culture Carrel (1923) pointed out that the growth curve of a
culture in a non-nutrient fluid was S-shaped, whereas if the fluid contained everything for the needs of the tissue it became parabolic. From the growth curves
obtained by measuring the areas of tissue cultures several investigators (BuchAnderson and Fischer, 1928; Ephrussi and Teissier, 1932; Love, 1931) have deduced
formulae to express the process of growth, but owing to the complexity of the
factors involved it is doubtful how valuable these expressions may be. They all
depend on assumptions as to the behaviour of the cells in the culture, not all of
which are equally valid. The obvious ways in which a culture increases in area are by
outwandering of the cells from the central implant and by multiplication of the
cells. It therefore becomes important to examine the growth in detail to determine
the relative proportions of the new growth which are due to these two main causes,
and also to discover what relationship, if any, exists between the migratory activity
of the cells and their capacity for division. Although great wandering activity often
occurs at the same time as intense cell division the two processes are probably
distinct activities on the part of the cells. For example, reduction of the oxygen
pressure in a culture has been shown by Ephrussi, Chevillard, Mayer and Plantefol
(1929) to cause a cessation of division before it stops the wandering of the cells.
Secondly, certain proteose-like substances described in a previous paper (Willmer
and Kendal, 1932) have the capacity of causing large areas of growth in cultures but
apparently have no very marked effect on the division rate of the cells. Such
factors as these make it seem probable that cell division does not necessarily go hand
in hand with intense motile activity on the part of the cells, and probably special
conditions have to prevail before cell division can take place freely. Measurements
of the area of a culture may therefore be exceedingly misleading unless at the same
time steps are taken to ascertain the extent of cell division within the culture.
Alternatively, the method of prolonged subcultivation gives an adequate means of
testing the true growth of a culture. If the tissue can be divided at definite intervals
and subcultured regularly without loss of size of the fragments, then it is an obvious
indication of the true growth of the colony, and the rate of growth can be judged
from the frequency with which the culture can be divided without loss. For example,
it is possible to divide cultures of fibroblasts into two every 48 hours if they are
growing in a medium of plasma and embryo extract; consequently the amount of
new protoplasm must be approximately doubled in this time. Another method of
investigating the growth of a culture is by counting the number of cell divisions
which are occurring. Usually only the cells in the peripheral area of new growth of a
culture are counted, but Fischer and Parker (1929) obtained some very interesting
data on the behaviour of cultures by counting the mitoses in serial sections of
cultures. With suitable care in the selection of comparable cultures and the time of
observation very valuable data have been obtained from mitotic counts. Until
recently they have only been made on fixed preparations, with the obvious disadvantage that it is only possible to judge of the condition of the culture at one given
stage in its history.
Studies on the Growth of Tissues in vitro
325
The results described in this paper have come from experiments designed to
investigate more thoroughly the mechanism of growth in vitro and if possible to
establish a more reliable method than has hitherto been used for making measurements of the growth. The results are necessarily of a preliminary nature and are
based on experiments of an imperfect character, but the basis of the method is
satisfactory, and with suitable modifications should form a reliable method of
judging of the growth activity of cells in vitro.
A method has been evolved by which part of the field of a culture has been
photographed at regular intervals over long periods of time, so that estimates can
be made of the relative importance of cell division and cell migration in the production of the new outgrowth. The duration of mitosis, the length of the resting
period between mitoses, the percentage of dividing cells, and the possibility of
divisions occurring rhythmically can all be investigated by this method. Also it is
possible to see if cells are degenerating rapidly, since it may be that rapid division of
cells does not always lead to rapid growth rate, for the cells may be short-lived. This
state of affairs was suspected by Fischer and Parker (1929) in the case of the growth
of a carcinome culture.
A similar line of attack has been opened up by Olivo and Delorenzi (1932) who
observed cultures continuously for periods up to 18 hours, and recorded the occurrence of mitosis, but the limitations of such a method are obvious. However, these
results are extremely interesting and will be discussed in conjunction with those
obtained photographically in the experiments here described.
EXPERIMENTAL METHODS.
The method adopted for this investigation was essentially to photograph at
5-min. intervals a field containing something over a hundred cells. Photography
began as soon as this number of cells had spread out into a suitable position and
continued until the culture showed evident signs of degeneration. For this work
hanging drop cultures of chick heart fibroblasts were chosen, and the fragments
were grown in a fluid medium. By this method it was hoped to obtain a single
layer of cells spread out evenly on the surface of the coverslip, so that all could be in
focus on the photograph at the same time. The method has some obvious disadvantages. It is only possible to photograph a portion of the field of the culture, and
this portion may be in some way abnormal. For example, it is well known that
hanging drop cultures depend for their activity very largely on the degree of contact
which they make with the coverslip, for the cells can only wander out on the surfaces
offered in the medium. However, by taking great care to have the coverslips
chemically cleaned, by choosing very similar fragments of tissue, and by keeping
the same relative proportion of medium to tissue it was found possible to obtain a
series of cultures all showing about the same area of outgrowth, and by choosing an
average culture from the series it was hoped that these sources of error would be
minimised. The results of repeating the experiments under similar conditions were
fairly encouraging. Fresh heart tissue from 9—12-day embryos was used throughout.
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Tissue which had been cultured for some time in vitro would probably have been
preferable except for the fact that it is very difficult to transfer tissue from a plasma
coagulum into a fluid medium with regular results. The fibrin carried over with the
tissue prevents the necessary contact with the glass and the growth is extremely
irregular. Consequently it was felt that the variations in growth rate inherent in
similar fragments of chick heart would be a less serious source of error than those
due to the transfer from the plasma coagulum to the fluid medium.
The necessity for photographing the cells at absolutely regular intervals for long
periods made it essential that the camera should work automatically, and for this
purpose a rather special mechanism was required. An electric contact was established at the required intervals, in these experiments every 5 min., which closed the
circuit between a small 12-volt motor and the necessary storage batteries. The latter
were used in preference to the main supply in order to maintain a more constant
speed in the motor. The motor was then so geared that it caused a spindle to make
one revolution in about 15 sec. This spindle controlled the further movements of the
apparatus. As soon as it started to rotate an eccentric cam caused an Isenthal
mercury switch (in parallel to the switch on the timing mechanism) to come into
action so that the motor continued to rotate after the contact on the timing mechanism
was broken. At the same time another Isenthal switch completed the circuit for the
light for the microscope. This was operated by a bevelled cam on the spindle so that
the duration of the exposure could be altered at will. In practice it was found
preferable to keep the exposure time constant, and vary the intensity of the light by
inserting a variable resistance in the circuit. After the exposure circuit was again
broken by the rotation of the control spindle, a cog-wheel on the latter engaged with
the rollers between which the photographic paper (which was used in preference to
film for reasons to be described below) was passed, and moved on the paper in
preparation for the next photograph. The cog-wheel on the control spindle only had
teeth over part of its circumference, so that in the first half of its rotation it did not
engage with the driving rollers, thus ensuring that the paper remained still while the
photograph was being taken, and only after this was finished did the teeth engage.
In order to be certain that this should take place and that the teeth should not lock
against each other the spindles were mounted in slots and kept together by means of
a spring. It was so arranged that the number of teeth on the cog on the control
spindle was just sufficient to turn the roller enough to clear the paper for the next
photograph. In order to facilitate examination and measurement on the photographs, these were taken directly on to cine-bromide paper 3 in. wide, and each
photograph was 3 x 4 in., this being considered more satisfactory than taking on to
cinema film with the subsequent necessity for projection, and the difficulty of
making permanent notes and records on the photographs. The photographs were of
course negatives, but this was not found to be any serious obstacle. The paper was
pulled on by two rollers and rewound on to a spindle which was driven by means of
a slipping clutch mechanism, so that the paper was just kept tight on the spindle
but did not drag through too much when the roll of exposed paper became large.
At first the mechanism was controlled by means of a clock which made electric
Studies on the Growth of Tissues in vitro
327
contacts at the required intervals, but this was not found to be sufficiently accurate,
since the contact sometimes failed or remained so long as to expose two photographs instead of one. Finally it was decided to revert to the ancient water clock.
A small bucket-shaped vessel was so balanced that it tipped over when filled to a
certain depth, and this was brought about at regular intervals by allowing water to
drip under a constant head of pressure. Each time the bucket tipped it threw over a
D
o
lamp
Motor
Circuit A
Circuit B
Circuit A. A, switch on tipping bucket; B, switch on control spindle; C, hand switch;
D, main switch. Circuit B. E, hand switch; F, switch on control spindle
C
D
C. Diagram of control spindle, i, engages with teeth on 2 after switch operated by 5 is opened.
Drives on the paper; 2, wheel with requisite number of teeth to move paper on 4 in.; 3, main
drive from gearing on motor; 4, cam for closing switch B at commencement of revolution and
breaking it at end of revolution, thus stopping the motor; 5, cam with bevel to close switch F for
time necessary for exposure.
D. Illustrating the mode of operation of switch F by cam 5. Switch B is operated in a similar
way by cam 4.
Fig. 1.
mercury switch which started the motor. It was found that this mechanism could be
set so that it tipped perfectly regularly and all the troubles inherent in the clock
contact were eliminated. The slight variations in the time of filling due to temperature changes, etc., were so small as not to be noticeable, and the apparatus has
worked with extreme regularity. The light for photography was provided first by an
ordinary 100-watt filament lamp, and later by a 6-volt 24-watt lamp built into the
substage of the microscope. This lamp was operated from storage batteries. The
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exposure, about 7 sec, was long, but the intensity of the light was not sufficient to
cause any injury to the tissues through this cause. A heat filter was inserted between
the lamp and the culture. The microscope was enclosed entirely in an incubator,
heated electrically, and kept at 390 C. It is essential to keep hanging drop cultures
on a horizontal stage so that the microscope was kept vertical and the light deflected
on to the photographic paper by means of a prism. A mirror could at any time be
inserted into the path of the beam, which was then reflected on to a ground-glass
plate at the same distance from the eyepiece as the photographic paper so that the
condition of the culture could be examined from time to time. The greatest difficulty
encountered in the photography was caused by the slight condensation which took
place on the inner surface of the hollow ground slide, since it tended to obscure and
distort the images of the cells. The general arrangement of the apparatus is illustrated
in Fig. 1.
EXPERIMENTAL RESULTS.
The most complete record so far obtained has been on a culture of the ventricular wall of the heart of a 12-day chick embryo in a medium consisting of an extract
of the rest of the embryo in 5 c.c. of Tyrode solution. The extract was prepared in
the usual way by grinding up the embryo, suspending the pulp in the Tyrode
solution and finally centrifuging for about 30 min. at 3000 rev. per min. In other
experiments although the extract has been prepared from younger embryos and
therefore was generally more dilute the figures show that it was always above the
strength which gives the maximum amount of growth. Photography was started
19 hours after the culture was made and was continued at 5-min. intervals for the
next 24 hours. The number of cells on the field was counted every hour by examination of the paper after development, and the number of mitoses which took place
was ascertained in the same way. The result is plotted in Fig. 2. The upper curve
represents the total increase in the number of cells on the photographic field, while
the lower curve represents the expected increase in the number of cells on the field
if they were only derived by division of existing cells, that is to say that the curve
represents the growth which can be accounted for by the mitoses recorded. It is
obvious that there is a wide discrepancy between these two curves which is explicable on the grounds that large numbers of cells migrate from the initial implant
and account for a very large fraction of the observed growth. It was later found
unnecessary to make counts of the cells present every hour and this rather laborious
task was reduced by taking very careful 4-hourly counts. The cell divisions are
easily recognised as they are generally conspicuous on at least four photographs,
and since the most readily sharply defined period of the division is telophase it was
this phase which counted as "mitosis." During division the optical properties of
the cell are markedly altered, so that in this condition the cells give very clear
images on the photograph. The curves are unfortunately rather too short to be able
to decide exactly to what type they conform. From 19 to 35 hours they are fairly
definitely parabolic, but there is some indication from the points obtained after
Studies on the Growth of Tissues in vitro
329
35 hours of growth that an inflection occurs about that time and that the curve is
really S-shaped. After 36 hours of growth the numbers of mitoses suffered a slight
decrease followed by a second increase, and this fluctuation has been observed in
other old cultures. It is symptomatic of a culture in an old medium. The curves
allow of the conclusion that growth is proceeding in a normal manner until about
the 36th hour of culture, after which point signs of degeneration begin to appear.
The numbers of mitoses become less constant and the rate of increase of the total
culture becomes less. Two experimental errors may creep in during the later stages of
the culture, although these are not regarded as serious. The very large number of cells
on the field at the end of the experiment means that on the side towards the central
550 r
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£ 200
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Age of culture in hours
Fig. 2. 12-day old chick heart fibroblasts in embryo extract. ®—© total number of cells on the
field; El—Q number of cells calculated from the number of cell divisions observed on the
photographs.
implant the layer of new growth may be more than one cell thick. This leads to the
possibility that rather too few cells may be recorded and that dividing cells are
being obscured. The latter source of error in a good series of photographs is
negligible: the former has to be borne in mind and in later work fields have been
chosen sufficiently far out to overcome this difficulty. But then it is a case of being
between the devil and the deep sea, for if fewer cells are present on the field the
statistical value of the figures obtained becomes less.
The actual numbers of mitoses which occurred each hour are plotted in Fig. 3,
and it is seen that they remained fairly constant throughout the period, but there is a
distinct maximum about 30 hours after culture. There is considerable variation in
the numbers, but this is to be expected since the total number of cells on the field is
E. N . WlLLMER
330
relatively small, and if the mitoses occur at random as they almost certainly do, a
more even distribution would be very unlikely. In all the cultures so far examined
there is no satisfactory evidence in favour of there being waves of mitotic activity,
except perhaps in the fluctuations already mentioned in old cultures. Normally,
however, all divisions occur more or less at random; with the exception that very
often two or three cells in the same area divide together. So far it has not been
possible to obtain a satisfactory answer as to why this should be so, but the photographic method can almost certainly be used to ascertain this point. There would
appear to be two possible explanations; either the cells have had a common origin
and being in very similar surroundings are probably ready to divide again at about
the same time (this was found to be the case in the observations of Olivo and
Delorenzi (1932); or that a cell when about to divide influences its neighbouring
cells to do likewise. The divisions are, however, so often nearly synchronous that
the former seems the more probable explanation. Further work with more frequent
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Age of culture in hours
Fig. 3. Total number of mitoses occurring per hour in embryo extract.
photographs of a smaller field would probably enable an answer to this question
to be obtained. The present series of photographs do not readily allow of the
daughter cells of one division being followed with absolute certainty until their
next divisions, although in a few cases this has been possible. This then leads on to
the question of the length of the interkinetic or intermitotic period.
The few observations which have been made show that this is exceedingly
variable. In one case a cell was observed to divide a second time at the end of 10
hours, which agrees reasonably with the earlier observations of Strangeways (1922),
who by following a cell by direct observation with the microscope saw a cell divide
for the second time after 12 hours. On the other hand several cells have been
followed for periods up to 20 hours without dividing. Strangeways (unpublished
data) too had kept one cell under observation for 49 hours and it had not divided.
This indicates, and further work only goes to make it more certain, that there is no
constant intermitotic period for the cells of a growing tissue. It should be noted,
however, that these observations have been made on tissue which is fresh from the
animal, therefore probably not homogeneous, and which may carry over much of
Studies on the Growth of Tissues in vitro
331
its variability into culture conditions. Tissues which have been grown for any length
of time in vitro possibly become more uniform, and it is conceivable that under such
standard conditions a typical fibroblast may have a definite interval between one
division and the next. This interval would of course vary from one medium to
another unless the cell could obtain all its requirements equally well from either.
These are but speculations, and the evidence so far seems to show that there is
little hope of judging of the growth capacity of tissues by measurements of the interkinetic period. Moreover, the evidence of Olivo and Delorenzi (1932) goes to show
that even after prolonged culture the duration of the interkinetic period is still far
from constant.
Photographs were not taken at sufficiently frequent intervals to allow of accurate
timing of the process of mitosis. Nor was the magnification large enough to be
absolutely certain of the beginning and end of the process. But, as previously
noted, a cell undergoing division was noticeably different from resting cells on at
least five photographs. This corresponds to a period of something over 25 min.,
which, allowing for the less conspicuous early stages of prophase, agrees satisfactorily with previous determinations of other workers. The gradual rounding-off
of the cell leading up to metaphase facilitates the counting of the divisions by
drawing attention to the cells which are about to divide. The mitotic times in the
media employed have not shown any very great variations (small ones could not be
detected by this method). When a culture is for any reason ceasing to grow, then
occasionally cells may be observed to round up as if to divide and then remain in
that condition often for an hour or more, before proceeding with the anaphase and
telophase. Old cultures fairly constantly show lengthening mitotic times.
By comparing the curves of Fig. 2 it is clear that in the case of an embryo
extract culture at least half of the apparent growth of the explant is in reality to be
explained by the outward migration of the cells—a process which is largely independent of their mitotic activity. The methods here employed throw very little
light on the mechanism of this migratory activity. In general it is centrifugal from
the implant, but, quite often cells may be observed moving at right angles to the
main direction, or even approaching the central fragment. Fig. 4 shows the paths
traversed by several cells. Two things become evident at once. First, cells that have
divided move apart rapidly, so that one of the daughter cells almost invariably
moves towards the implant. The direction, however, is soon reversed and the cell
generally wanders outwards again in the normal manner. Secondly, there is very
considerable irregularity in the rate of movement, both from cell to cell and from
hour to hour. Even the two cells formed by a division sometimes travel at widely
differing rates. The average speed in the culture examined from this point of view
appeared to be about 12 fj. per hour. Since the cells are for the most part orientated
for movement in a centrifugal direction it follows that the equatorial plate in cell
division is generally at right angles to the direction of the cell movement, that is to
say at right angles to the radii of the culture. This is fairly true when the cells are
closely packed together near the implant, but later when the cells become more
scattered, their uniform polarity is less obvious and then the direction of the equa-
E. N . WlLLMER
332
torial plate bears little relationship to the central implant. The rate of migration falls
off slowly as the culture ages, but this seems to be correlated with the age of the
culture rather than with its distance from the implant, which is perhaps evidence
that the cells are not moving down a gradient of metabolites as has been suggested,
for in that case it might be expected that the rate of migration would decrease as the
distance of the cell from the implant increased. On the other hand, in the time of the
experiment perhaps the concentration of the metabolite near the culture is such as to
cause nearly the maximum rate of movement of the cells, and this may continue far
out into the medium. In a larger medium preferably of plasma this point could be
Central fragment
of tissue
Fig. 4. Approximate tracks of cells migrating from the central implant. The points occur
at intervals of 1 hour. The numbers represent actual times.
put to the test further, for then a steeper and more uniform gradient would presumably be established and the cells have time to wander more freely into the
medium. The movement, however, in cells far out in the hanging drop of fluid
medium does not appear to be completely random, it always remains to some extent
under the directional influence of the central implant.
It has frequently been observed that cell divisions do not take place evenly
throughout the whole culture, but that they appear to be most frequent near the
periphery of the new growth, although cells completely isolated in the medium do
not often divide. Is this correlated with the density of the cell population, or has it
to do with the food supply, etc., in the medium, or with some other factor? Experi-
Studies on the Growth of Tissues in vitro
333
ment shows that it is primarily correlated with the density of the cell population,
although the latter may be influenced greatly by the conditions of the medium.
This correlation was established in two ways. In the first method, a circle of radius
0-5 cm. was drawn round the photograph of each dividing cell and every cell which
lay more than half within that circle was counted. This gave figures for the number
of cells within 30/x (scale: 0-5 cm. = 30^.) of every dividing cell, and the results are
plotted as a frequency curve (Fig. 5). This shows very clearly that most cells divide
when surrounded by about 15 or 16 others within a 30 JU, radius. On taking a large
number of fields at random it was found that the average density of the peripheral cell
population was 21 cells within the circle of radius 30/x. The same result was obtained
2 3 4 5 6 7 8 91011121314151617181920212223242526
Number of cells within 30/i radius of the dividing cell
Fig- 5- Frequency curve showing relationship between mitosis and the number
of cells in the vicinity, i.e. within a radius of 30/i of the dividing cell.
by the second method, in which the new growth was marked off in squares and the
number of cells in each square counted every 4 hours, so that the average density
of the population of cells in each square was known over 4-hour periods. All the
dividing cells within the squares were noted and it was therefore possible to correlate
the frequency of mitosis with the number of cells per square, and this again showed
that the cells divide most frequently when they are neither too crowded nor too
widely separated, and that the greatest frequency of mitosis occurred in a density of
cell population which was lower than the mean density. It is difficult in the present
state of knowledge to see exactly what this means. It seems probable that it is due
to the action of the cells on the medium, rather than to the mutual influence of the
cells upon one another.
E. N . WlLLMER
334
A few preliminary experiments "were made by the photographic method to
investigate the influence of the medium on the growth of the tissues, but the results
were not accurate or reliable enough to make the method of much value without
considerable modification. The main disadvantages of the method are inherent in
the coverglass technique of growing cells. In the first place only one culture can be
easily photographed at a time, and only part of that. Secondly, the amount of fluid
medium present is very small, and in spite of efforts to the contrary probably varies
considerably in quantity from culture to culture, at least in proportion to the tissue
present; and thirdly, the fluid is apt to evaporate slightly and condense on the
surface of the cavity slide, thus both altering the composition of the medium and at
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Age of culture in hours
Age of culture in hours
Fig. 7Fig. 6.
Fig. 6. Growth index over two 6-hour periods (20-26 hours and 26-32 hours) for cultures of
chick heart fibroblasts in embryo extract.
Fig. 7. Growth indices for two 6-hour periods of cultures in different media, i, 3, 4, embryo
extract, 5 c.c. Tyrode per embryo; 2, embryo extract, 1 c.c. Tyrode per embryo; 5, embryo extract,
5 c.c. Tyrode per embryo diluted with 5 c.c. Tyrode; 6, 7, embryo extract, 5 c.c. Tyrode per embryo
diluted with equal quantity of serum.
the same time obscuring the photograph. On the other hand, photography of
hanging drop cultures in fluid media, as long as the difficulty of condensation is
avoided, is the ideal method by which to obtain a clear picture of the cells, for they
spread out in one plane on the coverslip. Consequently several experiments were
tried in order to test out the possibilities of the method. The results are shown in
Figs. 6 and 7.
The activity of the cultures in the different media was studied both from the
point of view of increase in numbers of cells on the field due to cells wandering in
from the central implant, and also from the point of view of increase due to cell
divisions. The latter is of course the important factor in connection with actual
growth, and it will be seen that it is independent of the migratory activity of the cells.
The basis for comparison of the cultures rests on the following considerations.
Although the culture (Fig. 2) appears to be growing uniformly and regularly up
to the 36th hour, actually the growth rate is falling off all the time. To determine
Studies on the Growth of Tissues in vitro
335
points of this character it is necessary to obtain a satisfactory measure of the rate of
growth. Such a growth index has been obtained in the following way. It is necessary
to take into account the fact that the cell population is increasing as the result of two
distinct processes, cell division and emigration of cells from the central implant.
During the course of photography a very few cells leave the field of view. This
number is so small that it may be neglected, since a long enough time is not allowed
for cells to wander right across the field, and, the outwandering being generally
radial to the centre of the culture, very few cells leave at the sides of the field. Now
at first sight it would appear that any cell on the field is theoretically capable of
dividing, but those which come on late have less chance of dividing during the
photography than those which have been on all the time, and secondly, those which
divide during the period of photography cannot be considered as potential dividing
cells for at least the next 6 hours. In the method of expressing the growth rate an
attempt has been made to allow in some measure for these two considerations, since
it is probably best to express the growth capacity of a culture on the basis of the
number of cells which divide within a given time as a fraction or percentage of those
which might theoretically have divided. It is perhaps doubtful whether allowance
should be made for the fact that cells which have divided once are not likely to divide
again quickly, but it is now fairly well established that cells never, or at any rate
only very rarely, divide without an intermitotic period of more than about 7 hours.
In actual practice the number of cells which underwent mitosis during the period of
observation has been subtracted from the number of cells on the field at the end of
the experiment, and the mean has then been taken between this number and the
number of cells on the field at the commencement of the period. The number of
mitoses has then been expressed as a percentage of this mean figure, and the whole
divided by the duration of the observation in hours. In symbols, let a be the
number of cells present at the commencement, b those present at the end, x those
which divide during the period, and t the time in hours, then the expression for the
growth index is as follows:
x
a + (b — x)
2
100
x
~r-
t must always be less than the duration of the minimum intermitotic period, but on
the other hand, owing to the relatively slow rate at which cell multiplication occurs,
and since only a limited number of cells can be photographed at once, it is preferable
to make it as long as possible. Even so there is room for very considerable apparent
fluctuation of the growth rate which is almost entirely due to the methods of
measurement. For example, suppose the growth index of a culture works out, as is
frequently the case, at 1 per cent, of the cells dividing per hour, then if there are
only 100 cells on the field and two divisions happen to occur in the first hour and
none in the second, the growth rate over these two 1 -hour periods fluctuates from
2 to o per cent. This error is of course minimised by increasing the number of cells
on the field to the greatest possible extent consistent with photographic definition,
and by making the time factor as long as possible.
336
E. N . WlLLMER
Fig. 6 shows the decline in the value of the growth index after an interval of
6 hours in three cultures growing in embryo extract. The points A, B and C show
the growth index over the period 20-26 hours after culture, D, E and F during the
period from 26 to 32 hours. It is possible that this downward slope is somewhat
exaggerated in these curves since the most actively dividing region of the culture is
near the periphery, and this region occupies proportionately less of the photographic field as the culture progresses. This, however, does not account for the
whole decrease, which can be observed to occur even in the periphery itself. Incidentally this means that the intermitotic period steadily lengthens. For example,
calculations from the points on Fig. 6 show that the intermitotic period of cells
23 hours after culture is about 19 hours, whereas 6 hours later it works out at nearly
24 hours. Measurements of the intermitotic period therefore are not very helpful,
for if obtained in this way they give no more information than can be obtained from
the growth index, and by direct observation they are exceedingly variable. The
falling off in the value of the growth index must mean that the embryo extract alone
does not supply all the substances necessary for growth by cell division. The use of
the growth index would seem therefore to give valuable data as to the amount of
actual growth which is occurring in a culture over fairly short periods of time.
The migratory activity of the cells is expressed simply as the increase in number
of cells on the field which cannot be accounted for by cell division, divided by the
time of observation, i.e. the slope of the growth curve after allowing for the number
of mitoses occurring during the period. The number of cells which come into the
field depends on their activity and to some extent on the nearness to the source of
supply of fresh cells, namely, to the central fragment, so that actually except towards
the end of the experiment when the periphery of a culture is further removed from
the centre only a rough estimate can be obtained of the activity of the cells except
by direct measurements of the speed of locomotion of the cells in the various media.
These exact measurements of the rate of movement of the cells would, however, be
of little significance to correlate and compare with mitotic activity because the rate
of movement depends so very much on the local conditions, mechanical, physical,
and chemical, of the medium. Under more standard conditions of culture they
would undoubtedly be valuable. On the other hand, it is important to consider
whether a culture is increasing mainly by outwandering of cells or mainly by cell
division.
The highest rates of cell division occurred in cultures in which the medium was
composed entirely of embryo extract (curves 1, 2, 4, 3, Fig. 7), and in them after
20 hours of growth about 5 per cent, of the cells were dividing per hour. This
agrees well with figures estimated by Fischer (1925), for if the duration of mitosis be
taken as half an hour then 2-5 per cent, of the cells should be dividing at any one
time, which is of the same order as his figures. Delorenzi (1932) obtained rather
higher values (5 per cent.) using plasma and extract media. The point marked 5
(Fig. 7) was obtained from a culture in which the embryo extract had been diluted
with an equal quantity of Tyrode solution, and the growth rate is obviously very
much reduced. On the other hand, curve 2 shows the growth in an extract made
Studies on the Growth of Tissues in vitro
337
with only 1 c.c. instead of 5 c.c. of Tyrode, and the extra strength shows no significant difference in the growth rate. In the curves 6 and 7 the embryo extract was
diluted with an equal quantity of fowl serum. The serum was obtained by allowing
the blood to clot in a sterile tube, separating the coagulum from the walls of the
tube and allowing it to stand for 12 hours at room temperature in order to contract
and express the serum which was then pipetted off and centrifuged. It is obvious
that the division rate is not very much greater than when the extract was diluted
with Tyrode solution. A few mitotic counts subsequently made on fixed preparations from cultures in serum media have cast some doubt on these results, but it is
probable that the difference is really due to the method of preparation of the serum,
which varies in its growth-promoting power according to the treatment which it
receives. The variations are probably dependent on the fact that the leucocytes or
even the other blood cells may give rise to stimulating substances on prolonged
250 r
25Or
200
200
£ 150
150
o
jj 100
100
50
50
0
20
22
0
26 28
24 26 28
Age of culture in hours
30 32 34
Fig. 8. Curves showing the rate of increase in the number of cells on the field in different media,
which is due to inward migration only, and not to cell division. Two 6-hour periods (20—26 hours
and 26—32 hours).
standing or incubating. Then again the age of the animal from which it is obtained
may be an important factor (Carrel and Ebeling, 1922).
If Fig. 7 is compared with Fig. 8 in which the migratory activity of the tissues is
indicated, then it is observed that at least in the earlier stages of growth the order of
the curves is almost completely reversed; the increase in the number of cells on the
field takes place most rapidly when serum is present, and most slowly in those in
which division of cells is proceeding most actively, thus again making clear the
distinction between cell activity and cell division. Serum probably allows the cells
to behave more normally and permits them to effect better contact with the coverslip
and to wander more freely into the medium, but it seems to provide little in the way
of growth-producing substances, so that the implant spreads out quickly and widely
into the medium giving an erroneous impression of growth-promoting power, in
which property it is singularly lacking, at least when prepared by the method described above. In general, these results confirm those found by Carrel and Ebeling
(1923) for the behaviour of tissues in serum.
33^
E. N . WlLLMER
DISCUSSION.
Perhaps the most striking fact brought out in these investigations is the great
part which cell migration plays in increasing the apparent size of a culture, and the
independence of this migratory activity and the process of true growth by cell
division. Where the latter process is occurring as rapidly as possible in embryo
extract cell migration still accounts for nearly half of the total growth.
A hanging drop culture grows steadily but at a decreasing rate from about the
18th to 36th hour of cultivation, and during that time the number of cell divisions
on the field remains fairly constant, a maximum being actually reached at about the
30th hour. This maximum is presumably the result of a balance between the number
of cells on the field which could divide, and the decreasing rate at which the cells
are dividing. Practically these results are convenient because they allow cultures to
be taken between say 20 and 30 hours after implantation, and if cultures of similar
areas and activity are chosen the number of mitoses per culture will be fairly
constant. This had been observed to be the case by Spear (1928) who had made use
of the fact in his work on the effects of various physical agents upon mitotic activity.
For comparing the effects of the medium on the growth of cultures the method of
mitotic counts at fixed times is not so satisfactory as the photographic method. In
the first place unless plasma forms the support for the growing cells, which is often
undesirable, the areas of growth on the coverslip are not sufficiently comparable to
form an accurate basis for the mitotic counts; and secondly, although the growth rate
in embryo extract is just adequate to maintain the balance and give constant figures
between 20 and 36 hours, this will probably not be true for other media. In fact the
balance depends on the medium, and if the medium is altered the counts of mitosis
become far from comparable. For both these reasons then, a photographic method
which allows observations to be made over a considerable period of time is desirable.
The disadvantages of the photographic method as described above arise from
the variability of the cultures themselves, and the fact that only a small portion of a
single culture can be photographed at once. This, however, only makes the practical
details somewhat laborious, since the difficulty is largely overcome by repeating the
experiments. Hanging drop cultures are worse offenders in this direction than
flask cultures. Except for the fact that flask cultures necessitate the presence of
plasma, they would be more suitable for measurements of growth rate since the
conditions can be made far more constant owing to the greater volume of fluid
medium which is used, and in general growth in them is more uniform, so that the
results of different experiments are more comparable.
SUMMARY.
1. A method is described by which hanging drop cultures of chick heart
fibroblasts can be photographed at stated intervals for long periods of time.
2. The growth of cultures in embryo extract has been analysed and the actual
part played by outward migration of cells into the medium and by cell division has
been evaluated.
Studies on the Growth of Tissues in vitro
339
3. A formula is given for expressing the growth rate of a culture in terms of the
percentage of cells dividing per hour. This is termed the growth index.
4. Growth has been shown to occur uniformly, but at a decreasing rate for the
first 36 hours and then to decline quickly and to become somewhat irregular.
5. The actual number of cell divisions on the field in a culture in embryo
extract reaches a maximum at about the 30th hour of cultivation, but is fairly
constant from about the 20th to 36th hour.
6. Cells divide most frequently near the periphery, where there is an optimum
cell population density.
7. The period of mitosis remains fairly constant throughout the growth of the
culture, tending to increase when the culture is growing less rapidly at the end,
when the cells may stay for a considerable time in metaphase.
8. The intermitotic period is variable and increases greatly as the culture grows
older.
9. The effects of certain alterations in the composition of the medium are
described. Dilution of the extract with Tyrode solution caused a considerable fall
in the rate of cell division. The dilution of extract with serum instead of Tyrode
solution produced a similar decrease in mitotic activity, but tended to increase the
number of cells on the field.
10. The independence of cell activity and cell division is stressed.
The expenses of this research have been in part defrayed by a grant from the
British Empire Cancer Campaign.
REFERENCES.
BUCH-ANDERSON, E. and FISCHER, A. (1928). Roux Archivf. Entwicklungsmechanik, 114, 26.
CARREL, A. (1923). Journ. Exp. Med. 38, 407.
CARREL, A. and EBELING, A. H. (1922). Journ. Exp. Med. 35, 17.
(1923). Journ. Exp. Med. 37, 759.
EPHRUSSI, B., CHEVILLARD, L., MAYER, A. and PLANTEFOL, L. (1929). Annales de Physiologie et de
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EPHRUSSI, B. and TEISSIER, G. (1932). Arch.f. Exp. Zellforschung, 13, 1.
FISCHER, A. (1925). Tissue Culture, p. 175 (Heinemann).
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STRANGEWAYS, T. S. P. (1922). Proc. Roy. Soc. B, 94, 137.
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