J. Cell Sci. 12, 617-629 (1973)
Printed in Great Britain
617
THE EFFECTS OF ADAPTING HUMAN DIPLOID
CELLS TO GROW IN GLUTAMIC ACID MEDIA
ON CELL MORPHOLOGY, GROWTH AND
METABOLISM
J. B. GRIFFITHS
Microbiological Research Establishment, Porton, Salisbury, Wiltshire, England
SUMMARY
Two lines of human diploid cells, the Wi-38 and MRC-5, were adapted to utilize glutamic
acid in place of glutamine. This adaptation resulted in (a) more cells per unit culture area, (b)
an alteration in cell size and protein content, (c) a morphological change of the cells from
fibroblastic to epithelial-like, and (d) increased metabolic activity. Changes in the agglutinability of adapted cells by 3 lectins together with the results from polyacrylamide gel electrophoresis could be interpreted as a change in plasma membrane structure. Comparative studies
of unadapted, adapted and transformed cells showed that adaptation to glutamic acid produced
cells with a metabolism and amino acid uptake similar to those of transformed cells. These
changes were reversible and were not accompanied by any apparent karyological change. The
significance of these results for the study of density-dependent inhibition of growth is
discussed.
INTRODUCTION
Glutamine plays an essential role in cell metabolism and is involved in the synthesis
of amino acids, nucleic acids and cell protein (Kitos, Sinclair & Waymouth, 1962).
Unfortunately glutamine is very unstable in tissue culture media and its breakdown
can result in an inhibition of growth (Griffiths & Pitt, 1967). Glutamine can be deamidated in cell cultures by a non-enzymic reaction to pyrrolidine-carboxylic acid
(Bay, James, Raffan & Thorpe, 1949) and by the cells to glutamic acid and a-ketoglutaric acid (Kvamme & Svenneby, 1961). The replacement of glutamine with a
stable compound having the growth-promoting properties of glutamine is, therefore,
a logical step. This has been done repeatedly in cultures of heteroploid cells by
increasing the level of glutamine synthetase in the cell by adaptation procedures using
high concentrations of glutamic acid (Eagle, Oyama, Levy, Horton & Fleischman,
1956; DeMars, 1958; Paul & Fottrell, 1963; Griffiths & Pirt, 1967).
This report describes the results obtained with this adaptation procedure for 2 lines
of human diploid cells. Although these cells readily adapted to glutamic acid, morphological transformation occurred which, together with changes in metabolism, reduced
density-dependent inhibition of growth.
618
J. B. Griffiths
MATERIALS AND METHODS
Cell lines
The human diploid cells (HDC) were the Wi-38 and MRC-s lines, both kindly supplied
by the National Institute of Medical Research, Hampstead. These cells were both derived from
human foetal lung tissue and cultured by the procedure of Hayflick & Moorhead (1961). Cells
between passages 21 and 29 were used and chromosome analyses revealed that they were at
least 98 % diploid, both before and after adaptation to glutamic acid.
L-132 cells — an epithelial, heteroploid line derived from human lung material - (Davies &
Bolin, i960), and an SV40-transformed Wi-38 line (designated T/W1-38) were also used.
Cell culture
The cells were grown in Eagle's Minimal Essential Medium (MEM) (Difco) supplemented
with 10% foetal calf serum (Flow Laboratories). This medium is supplied glutamine-free and
glutamic acid or glutamine were added as necessary. Adaptation to glutamic acid was carried
out in Roux bottles; growth experiments in approx. 100-ml (4-oz) medical flats; metabolic
studies in 5-cm Nunc plastic dishes and morphological experiments on glass coverslips placed
in 9-cm Nunc plastic dishes.
Increase in cell number was measured by counting nuclei stained with crystal violet, and
cell protein by the modified Lowry method (Oyama & Eagle, 1956). Ammonia was measured
by a microdiffusion method (Conway, 1957).
Isotope labelling procedures
The following were obtained from the Radiochemical Centre at Amersham; a mixture of
uniformly labelled 14C-amino acids as precursors for protein synthesis, i/iCi/culture; thymidine6-T(n) (2 Ci/mM) for DNA synthesis, 10/iCi/culture; L-leucine-Cu (U) (10 mCi/mM) was
used to measure the intracellular concentration of leucine. All radioisotope techniques have been
described previously (Griffiths, 1972).
Cell agglutination and membrane analysis
The reaction of cells with concanavalin A (12-1200 fig/ml), wheat germ agglutinin, (121200 /tg/ml) and haemolymph of Limulus polyphemus (undiluted - 1:5oo dilution) was measured
in micro-assay plates over a period of 15 min at room temperature. Cells were harvested in a
calcium- and magnesium-free buffer with o-oi % versene, resuspended at 4 x io6/ml of buffer
and then diluted 1:1 with the agglutinin or buffer control in the micro-assay plates. Agglutination of the cells was checked by both macroscopic and low-power microscopic observation and
the presence or absence of agglutination was scored against the concentration gradient of the
lectin so that the minimum concentration required for agglutination could be recorded.
Agglutination with concanavalin A indicates the presence of a-methyl-D-glucopyranoside on
the cell surface (Inbar & Sachs, 1969), wheat germ agglutinin the presence of /9-iV-acetyl
glucosamine (Burger & Goldberg, 1967) and L. polyphemus haemolymph is believed to
indicate the presence of sialic acid.
The extracellular surface material collected from washing freeze-thawed suspensions of cells
was analysed by disk electrophoresis on 7-5 % polyacrylamide gel and stained with naphthalene
black 12 B (Amido Schwartz stain) for protein and the periodate/Schiff stain for carbohydrates.
Photomicroscopy
Cell cultures grown on coverslips were placed inverted on microscope slides using growth
medium as a wet mountant. Observations and photographs were made using a Zeiss Photomicroscope with phase-contrast illumination.
Cell growth in glutamic acid media
619
Table 1. Counts ( x io 6 per culture) of adapted and unadopted cells after 192 A in
culture from an inoculum of 0-5 x io 6 per culture (mean values of 3 replicate cultures are
given)
Medium
Cell
Wi-38
MRC-s
Wi-38 (GA/GM)
MRC-s (GA/GM)
Wi-38 (GA)
MRC-s (GA)
MEM
MEM (GA/GM)
MEM (GA)
270
3'44
275
4-SS
270
3-56
165
2-24
2-40
279
2-60
3-59
3-25
478
2-17
367
3'2O
473
Table 2. Yields of cell protein (fig) after 192 h growth and the protein content of adapted
and unadopted cells (mean values of 3 replicate cultures are given)
Medium
Cell
MRC-s
MRC-s (GA/GM)
MRC-5 (GA)
Total protein
Protein/106 cells
Total protein
Protein/io e cells
Total protein
Protein/109 cells
MEM
MEM(GA/GM)
760
220
945
208
850
180
720
202
950
198
750
205
MEM (GA)
675
300
730
260
940
262
Nomenclature
The normal medium with glutamine is referred to as MEM: with glutamine replaced by
glutamic acid (600 /tg/ml), MEM (GA); and with glutamic acid (600 /Jg/ml) and glutamine
(ioo^g/ml), MEM (GA/GM).
Cells adapted to grow in MEM(GA) are referred to as Wi-38 (GA) or MRC-5 (GA), and
in MEM (GA/GM) as Wi-38 (GA/GM) or MRC-s (GA/GM).
RESULTS
Adaptation to glutamic acid and growth yields of adapted cells
Cells grown in MEM were adapted to grow in glutamic acid media by the following
procedure: (1) A 75 % confluent cell sheet was transferred to a glutamine-free medium,
containing 3000/fg/ml glutamic acid, for 4 days. During this period there was an
increase of approximately 10% in cell population and no evidence of increased cell
death. (2) 75 % of the cells were then transferred to a similar medium but with only
1500 /tg/ml glutamic acid for 4 days. The cells grew to a semi-confluent density during
this period, an increase of approximately 20%. (3) This culture was then split equally
into MEM (GA) and MEM (GA/GM) and thereafter routinely cultivated in these
40-2
J. B. Griffiths
620
1-4
GM
10
o
<
12
24
Time, h
36
48
Fig. i. Ammonia production (/tg/ml) in MEM (GA/GM). O—O, ammonia produced
in the cell culture; A—A, ammonia produced in the cell-free medium at 37 °C. GM
marks the limit at which glutamine is theoretically totally utilized.
media. GA/GM cells divided at the same rate as the unadapted cells with a doubling
time of 23-26 h but GA cells grew more slowly with a doubling time of over 32 h.
Two passages later the growth potential of the adapted and unadapted cells in the
various media was compared (Table 1). Both Wi-38 and MRC-5 cells gave very
similar results even though the experiment with MRC-5 c e u s w a s carried o u t I O months
after the initial experiment with Wi-38 cells. The highest cell counts were obtained
with GA/GM cells in both MEM and MEM (GA/GM) and the lowest counts were
with GA cells in MEM (GA). However, because of differences in cell protein between
unadapted, GA and GA/GM cells the cell counts could be slightly misleading. Cell
protein measurements (Table 2) showed that the protein yield of GA/GM and GA
cells in their respective media were similar despite the 32% higher GA/GM cell
count. Adapted cells in MEM (GA/GM) had a slightly lower protein content than
unadapted cells (90% of control cells) but in MEM (GA) they had a significantly
higher protein content than unadapted cells (120-137% of control cells).
MEM (GA/GM) has a small amount of glutamine (100/ig/ml) added to a glutamic
acid medium and increases both the growth rate and cell yield of LS cells (Griffiths &
Pirt, 1967). This small quantity of glutamine was rapidly utilized, as judged by
ammonia production (Fig. 1) and the medium became glutamine-free within 12-16 h
and so did not interfere with the objective of using glutamic acid to stabilize the
medium. The problem of glutamine instability in tissue culture media is exemplified
by the data in Fig. 1 where the production of ammonia in the incubated cell-free
medium shows how rapidly glutamine was hydrolysed.
Cell growth in glutamic acid media
621
Table 3. The minimum concentration of lectins required to agglutinate cells
(jig/ml or dilution)
Concanavalin A
Wheat germ agglutinin
L.polyphemus
MRC-5
MRC-5
(GA)
MRC-5
(GA/GM)
100
400 (800) •
1/50(1/25)*
800 (400)*
400
1/10
200
400
1/20
T/W1-38
50
100
1/5 (i/2'5) #
• At least 3 experiments were carried out for each cell line and where the results differed
these values are given in brackets.
Structural alteration after adaptation to glutamic acid
When cells are harvested by trypsinization it is, normally, easy to disperse the cell
suspension into single cells by gentle agitation. However, from stage 2 of the adaptation procedure onwards the harvested cells had a tendency to clump together in a
mucilaginous mass which was extremely difficult to break down to clumps of less than
4-7 cells. This indicated changes in the membrane structure or the liberation of
extracellular membrane material.
The effect of 3 lectins (concanavalin A, wheat germ agglutinin and Limulus polyphemus lectin) was investigated to see if changes in the surface structure of adapted
cells could be detected. The minimum concentration of lectin necessary to agglutinate
the cells is given in Table 3. The T/W1-38 cell was included in this investigation to
determine whether changes in the agglutination pattern of adapted MRC-5 cells
tended towards that of the transformed state in Wi-38 cells. Adaptation to glutamic
acid had no effect on the reaction with wheat germ lectin but adapted cells reacted
differently from normal cells with the other lectins. With Limulus polyphemus the
GA and GA/GM cells were similar to the transformed cells (T/W1-38) which probably indicates a higher sialic acid level but with concanavalin A they showed a decreased agglutinability unlike T/W1-38 cells. If extracellular material was being produced then it could mask the agglutination sites. There are, therefore, indications of
cell surface changes on adaptation to glutamic acid but not too much significance can
be put on these results as recognition of agglutination was made difficult by the
impossibility of obtaining single-cell suspensions.
A further test to demonstrate differences between the extracellular surfaces of
unadapted and adapted cells was made by polyacrylamide gel electrophoresis. The
Amido Schwartz protein stain showed that MRC-5 (GA) cells lacked a band in the
IgG region present in the other 2 cell types and MRC-5 (GA/GM), although having
identical bands to the other 2 cell types in the fast moving region (e.g. a, ft globulins,
ft lipoproteins) had far greater amounts of material in these bands. There were no
differences with Schiff staining between the different cell types.
The results obtained from agglutination and electrophoresis studies demonstrate
that changes do occur to the cell surface on adaptation to glutamic acid but as the
investigation was only qualitative no attempt can be made to define these differences.
622
J. B. Griffiths
Table 4. A morphological description of adapted and unadopted cells in
various media after 72 h growth
Medium
Cell
Wi-38
MRC-5
Wi-38(GA/GM)
MRC-5 (GA/GM)
WI-38 (GA)
MRC-s (GA)
MEM
Normal fibroblastic
morphology showing parallel orientation (Fig. 3)
Large areas of
epithelial growth
among some fibroblasts. Haphazard
growth
Epithelial, pronounced nuclei and
atypical cells
MEM (GA/GM)
MEM(GA)
Epithelial appearance Fibroblastic, but
just becoming
individual cells
noticeable
slightly necrotic
Cell sheet completely epithelial
with pronounced
nuclei (Figs. 4-6)
Epithelial rather
than fibroblastic.
Haphazard growth
Fibroblastic rather
than epithelial but
cells dense, granulated and atypical
Semi-epithelial cells
Morphological alteration after adaptation to glutamic acid
It was noticeable in the growth yield experiments (Tables 1 and 2) that some
cultures were undergoing a morphological change. The cells were then grown on
microscope slide coverglasses under the growth conditions used for Tables 1 and
2 so that they could be observed microscopically. The results are described in Table 4
and shown in Figs. 3-6. Human diploid cells are elongated and show parallel orientation (Fig. 3) but the GA/GM cells grown in MEM (GA/GM) showed a complete
change from a fibroblastic to an epithelial-like morphology (Fig. 4). Wi-38 and MRC-5
cells showed signs of this alteration during their first passage in MEM (GA/GM) and
the epithelial-like cells reverted to the fibroblastic shape as soon as they were transferred back into MEM. In cultures of adapted cells the nuclei were more pronounced
(Fig. 6); the cells had lost their polarity and growth had become haphazard with cells
growing over each other (Fig. 5).
These changes in cell morphology were reversible and the typical fibroblastic
appearance would return after a single passage in MEM. There was no alteration to
the karyotype in any of these experiments. The phenomenon appears to be typical of
HDC rather than of a particular cell line since the results with MRC-5 cells, which
were obtained 10-12 months after the initial work with Wi-38 cells, were in total
agreement with each other.
The effect of adaptation to glutamic acid on cell metabolism
Adapted cells showed increased rates of both protein and DNA synthesis compared
to unadapted cells in MEM (Fig. 2). The rate of DNA synthesis in cultures of MRC-5
(GA) and MRC-5 (GA/GM) was similar, both being 50% higher than the control;
the higher rate of synthesis was also maintained for a longer period. A medium change
Cell growth in glutamic acid media
623
x 30
E
o.
20
.9 10
i
4
X
E
o
c
o
1 -
8.
24
48
72
96
Time, h
120
144
168
Fig. 2. The rates of protein and DNA synthesis in cultures of MRC-5 cells, MRC-5
cells adapted to glutamic acid and transformed cell lines (L-132 and T/W1-38).
A, MRC-s; D, MRC-s(GA); O, MRC-5(GA/GM); • , T/W1-38; • , L-132.
after 120 h evoked a similar response as DNA synthesis rose to a peak some 40%
higher in adapted cultures than in unadapted cultures. Protein synthesis was also
significantly increased in adapted cultures, MRC-5 (GA) cells reaching a peak 40%
higher and MRC-5 (GA/GM) cells 70 % higher than the unadapted cultures. A medium
change after 120 h caused a rapid increase in the rates of protein synthesis in cell
cultures and GA/GM reached a new peak 40% higher than GA and unadapted cells.
The rate of cell metabolism was greatly enhanced, therefore, in glutamic acid-adapted
cultures and comparisons were then made with 2 transformed cell lines (Fig. 2) as
transformed cells have a greatly increased growth and metabolic potential compared
to untransformed cells. The rates of DNA synthesis of adapted cells in fact reached a
624
J- B. Griffiths
Table 5. The intracellular leucine concentration (figjml cell water) in control, adapted
and transformed cell lines at various extracellular leucine concentrations and cell densities
Cell line
MRC-5(GA) 1MRC-s (GA/GM)
MRC-s
Extracellular leucine
rnnrpnlratinn
\
t
concentration
(/ig/ml)
1
2
1
2
Pre-confluent
Cell count ( x io*/culture)
6
I'O
12
25
5°
Threshold leucine
concn (1130)*
Post-confluent
Cell count ( x io e culture)
6
12
25
5O
Threshold leucine
concn (1130)*
1
2
L-132
T/Wi38
I-I2
A
^
I-I2
0-90
I-O5
o-95
1-18
1-25
80
75
65
145
135
90
170
300
600
28s
280
70
130
270
70
150
310
610
60
135
325
59°
580
59o
290
600
145
315
59°
600
10
10-5
n-5
n-5
12
II
9
1 92
2-15
26
i-95
55
2-40
45
i-86
2-30
53
95
85
50
95
35
70
120
200
380
165
325
180
235
375
130
310
17
20
17-5
22-5
45
75
140
280
23
27-5
135
75
10-5
2-70
65
2-28
135
260
100
520
345
12-5
!6S
55
190
• The extracellular leucine concentration required to maintain an intracellular leucine
concentration of 130 fig/ml cell water.
peak as high as that of the transformed cells but as with MRC-5 cells the rate declined
rapidly to a low resting value by 72 h whereas DNA synthesis in the transformed cells
was maintained at a higher rate for much longer periods. The rates of protein synthesis
in adapted cells, however, were more similar to the rates in unadapted cells than to
the rates in transformed cells, even though the rate of protein synthesis in adapted
cells was significantly higher than in unadapted cells after 48-96 h. Although the
adaptation of cells to glutamit acid brought about significantly higher rates of cell
metabolism this increase did not equal the metabolic potential of transformed cells.
Leucine uptake by glutamic acid-adapted cells in crowded (post-confluent) cultures
was significantly greater than in unadapted cells (Table 5). The index used for comparing leucine uptake is the extracellular leucine concentration required to produce
an intracellular level of 130 fig leucine/ml cell water (referred to as 1130). This concentration was found previously to be the minimum for maximum protein synthesis
(Griffiths, 1972). These values for unadapted, adapted and transformed cells at preconfluent and post-confluent cell densities are given in Table 5. In pre-confluent cell
cultures there is very little difference between these various cell lines but in postconfluent cultures the adapted cells require a significantly lower concentration of
leucine for the optimum intracellular level to be attained. Transformed cells (L-132
Cell growth in glutamic acid media
625
Table 6. Calculated threshold leucine concentrations (1130) (fig/ml cell water) for cell
densities of 1 and 2 x io 6 per culture
Cell line
Cell
density
I X IO6
2 x
10'
A
MRC-5
MRC-5(GA) MRC-5(GA/GM)
10
24
i7'5
"•5
18-5
L-132
9
11
T/W1-38
10
i4-5
and T/W1-38) are characteristically much less affected by cell crowding than are
untransformed cells but the data in Table 5 show that T/W1-38 cells are only marginally better than the glutamic acid-adapted cells. Differences in cell density between
the various cell lines make accurate comparisons difficult but it has been found that
a logarithmic plot of 1130 against the cell density gives a straight line. Thus 1130
values for cell densities of 1 x io 6 and 2 x io 6 per culture can be calculated and these
are given in Table 6. These results show that in post-confluent cultures adapted cells
lie between unadapted and transformed cells and again indicate that the metabolism
of adapted cells tends towards that of transformed cells.
DISCUSSION
The mechanism by which cultured cells can be adapted to utilize glutamic acid in
place of glutamine is well known (DeMars, 1958; Paul & Fottrell, 1963). High levels
of glutamine synthetase (D-glutamyltransferase) are induced by subjecting the cells
to a regime of high glutamic acid concentrations and this is maintained by keeping the
cells in glutamic acid. This enzyme is rapidly lost if glutamine (300 /tg/ml) is added
(DeMars, 1958) but Griffiths & Pirt (1967) found that maximum cell growth was
achieved by using glutamic acid (6oo/(g/ml) and glutamine at 100/fg/ml. This
mixture was also found to be suitable for human diploid cells and the low level of
glutamine is believed to be necessary for priming the glutamine synthetase reaction
(Griffiths, 1966).
The adaptation of human diploid cells to utilize glutamic acid had 4 effects: (1) the
growth potential of the cells was increased thus enabling them to produce significantly
higher yields; (2) the cell size and protein content were altered; (3) the cell morphology
was altered; and (4) the metabolic activity of the cells was increased. The change in
morphology from a parallel-oriented fibroblastic culture to a haphazardly growing
epithelial-like culture parallels the change which occurs during transformation of cells
by oncogenic viruses. Also, most human diploid cell lines are fibroblastic but most
human heteroploid cells are epithelial and have an increased growth potential (Eagle,
1965). The mechanism by which the state of viral transformation is maintained is
mediated through alterations to the cell surface but unless the karyotype is altered the
cell can revert to the behavioural pattern of normal cells (Pollack, Wolman & Vogel,
1970). Changes to the normal cell which accompanied adaptation to glutamic acid
included probable changes at the cell surface (for example, the mucilaginous clumping,
626
J. B. Griffiths
the morphological change and the evidence produced by gel electrophoresis and
agglutination studies) and these changes allowed the cell to behave in a manner more
characteristic of heteroploid than diploid cells. However, there was no change in the
karyotype and the cells quickly reverted to normal growth patterns when returned
to the glutamic acid-free medium. It is concluded that glutamic acid-containing media
caused a morphological transformation. The well documented example of morphological transformation is the effect of dibutyryl cyclic AMP on epithelial cells (Hsie &
Puck, 1971; Hsie, Jones & Puck, 1971; Johnson, Friedman & Pastan, 1971; Sheppard,
1971). However, glutamic acid had the opposite effect in that it changed the cell from
the fibroblastic to the epithelial form. These results are consistent with the idea that
membrane structure plays an important role in contact inhibition of growth.
Certain biochemical and metabolic parameters have been studied in normal and
transformed cells (Griffiths, 1970, 1971, 1972) with a view to defining differences
between them. The study of these parameters was extended to glutamic acid-adapted
cells to determine how important cell morphology is in contact inhibition of growth.
It has previously been found that normal cells have much lower rates of macromolecular synthesis than transformed (heteroploid) cells (e.g. the rate of protein and
DNA synthesis is 50-100% more in transformed cells) and this difference is magnified
when the cultures become confluent (Griffiths, 1972). Also, the ability of normal cells
to take up amino acids is greatly depressed when cell cultures become confluent with
the result that much higher concentrations of amino acids are required in the medium
to maintain an adequate intracellular level. Transformed cells are far less affected by
cell crowding in this respect (Griffiths, 1972). The results in Fig. 6 show that adapted
cells had a far higher metabolic activity than the unadapted cells and DNA synthesis
in particular was at the maximum rate for far longer periods in the adapted cells. Also,
leucine uptake was less inhibited in adapted cultures. The criterion used to compare
the uptake ability of cells is the external concentration of leucine required to maintain
an intracellular concentration in excess of 130/ig/ml of cell water. This concentration
allows protein synthesis to proceed at the maximum rate (Griffiths, 1972). The adapted
cells at densities of 2 x io 6 per culture required an external leucine concentration of
17-5-18-5 /ig/ml whereas unadapted cells required 24/tg/ml, an increase of 33%. In
terms of cultural behaviour this means that in a culture containing 2 x io 6 unadapted
cells leucine will become growth-limiting when it is 55% utilized but in cultures of
adapted cells at the same density leucine will not become growth-limiting until it is
66% utilized. In pre-confluent cultures of adapted and unadapted cells leucine would
not become limiting until about 80% utilized.
This work has shown how a relatively small alteration to the cell and its environment can bring about physiological changes similar to those associated with carcinogenesis in vitro. As glutamine has a central role in cell metabolism there may be an
indication here of the interfering action that oncogenic viruses have on cell metabolism.
I wish to acknowledge the invaluable technical assistance of Mrs I. K. Strutt and to thank
Dr D. C. Ellwood and Mr R. Gallop for supplying the lectins and for the disk electrophoresis
analysis.
Cell growth in glutamic acid media
627
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(Received 27 June 1972)
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J. B. Griffiths
Fig. 3. MRC-s cells showing normal polarized fibroblastic growth, x 1200.
Fig. 4. Wi -38 (GA/GM) cells showing the change to an epithelial-like morphology,
x
1200.
Fig. 5. MRC-5 (GA/GM) cells in MEM(GA) showing loss of polarized growth
resulting in haphazard growth, x 1200.
Fig. 6. Wi-38 (GA/GM) cells in MEM (GA/GM) at higher magnification showing
the prominence of the nuclei, x 2700.
Cell growth in glutamic acid media
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