IN VITRO

Vol. 10, No. 5 & 6, 1974

RESPONSE OF MAMMALIAN CELLS TO CONTROLLED G R O W T H

RATES IN STEADY-STATE CONTINUOUS CULTURE 1
R. SINCLAIR2
McGiU University, Montreal
SUMMARY

1. Mouse LS cells grow in completely mixed steady-state continuous suspension

"ehemostat") culture in defined medium.
2. The steady-state concentration of cells is maximal at a dilution rate of 0.30 to
0.35 day -1
3. Glucose can act as the hmiting substrate for LS cells under chemostat conditions_
4. The glucose oxidation rate per cell does not vary with dilution rate.
5. Maintenance energy is 19 picomoles of ATP per cell per day. Growth energy is
22 picomoles of ATP per cell.
6. Slowly growing cells contain more protein and less RNA per cell than rapidly
growing cells.
7. The "efficiency" of protein synthesis decreases in slowly growing cells, in whicb
a lower proportion of ribosomes is present in the form of polysomes or ribosomal
subunits.
8. Newly-made 18S RNA appears early in the cytoplasm of rapidly growing cells,
but is greatly delayed in slowly growing cells.
9. Pulsed additions of a limiting substrate to steady-state populations may lead to
synchronized cells that have a controlled interdivision time. Hence chemostat cultures
may be used to investigate the interdependence of events in the cell cycle.
In his introductory lecture to the Fifth International Symposium on the Continuous Culture
of Micro-organisms, Pitt (1) made the following
statement: "Chemostat culture is the only conceivable way to bring under full control, growing
populations of either single types of organisms of
mixed cultures, constituted from bacteria, algae,
protozoans, fungi, animal and plant tissue cells.
The ultimate objectives of such studies are to
discover all the mechanisms of living cells and
the properties of populations, and to invent new
cell behaviour by means of mutant organisms and
exploitation of metabolic regulation. Almost the
whole of the dynamic as opposed to the static
models of cell structure and function lie before
us awaiting discovery." The modeling approach,

that is, the theoretical assumptions and predictions as to the behavior of populations of cells in
chemostat culture is well advanced, and the degree of sophistication of the chemostat technique
continues to increase. The literature is now replete with descriptions of marvelously engineered
chemostats, cytostats, biostats, cytogenerators,
turbidostats, and nephelostats--systems that are
designed to grow cells for indefinite periods at
constant and controllable growth rates.
What are we learning about the dynamic
model of cell structure and function that should
accompany these theoretical and technical advances? It turns out that rather little use has
been made of these exciting tools to investigate
physiological alterations in eukaryotic cells grow,
Presented at the Twenty-fifth Annual Meeting ing at different rates, despite the fact that they
of the Tissue Culture Association in the Paul F. do provide a wide avenue to the study of regulaKruse, Jr. Memorial Symposium on Steady-State tion of gene activity through the manipulation
Culture of Cells.
of controllable environmental factors. Changes
Send requests for reprints to Dr. R. Sinclair,
Dept. of Biology, McGill University, Box 6070, in mammalian cells with growth rate have indeed
been examined, but the experimental systems
Stn. A, Montreal, H3C 3G1 Canada.
295

296

SINCLAIR

u~ed are highly biological and relatively uncontrolled, and include, for example, regenerating
liver, cells released from contact inhibition or
starvation, phytohemagglutinin-stimulated lymphocytes, virus-transformed cells, hepatomas of
wtrymg growth rate, and similar systems. Generally speaking, physiological profiles, for example, of the protein-synthesizing machinery, or of
the energy-produchlg systems, arc examined in
the quiescent state, and compared with cells that
have been stimulated to grow and divide at some
arbitrary and poorly defined rate. Not only is
the more rapid rate ill defined, it is often a transitory state, and one which the cell will not maintain indefinitdy. This again emphasizes the need
for polmlations of cells growing in controlled
steady-state, and for an examination of the
changing physiology of such cells.
The growth of cells in a ehemostat is controlled by nutrient limitation. Any growth rate
tess than the maximum is a result of starvation
conditions, of cells growing more slowly because
some limiting substrate is unavailable when
needed. Starvation however, is a relative term.
All the factors required to allow mammalian cells
to express their maximal growth potential are not
yet known, and we cannot be sure that even in
the richest medium there is not some factor, yet
unknown, which, when added (or removed), will
urge the cells to faster growth rates. This is a
problem through which the design of mammalian
cell growth media has already evolved; first, the
medium is designed to contain the minimal components to support growth, then supplements are
added to increase the rate of growth, aad finally
the quality of growth becomes more important
than the quantity, and media are constructed
for specific purposes.
The existence, then, of a limiting substrate,
does not present a problem; there will alw:tys be
a limit, whether it be the extrinsic nutrient, or
the intrinsic limitation in the capacity of the cell
to reproduce itself faster than a given rate. The
nature of the limiting substrate, however, is of
great significance, and may, as the microbiologists :dready know, have a profound effect on the
physiology of the cell.
The growth of mammalian cells in steady-state
continuous culture will be considered under five
topics, each of which develops from the preceding
one: (a) Do mammalian cells achieve steadystate in the ehemostat ? (b) Do the steady-st'~te

conditions achieved conform to the theoretical

predictions developed for microorganisms by
Monod, Novick, and Herbert and their associates? (c) Does the metabolic activity of the cell
change at tim various growth rates which can be
achiev(~ in the chemostat? (d) Do the cells remain structurally the same at different growth
rates'? (e) By observing alterations in the cells at
different growth rates, is it possible to form conclusions about the regulation of gone activity
from such experiments ?
The experiments that are reviewed here concorn completely mixed steady-state continuous
suspension cultures, and although they do not
qualify under the strict defnition of the "chemostat," this term is frequently used as an abbreviation, and its use will be continued in this article.
STEADY-STATE CONDITIONS IN THE CHEMOSTAT

Some of the earliest attempts to grow cells under chemostat conditions were reported by Cohen
and Eagle (2), who demonstrated that HeLa $8
cells could be grown continuously in a simple
system in normal medium, and under favorable
conditions could be kept in steady-state for as
long as 10 days. They also described considerable
oscillations in the cell population in extended culture, which they ascribed to undefined changes
in the nutrient medium. Work from our laboratory (3), described a "solera" culture in which
medium was added at a constant rate to a stirred
culture and, when the volume had reached 3 to
4 times the starting volume, the excess of cell
suspension was removed and the cycle repeated.
One such solera culture of strain L cells in defined medium was maintained in this way for
more than 300 d'tys. This was later developed
into a more convention'd constant volume arrangement (4), and it was shown that, from a
high starting concentration of cells, it was possible to reach steady-state cell populations in 5 to
6 days. Pirt and Callow (5), working with more
elaborately mech,mized equipment, showed that
ERK and strain L cells could also achieve steadystate conditions. This is best illustrated in a later
paper (6), which shows one culture in which the
dilution rate was shifted repeatedly, after steadystate had been confirmed. In every case, 4 to 8
days were required to reach constant conditions.
Perhaps the most painstaking analyses were
carried out by Moser and Vecchio (7), who main-

CELL BEHAVIOR IN STEADY-STATE CULTURE

rained cultures of P815Y mouse aseites mast
cells, in a modified cytogenerator for 50 to 60
days. Steady-state conditions were achieved only
after an average of 20 days, and the approach
to steady-state was characterized by regular oscillations in cell concentrations. More recently,
the nephelostat has been modified to permit the
growth of mammalian cells (8), and the developers of this equipment have demonstrated that,
when required to do so, the nephelostat is capable of maintaining HeLa cells in suspension at
constant concentrations for up to 5 days, the concentration being controlled by appropriate dilution with fresh medium. No information was
given, however, to indicate the dilution rates required to maintain these constant concentrations.
From the foregoing evidence, it may be coneluded that mammalian cells capable of growing
in suspension can probably reach steady-state
conditions within a limited range of dilution
rates. This range varies from just below the
maximal growth rate of the cells, usually not less
than a doubling time (t~) of 24 hr, down to
growth rates of about one-fifth of that, i.e. doubling once every 120 to 150 hr. It is generally
agreed that very slow growth rates have not yet
been achieved consistently; moreover, such
growth rates may well be accompanied by a significant proportion of cell deaths. Approach to
steady-state is often characterized by an oscillatory pattern, the analysis of which promises to
be fascinating, when the technical methods are
sufficiently sophisticated to obtain oscillations
with regularity.
CELL CONCENTRATIONVARIATIONS WITH
GROWTH RATE

Are the steady-state concentrations reached in

continuous culture in accord with the predictions
based on Michaelis-Menten kinetics, and best
developed by Herbert, Ellsworth and Telling
(9) ? This is a question asked by few investigators growing mammalian cells. Early work
from Pirt's laboratory (5) showed steady-state
cell concentrations with no obvious pattern; but
later, a more satisfactory relationship was obtained that displayed reasonable conformity, but
with considerably reduced concentrations and
low dilution rates (6). Despite the maintenance
by mouse ascites mast cells of prolonged steadystate cell concentrations, these concentrations
fluctuated with dilution rate in an inconsistent

297

manner, varying from 14 10~ cells per ml at

~t. = 32 hr, 18 10~ cells per ml at t~ = 53 hr,
9 10~ per ml at t. = 57 hr, and 1.8 10~
cells per ml at t, = 122 hr (7). In our own
laboratory., it was shown that conformity was
maintained for a limited number of dilution
rates, for strain L cells growing in defined medium (4). More recently, we have used LS cells
and have obtained further evidence that these
cells do, behave predictably in continuous culture,
and appear to display two modifications of the
classical microorganisms pattern. Fig. 1 illustrates cell number, DNA (as deoxyadenosine,
dA) and protein plotted against dilution rate, or
ratio, along the horizontal axis, translated into
doubling time along the upper axis. The cells are
LS cells, derived by Paul from mouse L cells, and
selected because of their poor attachment to glass
surfaces. The medium is the Birch and Pitt
formulation (10), containing the normal spectrum of defined components, although comparatively rich in amino acids. Its sole macromolecular constituent is low molecular weight methylcellulose. These results, which illustrate the behavior of semicontinuous cultures, have not required the use of any sophisticated, automated
equipment beyond a reliable alarm clock. Cultures were set up in magnetically stirred Florence
flasks, gassed continuously with 5% CO2 in air,
and portions of the cell suspension were replaced
w~th fresh medium at 8-hr intervals, to achieve
the dilution ratios noted along the abscissa.
Feeding at intervals was continued for 10 to 12
days until we were satisfied that steady-state had
been reached. It is evident that the steady-state
concentration of cells goes through a definite
maximum at about 0.33 day -1, with a pronounced
decline in the values on each side of this maximum. The near constant or hm:iz0ntal relationship, predicted and experimentally verified in
microorganisms, has disappeared. At high dilution rates the decline is not as steep as the predicted pattern, which requires a precipitous drop
to the critical dilution rate, above which onIy
washout occurs. This phenomenon has been
noted to a lesser degree with microorganisms and
has been attributed to wall growth (9, 11), and
Co the accumulation of toxic by-products (11).
We have not encountered significant interface
growth with this particular cell system, and
while experiments have not been carried out, to
examine the possibility of toxic by-products, it

298

SINCLAIR
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:FIG. 1. Steady-state concentrations of cell numbers, DNA, and protein at various dilution ratios. LS mouse cells were grown semicontinuously in defined medium, and appropriate
fractions were replaced at 8-hr intervals for 10 to 12 days. Points represent means of the
three samples taken during 1 day.
would certainly be difficult to visualize, in mammalian cells, a greater accumulation of toxins at
high than at low dilution rates. A more likely
explanation is a more fundamental one, based
on the model of cell growth proposed by Williams
(12). He regarded cell growth as a two-stage
process: uptake of nutrients into the cell, and
synthesis and processing of macromolecules from
the nutrient pool. On this basis, the steady-state
concentration of cells with respect to dilution
rate turned out to be a linear relationship, the
cell concentration declining steadily with increasing dilution rate. If nutrient uptake followed
Michaelis-Menten kinetics, and had superimposed on it a constant time period for macromolecular synthesis, then this may well lead to
a relationship closer to that found experimentally.
The decline found at low dilution rates has
been attributed to the reduced availability of
energy. Stated simply, as the cells grow more
slowly, more of the available energy must be

channeled into maintenance of the cell, and less

is available for growth processes. Since the rate
of growth is fixed, this leads to reduced steadystate cell concentrations. Theoretical analysis of
this possibility has been explored by Pirt (13),
and by Wase and Hough (14), and experimental
observations under different O~ concentrations
have been made by Kilburn et al. (15). By measuring the rates of glycolysis and oxidation in
batch cultures under different O~ tensions, it was
shown that, even at comparatively high growth
rates (e.g.t. ---- 1.15 day), some 65% of the cell's
energy production was used for maintaining the
cell, as distinct from the energy required to synthesize material for a new daughter cell. This
was contrasted with the bacterial cell which used
less than one-tenth of its energy production for
maintenance, despite the fact that it required a
much greater (by 10 times) amount of energy
per unit of dry weight for maintenance. Cells
growing more slowly will require an even greater
proportion of their energy production for main-

CELL BEHAVIOR IN STEADY-STATE CULTURE

tenance purposes, and further evidence on this
problem is presented below.
It must be added here that the decline in
steady-state cell concentrations at low dilution
rates may also be a result of cell death. Theoretical consequences of a constant loss of cell
viability have been discussed by Sinclair and
Topiwala (16), who introduced into the steadystate equation, the term --3,x, the rate of loss of
cells by death, and showed that the steady-state
concentration of cells will decline to an increasing extent at low growth rates. Mammalian cell
populations are always subject to a proportion
of cell deaths, but we have not found this a
problem with this cell system. Dye exclusion
routinely shows a viability of over 90%, and
even in the slowest cultures it has rarely fallen
below 85%.
ENERGY ~r

AND GROWTH ]:~ATE

Of the principal causes of the decline, we regard energy deprivation as predominant. This is
shown in Fig. 2, illustrating the steady-state
concentration of glucose and lactate in the
Td ,

growth medium. At low dilution rates, no glucose is detectable in the output medium until a
doubling time of 2 days; at shorter doubling
times the more rapidly growing cells are not
using all the available glucose, and the increasing
amount detected mirrors the declining steadystate concentration of cells. This is evidence that
mammalian cells can grow in steady-state, with
glucose as the limiting nutrient. Lactate is not
produced by slowly growing cells; the maximal
concentration is found in the culture in which
the steady-state cell concentration is also highest. These simple nutrient measurements may be
converted to metabolic quotients (Fig. 3), by
normalizing and multiplying by the dilution rate,
and reveal that the glucose consumption p~r
million cells per day increases almost linex:ly
with dilution rate, as does lactate output, ~ppearing to reflect the more rapid rate of gro~th
of the cells.
It turns out, however, that when the nmtabolic quotient of O~ consumption is measured,
this does not reveal the same linear increase
with dilution rate. Oxygen is supplied to the

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Fro. 2. Steady-state concentrations of glucose and lactate at various dilution ratios.

Growth conditions were the same as in Fig. 1, So, input concentration of glucose.

30{)

SINCLAIR
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Fro. 3. Metabolic quotients of total glucose used, lactate produced, glucose oxidized,
and oxygen used by LS mouse cells grown in suspension at various dilution ratios9
cells by continuous gassing with 5% CO~ in air,
and by using the Florence flask with a relatively
high surface to volume ratio, we find it is possible to maintain 80 to 90% oxygen saturation
in all the cultures except in the densest cultures,
where it may fall to 60 to 65%. Oxygen partial
pressures of 80 to 160 mm Hg usually allow cells
to express their maximal growth (17-19), and
the gassing method is considered to maintain a
nonlimiting supply of 02. Oxygen uptake rates
were measured by an external method. Samples
of cell suspension were transferred from the continuous culture to a standard Clark electrode
polarograph assembly, and after 3 rain equilibration with 5% C02 in air, the rate of O~ consumption was measured. Thus, for each steadystate population of cells, the maximum capacity
for O~ uptake was measured. This was found to
be remarkably stable (Fig. 3) and, except for
the slowest growing cells, remained constant at
4.0 to 4.2 micromoles of 02 uptake per l0 s ceils
per day (18). If we calculate the quotient of
glucose that is consumed, but which does not
appe.qr as lactate in the medium, that is, the

glucose which is presumed to be oxidized, this

results in a glucose oxidized quotient that is
also nearly constant, 0.67 to 0.74 mieromoles per
10~ cells per day. Assuming an uptake of six
molecules of 0~ per molecule of glucose oxidized,
these figures are surprisingly consistent.
It now becomes possible to calculate in a
completely different way the relative amounts
of energy used in maintenance and in growth.
The diagrams show that, since oxidation is relatively constant, extra energy used by the more
rapidly growing cells is obtained from the production of lactate, although this source amounts
to less than 5% of the total requirement of the
cell, except in the case of the most rapidly growing ceils. Assuming 38 molecules of ATP per
glucose molecule oxidized, and 2 molecules of
ATP from glycolysis, it is possible to calculate
the total energy production of the cell. Then,
E:x'Em+x'D'Ea
or

E / x -= E,~ 4- D'Eg

CELL BEHAVIOR IN STEADY-STATE CULTURE

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EzG. 4. Activities of glucose-6-phosphate dehydrogenase, cytoplasmic malic dehydrogenase,

and lactate dehydrogenase in LS mouse cells at various dilution ratios.
where E is the total energy produced; Era, the
amount used in maintenance; E~, the amount
used in growth; x, the cell concentration; and D,
the dilution rate. Thus, if E is plotted against
D, the intercept is a measure of E,~ and the
slope is a measure of E~. Regression analysis
shows the maintenance energy to be 19 micromoles of ATP per 106 cells per day, and the
growth energy, i.e. the energy to make 10~ cells,
is 22 micromoles of ATP. The same parameters
measured for LS cells grown in batch culture
under limiting concentrations of 02 were shown
to be 17 and 23 micromoles of ATP per 106 cells,
respectively (15). It is evident that, in more
rapidly growing cells, the residence time is
shorter, and less energy per cell is required for
maintenance, thus more is available for growth,
and glycolysis never generates a significant part
of the total energy in this system.

A number of energy-associated enzymes have

been measured within the cells, and these are
shown in Fig. 4. Cytoplasmic malic dehydrogenase (EC 1.1.1.37) (MDH) tends to decrease
with increased growth rate. Glucose-6-phosphate
dehydrogenase (EC 1.1.1.49) (G-6-PDH) remains constant at low dilution rates, but increases as the cell growth rate increases, and
probably reflects the increasing demand for penrose for nucleic acid synthesis. Lactate dehydrogenase (EC 1.1.1.27) (LDH) behaves in what
appears to be an anomalous manner, decreasing
by 4-fold as the growth rate increases. Why
should slowly growing cells, which are not producing lactate into the medium, have relatively
high concentrations of lactate dehydrogenase?
This would seem to be an artifact of the semicontinuous feeding schedule. Hourly analysis of
the medium after a normal proportional change,

302

SINCLAIR

has shown that lactate is immediately generated

into the medium after feeding, and in slowly
growing cells this lactate is later removed from
the medium by the cells. Thus, because they are
chronically glucose deficient, newly added glucose is immediately converted to quick energy
by glycolysis, and the lactate is then slowly
taken up again and oxidized. Therefore, the LDH
activity must be maintained to catalyze this
metabolic requirement, and it might be predicted
that in a fully continuous culture, LDH activity
would be much lower in slowly growing cells.
This behavior is reminiscent of earlier work
by Glinos et al. (20), who showed that strain L
cells maintained in stationary phase without renewal of medium, had an LDH activity of about
1.0 unit per mg protein, a value similar to that of
exponentially growing cells, and the activity increased to 8 to 10 times that value when the
medium was replaced each day. The latter cells
showed a similar release of lactate into the medium after feeding, followed by a later and more
gradual uptake of that lactate. Similar enzyme
activities in oxygen-limited batch cultures of LS
cells have been investigated by Self et al. (21),
who showed that G-6-PDH increased slightly
with increasing 08 tension and growth rate;
M D H decreased, and LDH remained nearly constant. In a study of Morris hepatoma tissues,
Criss (22) has shown that rapidly growing,
poorly differentiated hepatomas have M D t t activities that are lower than in the more slowly
growing highly differentiated hepatomas, which
in turn are lower than in normal liver. Similarly,
G-6-PDH increased with the rapidity of growth,
and LDH did not vary significantly between the
different hepatomas. The similarity between the
results from steady-state continuous cultures and
the much more biologically heterogeneous "minimal deviation" hepatomas, suggests that the
former may be used as a model system to examine metabolic shifts that occur as tissues
change their growth rate.
PROTEIN SYNTI-IESIS AND GROWTH RATE

With certain exceptions, the enzymes we have

measured tend to increase as the cells grow more
slowly, and so too, does the total protein content
of the LS cells (Fig. 1). This is true also of HeLa
cells growing in suspension in serum-supplemented medium, and is in sharp contrast to the
ribonucleic acid per cell, or per unit DNA, which
is seen to double between slowly growing and

rapidly growing cells (28). This is shown even

more dramatically when calculated as the rate
of RNA accumulation per unit DNA (the
steady-state RNA/DNA times the dilution rate),
which increases 10-fold within the range measured. The rapidly growing cells are, of course,
producing more protein per unit time than slowly
growing cells, and this relationship may be quantified by the "efficiency" of protein synthesis, a
parameter that has been related to growth rate
in a number of bacterial systems (23, 24). The
"efficiency" of protein synthesis is defined as the
output of protein (as a rate) relative to the
steady-state concentration of RNA, and is can
culated as the dilution rate times protein/RNA.
As seen in Fig. 5, this quantity remains relatively
constant at higher dilution rates, but falls off
markedly at low dilution rates. To investigate
this loss of efficiency, we have examined the cytoplasmic ribosomal profiles of the cells at different growth rates, and find that rapidly growing
cells have a greater proportion of polysomes, and
a greater number of ribosomal subunits relative
to monomeric ribosomes (Table 1). It may be
noted that the proportion of polysomes appears
to increase proportionately with growth rate,
and does not reflect the discontinuity illustrated
in the efficiency of the protein-synthesizing system referred to earlier. This system is, of course,
highly complex and is under a variety of controls,
of which the availability of ribosomal subunits
and the formation of polysomal complexes is only
one aspect, and certainly the data presented here
are sufficient only to emphasize an interesting
area for further investigation.
Similar relationships between the polysomal
content and the rate of growth have been reported in other experimental situations, for example, in ascites cells transferred to enriched
medium (25), and in HeLa cells released from
puromycin treatment (26). Microorganisms
grown in chemostat conditions also increase their
protein-synthesizing efficiency by an increased
number of polysomes at high growth rates (23,
24). The reduced rate of protein synthesis at low
growth rates means that, in these cells, a greater
proportion of the RNA present is unused, and
this has been referred to as "extra" RNA by
Koch (27), who has extensively discussed its role
in the life history of Escherichia coli.
The information discussed to this point has
been obtained without the use of radioisotopes.
It requires only brief reflection to appreciate

CELL BEHAVIOR IN STEADY-STATE CULTURE

303

S.O

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3.0

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016

lhG. 5. The "efficiency" (D protein/RNA, by weight) of the protein-synthesi~.ing system in HeLa cells grown in Eagle's minimum essential medium plus 10% calf serum, at
various dilution ratios. Different symbols represent separate experiments.
TABLE 1
COMPARATIVE PROPORTIONS OF RIBOSOMES, AS
POLYSOMES, MONORIBOSOMES~ AND RIBOSOMAL
SUBUNITS IN HELA CELLS AT VARIOUS D I L U TION R A T E S
Dilution
Rate
(day-L)

0.12
0.25
0.48
0.57

Polysomes

Monosomes

%
25.7
25.9
35.6
40.5

%
74.3
52.9
43.9
33.0

Ribosomal Polysome
Subunits Monosome

%
0
21.2
20.5
26.5

0.35
0.50
0.81
1.22

that the addition of such probes to a continuous

culture system generates a completely new set
of problems. These arise initially as practical
decisions which must be made, and quickly escalate into theoretical problems demanding ki-

netie analysis, which has not yet been made. Experimentally, the addition of a radioactive
compound and its cold carrier, unless completely
inert as a nutrient, will affect the delicately balanced steady-state conditions in the chemostat.
Further, the permutations of pulse and chase
become endless. Is the labeled compound added
to the growth vessel only? If so, is the dilution
continued or stopped ? Is the reservoir supplemented with an equal concentration of cold compound? Is the labeled compound added to the
reservoir only, and its concentration allowed to
build up in the growth vessel? Is the labeled
compound added to both growth vessel and reservoir ? How then can it be chased if this is desired? Each of these situations results in different kinetics of the passage of the radioisotope
through the organism, and of course will vary
enormously with the isotope of interest and the

304

SINCLAIR

radioactive compound into which it is incorporated.

These problems are not as acute in semicontinuous culture, and in order to investigate further the loss in ribosomal efficiency, we have
proceeded, undaunted, to add tritiated uridine,
and to measure its incorporation into RNA in
rapidly and in slowly growing cells (28). After a
45-min pulse, ribosomes and ribosomal RNA
were isolated by standard detergent-washing and
phenol extraction methods. It was observed that
the major incorporation was into the monosome
and subunit peak, or more specifically, into the
cytoplasmic 18S RNA of the rapidly growing
cells. By comparison, the incorporation of label
into rRNA in slowly growing cells in 45 rain was
insignificant. The addition of label for 12 hr resulted in 28S and 18S RNA of equal specific activity in rapidly growing cells but, even after
that time, in slowly growing cells, the 18S RNA
had a lower specific activity than 28S RNA
(Table 2). Thus, there appears to be a differential rate of processing of the two RNA species,
between fast and slow cells. Evidence yet to be
confirmed suggests that the nucleoplasm of the
rapidly growing cell contains a small proportion
of 18S RNA of high specific activity, in contrast
to the nucleoplasm of the slower cells, which contain more 18S RNA of much lower specific activity. Thus, control of the availability of new
cytoplasmic ribosomes may be through the
smaller subunit, controlled in turn by rate of
movement, or modification of the 18S RNA, in
the nucleoplasm of the cell.
These, then, are tentative examples of the
potential use of the continuous culture method,
through its capacity to generate biochemieally
useful amounts of cells with varying physiological
profiles, and illustrate the possibility of investigating fundamental biochemical and biological
processes.
Chemostat methods are usually considered to
reveal the behavior of populations of cells, and
their extensive properties under chosen conditions. They may also be used to investigate regulation in the individual cell by modifying the
system to generate synchronized cells. This may
be achieved by regulating the limiting substrate.
It has been shown theoretically (29, 30) that if
the limiting substrate is added to the chemostat
in a series of pulsed additions, the cell population
will become entrained, or synchronized in some

TABLE 2
[ 3 H ] - U R I D I N E D I S T R I B U T I O N IN CYTOPLASMIC

RNA AFTER 12 HOURS

Dilution
Rate

RNA
Class

lDgAa

Counts/
miu

Specificb
Activity

28S s.a.
18S s.a.

0.57

28S
18S
28S
18S

21.5
19.2
13.3
11.8

25,740
21,590
2,690
1,330

1197
1124
202
113

1.06

0.12

(s.a.)

1.79

Calculated as 20 O.D.26 o units = 1 mg RNA.

b Specific activity = cpm/t~g RNA.
fashion related temporally to the pulsed addition.
Thus, if a constant growth rate is established,
and the limiting substrate is provided in measured amounts at intervals equal to the doubling
time of that steady-state population, then after
six to eight additions, the cells should be synchronized, dividing once each interval. The result
is a system which generates synchronized cells
with a controlled division time, and which offers
the prospect of analyzing the events of the cell
cycle and their interrelationships, with much
greater precision than has been possible hitherto.
Although the technique has been applied successfully to yeast cells (29, 31), it has not yet been
used with mammalian cells, one reason being
that the limiting substrate has not before been
defined in mammalian cell chemostat experiments.
An attempt has been made to survey the current state of knowledge of the behavior of the
cell in steady-state suspension culture, and in
particular the physiological changes that occur
in response to alterations in growth rate, this
rate being controlled by the investigator. It has
been shown that the energy metabolism is surprisingly constant at different growth rates, but
the protein-synthesizing system can demonstrate
consistent shifts that may be used to speculate
on regulatory mechanisms within the cell. Other
possible uses have been discussed, attempting to
show that the chemostat is a versatile tool for
investigating both the colligative and intrinsic
properties of the growing cell.
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I wish to thank Dr. I. Daskal and Mr. P. B. Woodruff, some of whose

postgraduate work is reported here. T h e support of the National Research
Council of Canada, grant A3458 is acknowledged.