J. Cell Sci. 6, 739-749 (1970)
Printed in Great Britain
739
THE QUANTITATIVE UTILIZATION OF AMINO
ACIDS AND GLUCOSE AND CONTACT
INHIBITION OF GROWTH IN CULTURES OF
THE HUMAN DIPLOID CELL, WI-38
J. B. GRIFFITHS
Microbiological Research Establishment, Porton, Salisbury, Wiltshire, England
SUMMARY
There are many reports in the literature showing that contact inhibition of growth is affected
by the culture medium. A quantitative study of amino acid and glucose uptake by the human
diploid cell line, WI-38 was carried out to determine more precisely what effect nutritional
factors have on contact inhibition of growth.
Eagle's minimal essential medium (MEM) was found to support higher cell yields than
Eagle's basal medium (BME) and for growth to continue beyond 96 h a medium change was
essential. However, analysis of the used growth media showed that neither amino acids nor
glucose were fully depleted after 96 h. The rate of glucose utilization was in the range 65-100
/ig/mg dry wt./h and this agreed very closely with the results of other authors. The pattern of
amino acid uptake also closely resembled that for other cell lines except that the utilization of
cystine was higher. The nutritional requirements were further studied as the results from the
medium analyses failed to explain the growth-promoting activity of MEM. Daily medium
changes greatly increased cell yields even though the medium nutrients were not exhausted.
This effect was dependent upon fresh medium being used and the only medium component
found to be of importance was the amino acid complement. These results are discussed in
relation to the low saturation density of diploid cells in culture and a possible explanation is
proposed in terms of differences in the cell membrane between normal and altered cells.
INTRODUCTION
It has generally been accepted that Eagle's basal medium (Eagle, 1955) is nutritionally adequate for the cultivation of human diploid cells. However, there is a marked
trend towards the use of Eagle's minimal essential medium (Eagle, 1959) for these
cells and also many reports have shown that better and more prolonged growth is
achieved with modifications to the basal medium (Kruse & Miedema, 1965a; Baugh
& Tytell, 1967). The nutrition of human diploid cells has not received much attention,
due probably to the fact that the low saturation density of these cells in culture has
precluded the idea that the medium may not be nutritionally adequate.
Human diploid cells undergo contact inhibition or density-dependent inhibition of
growth (Stoker & Rubin, 1967). This appears as a retardation and finally cessation of
protein and nucleic acid synthesis and cell division as the cell sheet approaches and
reaches confluency (Levine, Becker, Boone & Eagle, 1965). Although this growthregulating mechanism has been extensively studied, the manner in which it operates
is not understood. Carter (1968) has proposed that for cell division to occur the plasma
740
J. B. Griffiths
membrane must remain unstable, and that the known facts about cell growth and
contact inhibition can be explained in terms of membrane stability. Although contact
inhibition of growth is usually attributed to a reaction initiated by cell-to-cell contact,
a number of observations indicate that it may be influenced by properties of the growth
medium. For instance, the substitution of glucose by galactose (Baugh & Tytell, 1967),
frequent medium changes (Rubin, 1966; Rhode & Ellem, 1968), the use of a large
medium volume in relation to the cell-covered surface (Stoker, Shearer & O'Neill,
1966) and the use of a continuous medium perfusion culture (Kruse & Miedema,
1965 a) have all reduced the effect of contact inhibition. These modifications may be
effective due either to simply overcoming a nutritional deficiency or to an unstabilizing
effect on the plasma membrane.
This report describes the quantitative uptake of amino acids and glucose during the
growth of the human diploid cell line, WI-38, in an attempt to find out whether
nutrient exhaustion influences contact inhibition of growth.
MATERIALS AND METHODS
Cell line
The human diploid cell line WI-3S was used. Stock cultures were grown in Eagle's basal
medium (Grand Island Biological Company) supplemented with 10% foetal calf serum (Flow
Laboratories Ltd.) and the culture protocol of Hayflick (Hayflick & Moorhead, 1961) was
followed. Cells between passage 21 and 29 were used. All the cell populations were at least 98 %
diploid as revealed by regular chromosome analysis.
Culture procedure
Two types of media were studied: Eagle's basal medium (BME) (Eagle, 1955) and Eagle's
minimal essential medium (MEM) (Eagle, 1959). Both media were supplemented with 10%
foetal calf serum and the glutamine concentration of MEM was doubled to 600 /tg/ml. In the
experimental cultures 10 ml medium in 4-oz medical flats were used and the medium was
changed after 96 and 192 h; cell counts and medium analyses were carried out at o, 96, 192 and
264 h. There were 6 replicate cultures for each sampling time: 3 were used for cell counts and
2 for dry weights. The inoculum was 1-2 x io4 cells/cm2 (5 x io5 cells per culture) in all cultures.
Measurement of growth
Counts of nuclei were made following removal of cells from monolayers by trypsinization and
disruption in citric acid. Counts are given as the mean and standard deviation of two counts on
triplicate cultures.
Cell dry zveight was determined by heating cell pellets at 95 °C for 2 h followed by desiccation
until the weight was constant. Values are given as the mean and standard deviation from two
cultures, each carried out in duplicate.
Amino acid analysis
Amino acids were determined by microbiological assay (Griffiths & Pirt, 1967). The assay
media were obtained complete from Difco Laboratories. Streptococcus faecalis, American Type
Culture Collection (ATCC) 9790 (Stokes, Gunness, Dwyer & Casvvell, 1945) was used for the
assay of L-histidine and L-threonine. Leuconostoc mesenteroides P-60, ATCC 8042 (Snell, Strong
& Peterson, 1937), was used for the determination of L-arginine, L-cystine, L-glutamic acid,
L-isoleucine, L-leucine, L-lysine, L-methionine, L-phenylalanine, L-tryptophan, L-tyrosine and
L-valine. Turbidimetric assay was applied, bacterial cell growth being measured in a Unicam
Nutritional requirements of WI-38 cells
741
SP-600 spectrophotometer at 675 nm. Determinations were carried out in duplicate for all
samples.
Glucose determination
A glucose reagent kit (Clinton Laboratories, California) was used. The method was modified
in order to lower the detection level, by increasing the sample (and standard) volume from 20 to
100 /tl.
Ammonia determination
Both the Conway microdiffusion method (Conway, 1957) and an Autoanalyser system using
Nessler's Reagent were used concurrently.
Glutamine hydrolysis
Residual glutamine concentrations were determined by measuring the ammonia evolved
after addition of glutaminase (P. L. Biochemicals, Inc.). The reaction mixture was 0-5 ml
culture medium; 0-5 ml i-o M phosphate buffer, pH 8-i; 0-5 ml borate buffer, pH 8-i; 0-5 ml
glutaminase (4 units/ml) incubated at 37 °C for 2 h.
Growth yield (Y)
This expresses the relationship between cell growth and the utilization of the amino acids and
glucose and is defined as
Y = Ax/As,
where Ax is the cell dry weight produced (/<g) and As is the quantity of amino acid or glucose
utilized (//g).
RESULTS
The ability of BME and MEM to support growth
WI-38 cells were grown in BME and MEM to compare cell production. The
results are shown in Table 1, where it is apparent that MEM will produce more cells
than BME (73 % more cells in MEM after 264 h). However, BME produced the
same number of cells as MEM when the concentration of amino acids was doubled.
Table 1. A comparison of the growth of WI-38 cells in Eagle's basal and Eagle's
minimal essential medium and the effect of supplements to these media
(The cell yields are given as number of cells per cm2 x io4.
The media were changed at 96 and 192 h.)
Basal medium (BME)
Time (h)
...
Control
x 2 amino acids
x 2 glucose
x 2 amino acids
and x 2 glucose
x 2 glutamine
Minimal medium (MEM)
96
192
264
96
192
264
3-2 ±0-21
4-810-21
4-210-24
4-210-22
4-4 ±0-26
7-9 + 0-37
5-410-37
6-110-40
5-2 ±0-27
—
—
—
4-4 + 0-26
3-4 + 0-22
—
—
7-4 ±0-37
7-510-40
—
—
8-0 + 0-37
8-310-37
•—
—
—
—
—
5"4i°'37
7-8 + 0-40
9-810-33
742
J. B. Griffiths
This is to be expected, as the major difference between the two media is the increased
concentration of amino acids in MEM. Additional glucose also produced more cells in
BME but this could be explained by the sparing effect that glucose has on amino acids.
Cell yields in MEM were not significantly affected by an increase in the concentration
of amino acids or vitamins, but a twofold increase in glutamine concentration raised
the yield by 23 %. Unless the medium is changed after 96 h in both BME and MEM
cultures, very little growth occurs in the period 96-192 h.
The results summarized in Table 1 give clear indications that BME is growthlimiting and that this could be overcome by increasing the concentration of amino
acids and glutamine. Cell yields below io5/cm2, therefore, can be attributed to a
nutritional deficiency. This result would explain the wide variations in the yields of
WI-38 cells that have been reported and also it shows the need for defining the nutritional requirements of a cell before growth-regulating mechanisms are studied.
The uptake of amino acids and glucose
The residual concentrations of amino acids and glucose were determined in BME
and MEM after 96, 192 and 264 h of growth of WI-38 cells. The medium was
changed at 96 and 192 h.
Table 2. The percentage utilization of the amino acids and
glucose in WI-38 cell cultures
(The media were changed after 96 and 192 h and this divided the
culture period into 3 distinct phases.)
BME
Time (h) . . .
Arginine
Cystine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Tryptophan
Tyrosine
Valine
Glutamine
Glucose
MEM
0-96
96-192
192-264
0-96
23
44
54
23
62
24
43
29
26
22
10
23
96-192
192-264
34
31
24
5°
49
53
17
8
11
11
33
33
33
33
21
27
29
28
33
18
26
20
IS
16
18
18
19
25
19
6
19
3i
12
24
5
4
11
6
22
33
5
4
IS
13
13
19
18
9
10
9
9
10
12
20
20
21
21
S3
56
65
49
8
J
7
59
81
78
80
63
98
46
98
1
The extent to which the various nutrients were utilized is shown in Table 2. The
results were contrary to those expected as no nutrient was found to be sufficiently
utilized to become growth-limiting after 96 h growth. If the uptake values for the
different growth periods are added together it can be seen that several nutrients,
notably cystine, glutamine and glucose would have become exhausted after 192 h
Nutritional requirements of WI-38 cells
743
without a medium change. Arginine, isoleucine and leucine would all be approximately 90 % utilized after 264 h without a medium change in BME, and arginine and
leucine to the same extent in MEM. All the other amino acids were present in high
residual concentrations. It should be stressed that an amino acid would become growthlimiting before it was 100% utilized as a certain residual concentration in the medium
would be needed to keep the intracellular metabolite pool in equilibrium with the
external environment.
Under the conditions of this experiment all amino acids were present in excess
throughout the culture period but glucose was growth-limiting in MEM, and very
nearly so in BME after 192 h and 264 h. Without a medium change, glucose would
limit cell growth to 5-6 x io4 cells/cm2 and if the glucose concentration was increased
then glutamine would limit growth to 6-6 and 6-8 x io4 and cystine to 7-1 and 7-8 x io4
respectively in BME and MEM. These results indicated that glucose could be beneficially replaced by galactose as Baugh & Tytell (1967) have shown that galactose is
utilized less rapidly than glucose and supports higher populations of WI-38 cells.
However, in this laboratory the use of galactose has failed to cause any increase in
growth.
Individual pattern of nutrient utilization
The quantity of the essential amino acids and glucose utilized during cell growth
together with their growth yields (Y) (that is, the ratio of the amount of cell dry weight
produced to substrate used) is given in Table 3. Although only the data obtained after
192 h of growth are given, the relative order of magnitude is approximately identical at
all 3 sampling times. Arginine, leucine, isoleucine and cystine were the most highly
utilized amino acids, followed by lysine, histidine and valine. The growth yields of all
nutrients decrease as the culture progresses indicating a higher utilization rate per
cell as the culture ages. This phenomenon of diminishing returns, demonstrated by
the summarized values given in Table 4, is common to cell-culture systems when
prolonged by additional feeding. In this study the over-all cell growth rate was slow
and the cells spent long periods in a semi-stationary state. Griffiths & Pirt (1967)
demonstrated that the amino acid utilization rates were high at low growth rates due
to maintenance functions and this would explain why the most economical utilization
of nutrients occurred during the first 96 h of growth compared with the later growth
periods (Table 4). The growth yields are also much lower in MEM than BME, indicating that a higher turnover of nutrients occurs in the more enriched medium. This
is also a common phenomenon in cell cultures and can be attributed to the effect of
the law of mass action on the cellular enzymes.
The utilization rate of glucose (calculated from the data in Tables 2 and 3) was in
the range 65-100//g/mg dry wt./h throughout the culture period. This value compared closely with the results of other authors for both WI-38 cells (Kruse & Miedema,
19656) and for altered or malignant cells (Zwartouw & Westwood, 1958; Bryant,
Schilling & Earle, 1958).
48
C li L 6
744
jf- B. Griffiths
Table 3. The utilization of amino acids and glucose by WI-38
cells during 192 h of growth in culture
(The media were changed after 96 h and these figures are the sum
of the utilization in the first two phases of the culture.)
BME
Arginine
Cystine
Histidine
Isoleucine
Leucine
Lysine
Methionine
Phenylalanine
Threonine
Tryptophan
Tyrosine
Valine
Glutamine
Glucose
MEM
Yi
/ig/ml*
Ft
/ig/ml*
n-5
77
127
90
7-0
9-9
57
25'4
19
4-2
16-0
56
i7'5
8-o
S'i
II-I
4'°
3 -o
22-3
297
7-o
127
i-5
4-0
59'5
12-0
4-4
223
II-I
6-5
18-0
8-o
0-31
0-06
290
1505
7-0
15-3
25-S
33-0
5'2
3'2
19-0
7-0
5-6
15-3
26-8
8-9
24-4
16-5
5-9
40
508
O-20
1920
0-06
* Ten ml medium used per culture
f Growth yield (Y) =
cell dry weight produced (/tg)
amino acid or glucose used (jig)
Table 4. The increase in cell dry weight and the growth yields (Y)
of WI-38 cells for the amino acids and glucose
BME
MEM
A
Time (h)
0-96
...
Increase in cell
dry weight (/tg)
Y amino acids*
Y glutamine
Y glucose
935 ±18
1-19
0-46
o-io
96-192
192-264
0-96
96-192
192-264
1362 ± 29 1761 ±35 1028± 16 1690! 30 2270 + 44
O-87
O3I
072
028
0-06
0-05
066
0-27
0-08
049
0-44
O-2O
0-19
0-05
0-06
* These values were calculated from the total quantity of all 12 amino acids utilized.
Utilization of glutamine
Glutamine was very highly utilized in these experiments (Table 3). Since it is very
labile in cell culture media, as a result of which growth-limitation often occurs even
though glutamine is initially present in excess of the cells' requirements, the routes by
which it is utilized were studied. This was done by measuring the ammonia released
in (1) incubated cell-free media—this represents the glutamine spontaneously hydrolysed to pyrrolidone carboxylic acid; (2) the culture—the additional ammonia released
due to cellular action represents the glutamine converted to glutamic acid by gluta-
Nutritional requirements of WI-38 cells
745
minase action; and (3) culture media treated with glutaminase. The difference between this latter value and the expected value (theoretically calculated from the initial
glutamine concentration) should represent the glutamine incorporated directly into
the cells for protein synthesis and amide donor reactions. The results of these measurements are given in Table 5.
Table 5. The quantity of glutamine (/ig/ml) that is utilized in the formation of various
metabolic products, incorporated directly by the cells or utilized as glutamic acid in cultures
of WI-38 cells
MEM
BME
Fate of glutamine
Pyrrolidone carboxylic
acid*
Glutamic acid*
Utilized directly*
Utilized as glutamic
acidt
0-96 h
96-192 h 192-264 h
24
25
18
74
66
59
93
82
75
51
81
45
0-96 h
96-192 h
192-264 h
7i
72
53
130
137
178
107
172
15875
58
48
* The derivation of these values is described in the text.
f This value is the difference between the glutamic acid formed, as measured by ammonia
liberation, and the residual glutamic acid concentration found by microbiological assay.
Glutamine is used excessively in cell cultures (shown by the very low growth yields
in Tables 3 and 4) but under the conditions of these experiments it was not growthlimiting (Table 2) despite its high wastage rate. It has often proved worth while to
adapt cells to utilize glutamic acid in place of glutamine (Paul & Fottrell, 1963;
Griffiths & Pirt, 1967) as this aids the control of the environment by stabilizing the
medium. WI-38 cells were successfully adapted to utilize glutamic acid with no loss
in the cell yields but this was accompanied by a change in cell morphology from fibroblastic to epithelial. The reason for this alteration is not understood but its implications are under study.
Enhancement of growth by medium changes
The increased cell yields in MEM cannot be explained by the results presented so
far as neither glucose nor any amino acid was found to be exhausted, but the difference
in composition between BME and MEM indicates that it should be due to the increase in amino acid concentration. Further increases in the concentration of the most
highly utilized amino acids were found to stimulate further growth (Table 6), especially a mixture of arginine, isoleucine and leucine. The possibility of vitamin
limitation was also investigated but an increase in vitamin concentration had no effect
on cell growth (Table 6).
Kruse & Miedema (1965 a) greatly increased the yield of human diploid cells by
using a continuous perfusion culture. In an attempt to approach this rich nutritional
state the medium was changed at daily intervals over periods of up to 1 week. In the
experiment recorded in Table 6 the medium was changed 4 times and a count of over
48-2
746
J. B. Griffiths
5
2
2-i x io cells/cm was obtained. In the absence of a growth-limiting nutrient this
result is difficult to explain. It was considered important to establish that it was a
nutritional effect, as a medium change would alter the state of a culture in many ways;
for example, the composition of the gas phase would be altered and the cells would be
subjected to the stimulus of medium movement. The use of 72-h conditioned medium
Table 6. Medium supplements and culture conditions which increase
the yield of WI-38 cells after 168 h in culture
Cell yield per cm 2
Increase from
control (%)
( X IO 4 )
Increase in concentration:
Glucose ( x 1-5)
Cystine ( x 1-5)
Arginine "j
Isoleucine j- ( x 1 -5)
Leucine J
Vitamins ( x 2)
Daily media changes:
Fresh medium
Conditioned medium*
Conditioned medium + serumf
8-3 ±0-30
8-8 ±0-37
3
9
9-7 + 0-33
20
8-o±o-33
0
2i-4±o - 56
8-8 ±0-30
12-9+ 0-37
140
0
47
* Medium obtained from 72-h culture.
f 72-h culture medium + 5 % fresh serum.
Table 7. The quantity of nutrients which would be utilized to produce a
million cells (610 jiig dry wt.) during the first 96 h of culture
Nutrient (jig)
BME
MEM
Arginine
Cystine
Histidine
Isoleucine
Leucine
Lysine
Methionine
26
37
33
178
46
46
65
89
20
54
18
12
76
18
Nutrient (jig)
Phenylalanine
Threonine
Tryptophan
Tyrosine
Valine
Glutamine
Glucose
BME
MEM
6-5
65
12
5-2
13
14
18
47
20
12
870
145°
4000
5100
for the medium changes did not increase growth at all (Table 6) and showed the effect
to be dependent on fresh medium being used. Another factor to be considered at this
point was serum, as it was the only medium component not investigated for growthlimitation. Serum was not suspected of limiting growth as previous work has shown
that it did not significantly affect growth in the concentration range 7-20% (J. B.
Griffiths, unpublished data). Fresh serum was added to conditioned medium for
some medium changes, however, and was found to stimulate some growth, although
not compensating for fresh medium (Table 6). The free nutrients present in the serum
would account for the response it gives.
It must be concluded, therefore, that although certain nutrients are nowhere near
Nutritional requirements of WI-38 cells
747
becoming exhausted, their concentration falls sufficiently to cause depression of cell
growth after 72 h, and that there is a need for regular medium changes—or continuous
perfusion. The data obtained in this study also provide a useful basis on which
adequate growth media for WI-38 cells can be prepared (see Table 7).
DISCUSSION
It was surmised at the beginning of this investigation that although nutritional
deficiency was not the real cause of contact inhibition of growth, it may contribute to
the low yields of human diploid cells. Evidence for this was immediately forthcoming
from the empirical observations summarized in Table 1. It was an enigma, therefore,
that after analysis of the used growth media no amino acid was found to be growthlimiting in BME (Table 2), even though the cell yield was far less than in MEM. The
growth-promoting effect of MEM cannot therefore be explained as nutrient exhaustion in BME, so an explanation must be sought in either amino acid interrelationships or the equilibrium relationships between the cells and their environment.
Although the pattern of amino acid uptake of a wide variety of cell lines is remarkably
similar, each cell line has a characteristic utilization pattern. Usually arginine, leucine
and isoleucine are used in the greatest concentration followed by lysine, valine and
phenylalanine (Sinclair & Leslie, 1959; Kagawa, Kaneko, Takaoka & Katsuta, i960;
McCarty, 1962; Griffiths & Pirt, 1967). A comparison of the quantitative uptake by
WI-38 and LS cells (Griffiths & Pirt, 1967) shows a very similar uptake except that
cystine and histidine are more highly utilized by WI-38 cells. The uptake of cystine
constitutes a characteristic difference between WI-38 and other cell lines. Jacobs
(1966) also found cystine to be of special importance in the nutrition of WI-38 cells
as he demonstrated that these cells would grow in a simplified medium without the
addition of any amino acid except cystine and glutamine. In view of this special
requirement for cystine it is important that its concentration be increased in growth
media for WI-38 cells.
The yield of WI-38 cells in the media that were studied was very low (1*5—2-o x io5/
ml only), unless the medium was changed. The size of this increment in cell numbers
must be of significance as a similar value has been obtained in 4 different culture
systems. These are the stationary monolayer culture, large-scale roller cultures, the
homogeneous micro-carrier culture (van Wezel, 1967) and in cultures nutritionally
enriched by daily media changes. In this last system there was an increase in cell yield
of 1-9 x io5 cells/ml/medium change and thefinalyield was 2-1 x io 5 cells/cm2, a value
approaching that obtained by Kruse & Miedema (1965 a) in their perfusion culture.
Therefore, even with no nutrient found limiting, regular or continuous media changes
were necessary to obtain a high cell yield. For cell production to continue beyond a
certain level there is an absolute requirement for a medium change and the results
shown in Table 6 indicate that the only factor of importance is the amino acid content
of the medium. Growth limitation due to toxic metabolites released during cell growth
is always a factor to be considered but the demonstrations of Todaro, Lazar & Green
(1965) and Schutz & Mora (1968) provide enough evidence for the possibility to be
748
J. B. Griffiths
excluded, apart from the fact that this is highly unlikely to occur with the low cell
populations being obtained.
A characteristic difference between diploid and heteroploid cells is the low cell
yield due to contact inhibition of growth of diploid cells. Another consistent difference
between these two cell types is found in the structure of their plasma membranes
(Abercrombie & Ambrose, 1962; Defendi & Gasic, 1963; Kraemer, 1966). An
explanation as to how continuous medium perfusion or frequent medium changes
allow diploid cells to grow in multilayered sheets to densities approaching those of
heteroploid cells should be sought, therefore, in terms of the plasma membrane,
especially if growth control is associated with membrane stability (Carter, 1968).
An explanation that meets this criterion is that there is a difference between diploid
and heteroploid cells in their ability to accumulate nutrients intracellularly. Cells
actively take up medium components against a concentration gradient. The ability of
HeLa cells to concentrate nutrients intracellularly and the threshold concentrations
of amino acids required to initiate protein synthesis were measured by Eagle, Piez
& Levy (1961). A similar study with diploid cells may reveal a different capability for
these functions and show a need for higher extracellular concentrations of metabolites.
This would become of greater importance as the population density increased and the
cells became more compacted, so reducing the area of the cell surface in contact with
the environment. The thesis that initiation of inhibition in crowded cultures could
depend upon the uptake of a nutrient (or nutrients) into the intracellular pool which
would then regulate growth through a metabolic control system involving synthesis of
macromolecules is being studied.
The results contained in this report have pointed out certain characteristics of
WI-38 cell cultures that may have been overlooked and show that the growth-regulating mechanism of diploid cells is affected by the concentration of extracellular
metabolites.
I wish to thank Mrs I. K. Strutt and Miss H. Jack for their invaluable technical assistance.
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[Received 7 August 1969)