Download growth of human diploid fibroblasts in media with different amino

J. Cell Sci. 14, 671-680 (1974)
Printed in Great Britain
671
GROWTH OF HUMAN DIPLOID FIBROBLASTS
IN MEDIA WITH DIFFERENT AMINO ACID
COMPOSITION
J. LITWIN
Department of Applied Microbiology, Karolinska Institutet, 10401
Stockholm 6o, Sweden
SUMMARY
Human embryonic diploid lung fibroblasts were capable of growing in a medium consisting
of Eagle's MEM vitamins plus 0 1 /tg/ml biotin, Earle's salt solution, 4 mM glutamine, 0 4 raM
cystine, 0-5 mM tyrosine, 10 % undialysed calf serum and 1 mM pyruvate. The growth rate and
percent cell attachment in this medium was slightly less than in Eagle's MEM but the longevity
was about the same. Addition of 0-5-1 -o mM histidine stimulated growth but addition of 0 2 mM
phenylalanine, methionine or valine decreased longevity markedly.
Maintaining a constant pH of about 7-5-7-6 with 20 mM of the organic buffers HEPES, T E S
or T R I C I N E yielded a growth rate equal to or better than that of cells growing in unbuffered
medium. Some buffers gave good growth initially but eventually the longevity decreased
(2omMBIS-TRIS, 2omMBICINE, iomM TRIS). In almost all cases the initial exposure to the
buffers yielded the best growth results.
INTRODUCTION
The amino acid requirements of almost all mammalian cells, diploid or heteroploid,
grown in vitro have been shown to be served by 13 amino acids (Eagle, 1955 a-c, 1959;
Eagle, Oyama, Levy, Horton & Fleischman, 1956; Eagle, Oyama & Levy, 1957),
although the relative utilization of each amino acid may be different with each cell
strain (McCarty, 1962). These amino acids are: glutamine, arginine, histidine, lysine,
tryptophan, valine, leucine, isoleucine, methionine, threonine, phenylalanine, tyrosine
and cystine. Attempts to eliminate any single amino acid from these 13 results in a
rapid cessation of growth (Eagle, 1955,a,b; Eagle et al. 1956, 1957; Freed & Schatz,
1969; Litwin, 1972). However, Jacobs (1966) described a medium containing only
glutamine and cystine as amino acid source, which could support the growth of human
embryonic diploid lung fibroblasts. This report seemed incongruous with what had
been published earlier on the nutritional requirements of human cells (Eagle et al.
1957) and stimulated the present survey on the effect individual and combinations of
amino acids have on growth and longevity of these cells in media deficient in essential
amino acids.
672
J. Litwin
MATERIAL AND METHODS
Cells
Human embryonic diploid lung fibroblasts (HEDLF) cells were grown from the trypsinized
lung tissue of human embryos aborted in the third month of gestation. Two independent strains
were used with similar results. Most experiments were started with cells that were in their 7th
passage, approximately 20 to 25 cell divisions old. The growth patterns of these cells are similar
to those described by Hayflick & Moorhead (1961) and Hayflick (1965).
Medium
The growth of HEDLF cells in Eagle's minimum essential medium (MEM) (Eagle, 1959)
was used as comparison for the test media. The components common to all the test media were
Earle's salt solution and the vitamin mixture used in MEM but containing o-i mg/1. biotin
(Eagle, 1955 c). These 2 components will be referred to as VS (vitamin, salts) in the text. Earle's
salts were modified by using 09 % NaCl instead of 068 % and twice the glucose concentration
recommended. These changes were found to yield slightly better growth of HEDLF cells in
MEM. Glutamine was used at a concentration of 4 mM in all cases and only the L-isomers of
amino acids were used (Sigma Chemical Co., St Louis, Mo., U.S.A.). The media were supplemented with 10% calf serum, 1001.U./ml penicillin and 100/tg/ml streptomycin. Sodium
bicarbonate was used to adjust the pH. The serum was neither dialysed nor inactivated by
heating. The initial pH of the media in most experiments varied between 70 and 7-2. The day
following the initiation of the cultures the pH was adjusted to about 7-5 with NaHCO3.
Culture conditions
The cells were grown in Jena glass culture bottles with a growth surface of 46 cm2. Each
bottle was seeded with 2 x io5 cells in a volume of 20 ml medium. The following day the number
of cells which attached to the glass surface was estimated by counting the attached cells in
randomly selected fields of view with an inverted microscope (Litwin, 1971). A more accurate
estimate of the number of cell divisions occurring with each passage could be obtained by using
the number of attached cells as the starting cell density instead of the number of cells added to
the culture vessel. The average attachment value given in the text was calculated from the percent
attachment from all passages over the entire in vitro cultivation of each culture. When the cells
grew to a confluent monolayer they were trypsinized off the glass in 0-25 % trypsin at room
temperature, centrifuged, resuspended in 5 ml of fresh medium of the type used for their growth
and counted with a haemocytometer. In more recent experiments an electric cell counter was
used (Celloscope, Sweden). When most cultures reached senescence they were incubated for
about a month with weekly changes of medium. If the cell layer did not become confluent
during this time the cells were counted and discarded. To eliminate variations in growth due to
inconsistent dishwashing the same bottle was used for all passages without intermittent washings
in all experiments except those mentioned in the text. Prior experience has shown that this procedure gives as good growth as with cultures grown in clean bottles with each passage, and has
no influence on the longevity of diploid fibroblasts.
RESULTS
Although HEDLF cells stopped growing within 2 to 4 cell divisions when any one
of the amino acids in Eagle's MEM was omitted (Litwin, 1972), when all of the amino
acids, with the exception of glutamine, were withdrawn some cells could be observed
in mitosis after as long as 1-2 weeks of incubation. A survey was made of which amino
acid or combination of amino acids would stimulate growth when added to a basal
medium containing Eagle's vitamins and Earle's salts (VS) plus 4 mM glutamine,
1 mM sodium pyruvate and 10% calf serum. The following combinations of amino
Atnino acids required for diploidfibroblastgrowth
673
acids at a concentration of 0-2 mM, except cystine at o-i mM, permitted some cell
growth: cystine, tyrosine + cystine, phenylalanine 4-cystine, phenylalanine + cystine +
lysine, phenylalanine + cystine+ threonine, phenylalanine + cystine + valine, tyrosine
+ cystine+ histidine, tyrosine + cystine+ threonine. In these experiments clean
bottles were used for each passage and growth was relatively poor. When these combinations were investigated in greater detail, we began to re-use the same bottle with
each passage and the growth results improved considerably.
Using the base medium VS + 1 mM pyruvate very little growth occurred with only
60 r
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120 140
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80 100 120 140 160 180
Days
Days
Fig. 1
Fig. 2
Fig. 1. The effect of varying some of the conditions in the medium VS + 1 mM sodium
pyruvate on the growth of H E D L F cells, x • • • • x , 4 mM glutamine added (20%);
O
O, 0-2 mM tyrosine+ c 1 mM cystine, no glutamine (18%); x
x,
4 mM glutamine + 0 2 mM tyrosine + o-i mM cystine (38%); A
A, 4 mM glutamine + 0-5 mM tyrosine+ o-1 mM cystine (62%); • •
, 4 mM glutamine+
1-5 mM tyrosine + o-i mM cystine (62%). The values in parentheses represent the
average percentage cell attachment to the glass surface.
Fig. 2. The effect of varying some of the conditions in the medium VS + 4mM
glutamine, o-i mM cystine, 1 mM sodium pyruvate on the growth of H E D L F cells.
x
x , Eagle's MEM control (69%); A
A, medium given above (45%);
# # , 0 2 mM tyrosine (42%); O ' • " " O,o-2 mM phenylalanine (50%); •
Q,
0-5 mM phenylalanine (57%); H - V
, 1-5 mM phenylalanine (45%). The values in
parentheses represent the average percentage cell attachment to the glass surface.
674
J- Litwin
glutamine or cystine +tyrosine added (Fig. 1). However, significant growth was seen
when all 3 were present. The optimal concentration of tyrosine appeared to lie between
0-5 and 1-5 mM. With 2 mM tyrosine the cells degenerated after 6 or 7 cell divisions.
The base medium of VS + 4mM glutamine + o-1 mM cystine+1 mM pyruvate
produced good growth initially but the cells stopped growing after 34 cell divisions
(Fig. 2). This base medium plus 0-2 mM tyrosine gave as good a longevity (58 cell
divisions) as cells grown in Eagle's MEM (57 cell divisions), although the growth rate
of the former slowed inexplicably between 18 to 30 cell divisions. When 0-2 mM
phenylalanine was used instead of tyrosine the growth became slower after 25 cell
divisions and eventually stopped after 44 cell divisions. Higher concentrations of
50
50
r
40
40
30
30
20
<-> 20
10
10
i
i
20
1
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40
60
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180
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Days
Days
Fig. 3
Fig.[4
i
t
i
i
120 140
Fig. 3. The effect on the growth of HEDLF cells of 0-2 mM of various essential amino
acids added to medium containing VS + 4tnM glutamine, 0-4 mM cystine, 0-5 mM
x , Eagle's MEM control (69%); #
0,
tyrosine, 1 mM sodium pyruvate. x
medium given above, contaminated after 31 cell divisions (43 %); O " " • " O, lysine
(40%); A
A, arginine (37%); •
• , methionine (58%); +
+,
phenylalanine (27 %). The values in parentheses represent the average percentage cell
attachment to the glass surface.
Fig. 4. The effect on the growth of HEDLF cells of 0-2 mM of various essential amino
acids added to medium containing VS + 4mM glutamine, 0-4 mM cystine, 0-5 mM
x, histidine (44%); O " • " • O, threonine
tyrosine, 1 mM sodium pyruvate. x
(44%); AA, tryptophan (34%); • - — • . isoleucine (37%); •
•,
leucine (34%); H
h, valine (36%). The values in parentheses represent the
average percentage cell attachment to the glass surface.
i
160
i
i
180
Amino acids required for diploidfibroblastgrowth
675
phenylalanine reduced growth rate and longevity; with 2 mM the cells degenerated
after 9 cell divisions.
A study on the influence of various concentrations of cystine in VS containing 4 mM
glutamine, 0-5 mM tyrosine and 1 mM pyruvate showed that the optimum concentration was 0-4 mM. Cystine was toxic at o-8 mM (data not shown). Therefore, the following medium is denned: VS + 4111M glutamine + 0-4 mM cystine + 0-5 mM tyrosine
(VSGCT medium) containing 1 mM pyruvate.
Table 1. The effect of various organic buffers andpH on the
growth and longevity of HEDLF cells
VSGCTH medium was adjusted to pH 72 initially. After overnight incubation the medium
was adjusted to pH 7-6. One culture with 20 mM TRICINE was adjusted initially to pH 79 and
kept at this pH.
Buffer
Total no.
of cell
divisions
Cell
divisions at
senescence
Day
senescence
occurred
Cell division
time,
days*
Total
days
Average
% cells
attached
Sodium bicarbonate
021
140
3°
i4°t
3O
43
alone
0-37
10 mM HEPES +
33
31
85
38
147
10 mM TES
29
29
0-25
20 mM TRICINE
56
"3t
113
141
0-17!
172
20 mM TRICINE,
24
24
41
pH79
18
100
SO
17
10 mM TRIS
o-34
38
18
20
0-37
100
46
20 mM BIS-TRIS
49
0-29
18
90
48
20 mM BICINE
19
63
—
—
i8§
026
70
20 mM HEPES
41
1
119
0 2
10 mM HEPES
24
56
24
35
28
28
026
106
io6f
20 mM TES
53
019
24
126
40
10 mM MES
126J
24
16
019
119
16
84
20 mM MES
5°
* Calculated from the ratio of the number of cell divisions at senescence to the time senescence
occurred.
f Culture contaminated at senescence.
X Cell div./day = 023 if one does not include the long lag period of 63 days.
§ Culture discontinued accidentally.
Pyruvate appeared to have a stimulatory influence on growth of HEDLF cells in the
various deficient media studied (data not shown), although it did not influence cell
growth in Eagle's MEM. One and 2 mM pyruvate gave similar results but 10 mM was
inhibitory.
The influence of other amino acids added to VSGCT medium is seen in Figs. 3 and
4. In this experiment 0-2 mM histidine yielded better longevity and 0-2 mM threonine
gave as good results as Eagle's MEM. Isoleucine reduced longevity without a decrease
in growth rate, but arginine and leucine reduced the growth rate as well as longevity.
A greater reduction in longevity was found when 0-2 mM methionine or valine was
added to the medium even though the growth rate for the first 3 to 4 passages was good.
However, phenylalanine was the most inhibitory to both growth and longevity.
44
CE L 14
676
J. Litwin
When histidine was re-examined, growth at concentrations from 0-2 to 2-0 mM was
similar and 0-5 mM was selected for use in future experiments (VSGCTH medium).
However, cell growth in these media varied considerably in different experiments.
This variation also would occur, but to a lesser extent, with cultures growing in Eagle's
MEM. One possible explanation of this variation may be that cells growing in deficient
medium are very sensitive to slightly inhibitory substances which may be present in
varying concentrations in different batches of serum (Litwin, 1970). Another factor
which can affect growth is the rapid changes in pH which occur while the cells
are actively growing, requiring the frequent addition of bicarbonate. The buffering
capacity of the media studied was much less than MEM because they contained so few
amino acids. Therefore, some of the various buffers recommended by Williamson &
Cox (1968) and demonstrated by Ceccarini & Eagle (1971) and Eagle (1971) to increase
the maximum cell density of human diploid cell growth at pH 7-6 were added to
the medium to help stabilize the pH. The buffers were adjusted initially to pH 7-2
regardless of the pKft because it was found that better cell attachment was obtained at
this pH than occurred at higher pH levels. After overnight incubation the pH was
adjusted to 7*5—7'6 with bicarbonate and kept close to this level by daily inspection and
adjustment when necessary. The results in Table 1 show that the addition of some
buffers indeed increased the growth rate of HEDLF cells in VSGCTH medium
(10 mM HEPES+ 10 mM TES, 20 mM BIS-TRIS, 10 mM TRIS). Some buffers gave
good initial growth but the longevity was shortened (20 mM BIS-TRIS, 10 mM TRIS,
20 mM BICINE). TRIS at 20 mM was inhibitory to growth after only 10 cell divisions
(50 days incubation) but during the first 2 passages produced good growth. Most
buffers yielded the same growth rate as unbuffered VSGCTH medium, but in no case
was the longevity of the cells increased. The cells required about 60 days to adapt to
20 mM TRICINE adjusted to pH 7-9 at the start of the cultures, but they grew without
any lag at pH 7-6. Once the cells were adapted to or selected by the higher pH, they
grew at a rate comparable to unbuffered VSGCTH but stopped at an earlier age. It
appeared that only TRICINE, HEPES and TES could be used over extended periods
of time without causing a decrease in longevity.
The average attachment of cells on the glass surface after overnight incubation is
given in the various figure legends and Table 1. The range in most of these experiments varied between 10 and 100%. In most cases, the cell attachment in the various
deficient media was less than in Eagle's MEM (69 %). The presence of glutamine and
cystine in the media appeared to be necessary for optimal attachment. No other amino
acid and none of the buffers produced a significant increase in cell attachment.
DISCUSSION
The amino acid requirement of cells growing in vitro have long been divided into 2
groups: essential and non-essential. An amino acid is considered essential if cells are
unable to grow in its absence. Eagle (1963) and Eagle & Piez (1962) have shown that
cells are capable of synthesizing most of these essential amino acids but at concentrations
too low to compete with dilution when the ratio of medium to cell volume is large. The
requirement for many of these amino acids, therefore, becomes population dependent,
Atnino acids required for diploidfibroblastgrowth
677
4
but in most cases the cell population would have to be of the order of 2-4 x 1 o cells/cm2
(0-5-1 x io 5 cells/ml) or higher depending on the experimental conditions. However,
the range of cell attachment in our experiments varied between 4X io 3 and 4X io 2
cells/cm2 or io4 and io 3 cells/ml. Thus, the cell density on the glass surface at the beginning of each passage was low enough so that the amino acid synthesis in the cells
probably could not compete against the dilution factor of the medium. Furthermore,
the media used in our experiments were deficient in more amino acids than media used
in the experiments reported by Eagle & Piez (1962).
It is most probable that the amino acids supplied by the serum played a significant
role in the ability of HEDLF cells to grow in the various deficient media studied. The
serum was not dialysed in our experiments because dialysis also removes smallmolecular substances, other than amino acids, which may stimulate cell growth
(Eagle, i960; Griffiths, 1972; Ham, 1963; Metzgar & Moskowitz, i960; Sato, Fisher &
Puck, 1957). In addition one of the purposes of this study was to develop a simplified
medium which would be of practical use in laboratories which would not routinely use
dialysed serum for large-scale cell cultivation. It was found earlier (Litwin, 1972) that
upon removal of any one of the amino acids in MEM containing undialysed calf serum,
all cell growth ceased within 2-4 cell divisions, in agreement with the results of Eagle
(1955 a, b,d) and Freed & Schatz (1969) using dialysed serum. Thus the amino acids in
the serum were not at high enough concentration to rescue the cells from inhibition of
growth under these conditions. Jacobs (1966) who reported good growth of human
diploid fibroblasts in a medium containing only glutamine and cystine used undialysed
serum, also. However, we did not obtain as good growth with these 2 amino acids as
was claimed by Jacobs (1966). The free amino acids available in serum, the continual
release of amino acids and peptides into the medium by serum proteolytic enzymes
(Piez, Oyama, Levintow & Eagle, 1960), the ability of cells to concentrate many of these
amino acids (Eagle, 1959; Eagle, Piez & Levy, 1961; Piez & Eagle, 1958) and the
synthesis of amino acids by these cells may explain how the diploid fibroblasts were
capable of growing in a medium containing only 3 of the 13 essential amino acids. Thus,
it was apparent that HEDLF cell growth similar to that in Eagle's MEM was obtained
in the presence of 4 mM glutamine, 0-4 mM cystine and 0-5 HIM tyrosine plus the amino
acids supplied by 10% calf serum at the following concentrations estimated from the
serum amino acid values given by Piez et al. (i960) and Jacobs (1966): o-oi6mM
threonine, 0-025 m M v a u n e » 0-002 mM methionine, 0-007 m M isoleucine, 0-013 mM
leucine, 0-006 mM phenylalanine, 0-017 mM lysine, o-oi mM histidine, 0-008 mM
arginine, and 0-0003 mM tryptophan. The cells must be able to concentrate these amino
acids to a level which will support protein synthesis, O-OI-O-I mM (Eagle et al. 1961).
The function of glutamine, cystine and tyrosine under the conditions stated above is
not clear, but they may have a sparing effect on the cell requirement for the other
amino acids. The question that remains to be answered is: under these conditions how
low can the concentration of the other amino acids be made before they become growth
limiting ? However, some of these results suggest that the balance of amino acids in the
medium is a more important factor than their concentration.
The addition of other amino acids to the VSGCT medium gave highly variable
44-2
678
J. Litwin
results. Histidine and to a lesser extent threonine was stimulatory for growth and
longevity but methionine, valine and isoleucine reduced longevity without affecting
the growth rate. Arginine and leucine reduced both growth rate and longevity, but
phenylalanine was the most inhibitory of all the amino acids tried. The effects of these
amino acids emphasize the value of following the growth response of HEDLF cells
over long periods of time. Most of the effects did not become apparent before 30-60
days of cultivation (at least 3-4 passages).
The decreased buffering capacity of the deficient media permitted rapid changes in
pH during the growth period, which probably affected cell growth adversely. Since
Eagle (1971) showed that the optimum pH for human diploid fibroblast growth was
7-6, we adjusted all buffers tried to this range after allowing the cells to attach to the
glass at pH 7-2. In agreement with Eagle (1971), TRICINE, TES and HEPES gave the
best growth results over long periods of cultivation. The maintenance of a stable and
optimum pH had no influence on cell longevity.
It is known that withdrawing any of the 13 amino acids from Eagle's MEM results in
rapid cessation of growth (Eagle, io.55#-c; Eagle et al. 1957; Freed & Schatz, 1969;
Litwin, 1972) and that good growth occurs in medium containing only VS + glutamine,
cystine and tyrosine. It can be predicted, therefore, that as one continues to add amino
acids to these 3 there should arise mixtures which will be growth inhibitory. A partial
growth inhibition was already observed with phenylalanine, methionine, valine and to
a lesser extent leucine, isoleucine and arginine. The fact that some amino acids such as
histidine and threonine improved growth and longevity suggests that we may be able
to find eventually the optimum amino acid composition for HEDLF cells.
The influence of amino acid balance on cell longevity may be of some importance in
view of the recent findings and theories on the relationship between cell ageing and
mistakes in protein synthesis (Lewis & Tarrant, 1972; Orgel, 1973). Holliday &
Tarrant (1972) had shown that certain enzymes in ageing human fibroblasts were more
thermolabile than the same enzyme in younger cells. Furthermore, Gershon & Gershon
(1970, 1973) had shown that significant amounts of inactive enzymes are produced in
ageing organisms. One possible way in which these mistakes could occur is that the
wrong amino acids are selected for certain proteins. Lewis & Holliday (1970) reported
that the amino acid analogue ethionine increased the rate of ageing in a mutant of
Neurospora. Since many amino acids found in cells are structurally similar, differing
only by a methyl or hydroxyl group, it is possible that as cells age their mechanism for
selecting amino acids becomes less sensitive and cannot differentiate between very
similar molecules. Thus, increasing the concentration of some amino acids may
accentuate the mistakes made by ageing cells and reduce longevity, as we have seen
occurs with phenylalanine, methionine, valine, leucine, isoleucine and arginine. On
the other hand, if an amino acid is particularly important for the cell then an excess may
reduce the incidence of mistakes and increase longevity, as occurred with excess
tyrosine and histidine in Eagle's MEM (Litwin, 1972).
Further information on the effect of amino acid balance on the growth and longevity
of human diploid fibroblasts may be of value in studying the factors responsible for
cell ageing.
Amino acids required for diploid fibroblast growth
679
The author gratefully acknowledges the excellent technical assistance of Mrs Annette Litwin
and Mrs Ann Bjork.
This work was supported by a grant from the Swedish Technical Research Council.
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(Received 8 August 1973)
LITWIN,