Download necessary amino acids and vitamins

J. Cell Science 40, 281-291 (1979)
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NECESSARY AMINO ACIDS AND VITAMINS
FOR THE GROWTH OF HUMAN DIPLOID
FIBROBLASTS
JACK LITWIN
National Bacteriology Laboratory, 105 21 Stockholm, Sweden.
SUMMARY
Only 2 amino acids and one vitamin were found to be essential for the growth of human
embryonic diploid lung fibroblasts when 10 % undialysed calf serum was used as a medium
supplement. These amino acids were either glutamine + cysteine or serine + homocysteine.
Replacing cysteine or homocysteine with cystine or homocystine, respectively, reduced growth.
The growth rate in the glutamine + cysteine medium was slightly less than that in Eagle's
medium, but the in vitro life time was similar.
Folic acid was the only vitamin needed to support cell growth in vitro. The addition of other
vitamins had no stimulatory effect with the possible exception of nicotinamide.
When other amino acids were added to glutamine + cysteine none showed stimulatory effects
but tryptophan was either toxic or inhibitory for the 3 human diploid strains examined. Serine
was inhibitory for WI-38 but not for MRC5 cells.
Subtle nutritional differences appear to exist between fibroblasts of the same type obtained
from different embryos.
INTRODUCTION
A previous report (Litwin, 1970) had shown that increasing the overall concentration
of amino acids in Eagle's minimum essential medium (MEM) had no effect on the
growth rate or longevity of human diploid fibroblasts. However, when the concentration of individual amino acids was increased by 1 mM, cystine became highly inhibitory, isoleucine produced a marked decrease in longevity while tyrosine or histidine
increased longevity significantly. The other amino acids had little or no effect (Litwin,
1972).
Subsequently it was found that of the 13 amino acids in MEM only glutamine and
cystine were necessary to support growth (Litwin, 1974) in agreement with the results
of Jacobs (1966). Tyrosine and histidine stimulated growth and increased longevity,
whereas phenylalanine, methionine, tryptophan, valine, leucine and isoleucine inhibited growth or decreased longevity.
During the course of the above experiments it was observed that glutathione or
cystathionine could support growth of human fibroblasts without the addition of any
amino acids. The effect is described below.
282
J. Litwin
MATERIAL AND METHODS
Cell strains
Most experiments were performed with a strain of human embryonic diploid lung fibroblasts
(HEDLF) cells isolated in this laboratory and referred to as the ' S' strain. These cells were
frozen after approximately 15 population divisions and stored in liquid nitrogen. They show
human diploid karyology and the limited life-span described by Hayflick & Moorhead (1961)
and Hayflick (1965).
Experiments were performed also with HEDLF strains WI-38 and MRC 5 obtained from
Dr Hayflick and Dr Jacobs, respectively (Jacobs, Jones & Baillie, 1970; Jacobs, 1976). Both
strains were frozen at about the 20 to 22 population doubling level.
Media
Eagle's MEM supplemented with 10% calf serum, 4 mM glutamine, 1 mM Na pyruvate,
100 /tg/ml streptomycin and 100 U/ml penicillin was used as control. The experimental medium
was made with Earle's buffer modified in the following way: the NaCl concentration was increased to 0-9 % and the glucose concentration to 2 %. The optimal NaCl concentration for
HEDLF growth and longevity was found to lie between 0-9 and I-I % (unpublished results).
The vitamin solution was the same as used in Eagle's basal medium except where specifically
described in the text. All media contained 1 mM Na pyruvate and the antibiotics given above.
The L-cysteine stock solution was made up and sterilized by filtration each time new medium
was prepared.
The calf serum was undialysed and was not heat-inactivated.
Passage procedure
The cultures were started from frozen ampules. The cells were distributed in Jena glass
bottles with a surface of 46 cm J and allowed to grow out to a confluent monolayer in MEM.
Those bottles which permitted good and equal growth were selected for the experiment. The
cells were suspended with 200 /ig/ml crystalline trypsin (Sigma) in phosphate-buffered saline
adjusted to p H 8 by adding Tris-HCl buffer to a final concentration of o-i %, centrifuged,
counted with an electronic cell counter (Celloscope, Sweden) and 2 x 1 0 ' cells were added back to
the same bottles together with 20 ml of the test medium. The following day the number of cells
which attached to the glass was estimated with an inverted microscope by measuring the average
number attached cells per field-of-view. This value was used to calculate the number of population divisions. The percent attachment values from all passages in a given series were averaged
and this value is given in all the Tables.
When the cells grew to a confluent monolayer they were trypsinized, centrifuged, counted,
and 2 x io 6 cells were added back to the same bottle with the same type of medium they had
grown in previously. By re-using the same bottle with each passage the problem of dissimilar
growth results due to dish washing was eliminated. These 'conditioned surfaces' allowed cells
to attach and spread out faster than on clean surfaces but they had no influence on the percent
attached cells, growth rate or longevity of H E D L F cells. In most cases senescent cultures were
incubated for about 1 month before discarding.
The p H was adjusted by the periodic addition of Na bicarbonate. The media were changed
once a week in the event the culture was not passed.
RESULTS
When, the tripeptide glutathione or the dipeptide cystathionine was added to MEM
it produced no significant effect on growth (unpublished results). However, when used
as the only amino acid source, good growth was obtained (Table 1). The combination
of glutathione and cystathionine had no synergistic effect. The addition of cysteine,
cystine or tryptophan to glutathione was either toxic or highly inhibitory. Leucine,
Amino acids and vitamins for diploid cell growth
283
methionine, phenylalanine, serine or alanine were inhibitory to a lesser extent while
tyrosine, histidine, lysine, glycine, asparagine or taurine increased longevity. The
component amino acids of glutathione (glutamine, cysteine and glycine) are shown
to replace the growth activity of this tripeptide; substituting cystine for cysteine reduced growth.
Table 1. The effect of various amino acids on the growth and longevity of human diploid
fibroblast strain S
Media
P.D.
D.C.
G.R.
%A
298
047
Eagles MEM
63
i34
161
0-24
34'6
VS + Gluta.
59
328
0-26
VS + Cysta.
i73
47
VS + Gluta.:
0-25
+ Cysta.
35-9
i74
53
036
305
+ glutamine
i35
5i
0
0
—
—
+ cystine
o-i
42
82
+ cysteine
54
027
223
507
+ tyrosine
55
209
031
56
+ histidine
44'4
160
025
344
52
+ arginine
407
025
167
58
+ lysine
4-8
O-II
69
+ tryptophan
54
038
69
64
+ leucine
i5'S
J
27-0
0-24
+ isoleucine
45
43
0-24
+ valine
36-5
174
47
O22
+ threonine
2S-4
47
I4S
60
O-29
+ methionine
91
13-6
+ phenylalanine
i3S
75
75
O'33
O-23
243
53
43-3
+ glycine
0-23
153
47
25-9
+ proline
0-30
37-8
145
49
+ hydroxyproline
023
244
48
50-9
+ asparagine
148
0-26
52
319
+ aspartic acid
O-28
+ glutamic acid
174
35-i
59
199
O-22
55
113
+ serine
2I O
127
O-2I
+ alanine
53
O-23
52
243
43'9
+ taurine
O-3I
152
VS + glutamine 4- cysteine + glycine
47
393
223
O-I2
VS + glutamine + cystine + glycine
5°
237
All media contained 10% undialysed calf serum, 1 mM Na pyruvate, Eagle's vitamins (V)
and modified Earle's salts (S). The concentration of each amino acid as well as of cystathionine
and glutathione was 0-2 mM with the exception of glutamine, 2-0 mM. The growth rate was
calculated by taking the highest point on the linear part of the growth curve and dividing
population divisions by time in culture at that point. Abbreviations: Gluta., glutathione; Cysta.,
cystathionine; P.D., population divisions; D.C, days in culture; G.R., growth rate; %A,
average percent attachment.
Glutathione had similar growth-promoting activity over a concentration range of
0-05 to i-o mM (Table 2). Only the combination of glutamine and cysteine permitted
growth to the same level as with glutathione. Cysteine had similar activity over a concentration range of o-oi to 0-2 mM; no cell growth occurred at concentrations above
284
J. Litvrin
0-4 mM. Addition of glycine did not improve growth. Asparagine could replace glutamine.
The growth-promoting effect of cystathionine could be replaced by serine + homocysteine, but not by homoserine + cysteine; homocystine was less effective than
homocysteine.
Table 2. The effect of various amino acids and peptides on the growth and longevity of
human diploid fibroblast strain S.
Media
P.D.
Eagle's MEM
VS + glutathione, mM:
o-oi
53-7
386
D.C.
G.R.
%A.
149
0-62
76
0-27
0-37
64
57
41-0
142
142
142
0-29
55
43-8
177
390
142
032
030
42-2
64
76
68
0-05
o-i
0-2
0-5
i-o
49 "4
2-0
35-3
176
148
51
5O
5-5
54
VS + 0-2 mM glutamine
VS + o-2 mM cysteine
VS + 0-2 mM glutamine:
+0-2 mM glycine
+ 0-2 mM cysteine
+ 0-2 mM cysteine + 0-2 mM glycine
VS + o-5 mM glutamine + 0-2 mM cysteine
+ 0-2 mM glycine
V S + i-o mM glutamine+ 0-2 mM cysteine
+ 0-2 mM glycine
VS + 4-O mM glutamine + 0-2 mM cysteine
+ 0-2 mM glycine
VS + i-omM glutamine+ o-oi mM cysteine
+ o-2 mM glycine
V S + i-o mM glutamine+ o-i mM cysteine
+ 0-2 mM glycine
VS + i-o mM asparagine+ o-2 mM cysteine
VS + o-2 mM cystathionine
VS + o-2 mM serine + 0-2 mM homocysteine
VS + o-2 mM serine + 0-2 mM homocystine
VS + 0 2 mM homoserine+ o-2 mM cysteine
o-33
0-32
0-24
o-io
54
60
60
—
0
0
—
46-6
192
49
390
177
0-26
0-26
395
178
0-26
60
38-8
149
032
58
316
192
O"2O
48
38-4
167
0-25
36
38-4
167
341
117
142
167
0-25
0-29
43
63
o-33
0-30
O-2O
5°
33-5
47-2
24-9
0
141
0
56
5°
39
All media contained 10 % calf serum, 1 mM Na pyruvate, Eagle's vitamins (V) and modified
Earle's salts (S). The abbreviations are the same as in Table 1.
Preliminary experiments on the vitamin requirements for HEDLF growth showed
that of the 8 vitamins presented in MEM, folic acid could account for all the growthpromoting activity. The effect of various concentrations of folic acid in media with
different amino acid compositions is shown in Table 3. With MEM amino acids, folic
acid yielded similar growth over a concentration of 1-50 /tg/ml. In a medium containing only glutamine and cysteine as amino acid source the best result was obtained
with 1 /ig/ml.
The addition of other vitamins to a medium containing MEM amino acids + 2
Amino acids and vitamins for diploid cell growth
285
ml folic acid showed that none increased the growth rate or cell longevity with the
possible exception of nicotinamide (Table 4).
A simultaneous comparison of 3 HEDLF strains growing in different media but the
same batch of calf serum (Fig. 1) showed that all strains grew to about the same
extent in MEM and medium containing only glutamine+cysteine + complete vitamins. There was a marked difference in the growth in glutathione medium and only
Table 3. The effect of various concentrations offolic acid as the only vitamin present in the
medium on the growth and longevity of human diploid fibroblast strain S
Media
P.D.
D.C.
G.R.
'%A.
Eagle's MEM
Eagle's amino acids, no vitamins
Eagle's amino acids + folic acid, /tg/ml:
576
64
126
°59
49
0-19
79
49
38-7
49"4
57-2
O'l
IO
2'O
48-7
57'4
5'°
IO
156
0-25
171
161
0-37
222
0-41
039
O'4I
156
53
39
53
58
55
25
599
161
043
5°
62-9
197
0-37
51
42
32
49
—
—
59
54
IO
549
161
218
171
191
191
037
0-40
044
5'O
43 8
66-4
54-8
57-4
i mM glutamine + o-2 mM cysteine, no vit.
i mM glutamine + O'2 mM cysteine + folic acid, /tg/ml:
o-i
IO
2'O
63
0-45
50
039
47
All media contained i o % calf serum, i mM[ Na pyruvate and modified Earle'8 buffer. The
abbreviations are the same as in Table i.
MRC5 was capable of growing in cystathionine medium in this experiment. The WI-3
cells grew least well in the other media and not at all in serine + homocysteine + folic
acid. With all the strains it was found that homocysteine could not replace cysteine nor
did homocystine replace homocysteine (data not shown).
All attempts to grow these 3 HEDLF strains in glutamine + cysteine + folic acid
(GCF) medium containing 10% dialysed calf serum failed. The cells degenerated in
the initial passage without appearing to go through any divisions.
A comparison of the growth of MRC5 and WI-38 in media containing GCF and
one other amino acid (Tables 5 and 6, respectively) showed that MRC5 grew to a
higher longevity at a faster rate than WI-38. Their response to certain amino acids was
slightly different but no amino acid stimulated growth and longevity.
19
CEL40
J. Litwin
286
DISCUSSION
The results given above suggest that only 2 amino acids and 1 vitamin are absolutely
essential for the growth of human diploid fibroblasts when undialysed calf serum is
used as a medium supplement. These amino acids may be either glutamine + cysteine
or serine + homocysteine. Replacing cysteine or homocysteine with cystine or homocystine, respectively, reduced growth which suggests that the reduction of cystine to
cysteine may be a rate-limiting reaction for cell growth. Since no single amino acid or
Table 4. The effect of various vitamins on the growth and longevity of human diploid
fibroblast strain S
Media
P.D.
D.C.
G. R.
%A
Eagles MEM
AA.S.FA.
AA.S.F.A:
+ 1 /ig/ml biotin
+ 1 /tg/ml Ca pantothenate
4- 1 /ig/ml choline chloride
+ 1 /ig/ml I-inositol
+ 1 /ig/ml nicotinamide§
+ 1 /tg/ml pyrodoxal
+1 /tg/ml thiamine
+ 1 /tg/ml riboflavin
+ 10/tg/ml vit. C
+ 100 /tg/ml vit. E§
+ 1 /tg/ml vit. B l
+ 1 /ig/ml vit. Ks
479
467
144
160
°45
046
61
72
496
47-1
49-2
476
57'i
151
131
0-41
61
043
039
66
508
500
519
151
161
0-44
0-42
0-42
0-40
57
67
66
66
477
i53
i33
036
044
60
52
326
104
o-3S
40-5
37-0
42-8
41-8
41-6
33"5
i39
i33
039
103
042
76
65
67
64
69
+ 1 /tg/ml vit. B,j
146
160
146
°'33
70
0-44
+1 /tg/ml cholcalciferol
138
0-42
70
+1 /tg/ml ergocalciferol
133
81
0-37
+ 10 /tg/ml /?-carotene
i33
7-2
025
76
+ 10 /ig/ml retinal acetate
63
47-0
68
+ 10/tg/ml vit. Kx
138
o-43
The media contained Eagle's MEM amino acids (AA), modified Earles' salts (S), 2 /tg/ml
folic acid (FA), 1 mM Na pyruvate and 10% calf serum (AA.S.FA). The other abbreviations
are the same as in Table i.
§ Culture became contaminated after time indicated in Table.
Fig. 1. The effect of various media on the growth and longevity of MRC5, (A, B) WI-38
(c, D) and the S strain of HEDLF cells (E, F),
In A, c, E, E—E, Eagle's medium; x—x, glutamine +cysteine; O—o, glutamine +
cystine; G—c, glutathione; c—C, cystathionine. In B, D, F; E
E, Eagles' amino acids
+ folic acid; x—x glutamine +cysteine + folic acid; s—s, serine + homocysteine;
s
S, serine + homocysteine + folic acid. Pop. div., population divisions. Glutamine
was present at a concentration of 1 mM except in Eagle's medium which contained
4 mM. The other amino acids mentioned were present at 0-2 mM in the media. The
media with folic acid contained 50 /tg/ml folic acid as the only vitamin. In A, C, E the
media contained Eagle's vitamins as did serine + homocysteine medium in B, D, F. No
growth occurred in cystathionine medium with WI-38 or S cells. WI-38 did not grow
in serine + homocysteine + folic acid. Those curves which end abruptly without
bending to the right were terminated by contamination but the cultures all showed
morphological signs of senescence.
287
Amino acids and vitamins for diploid cell growth
50'
50'
40-
40
=5 30
30
Q.
20-
20
10'
10
20 40 60 80 100120140160
20 40 60 80 100 120140160
50-
50-
40-
40
•I 30-
30
20'
20-
10-
10
20 40 60 80 100120140160
20 40 60 80 100120140160180 200
50-
50
40-
40
30-
30
20-
20
10-
10
20 40 60 80 100 120140160
Time, days
20 40 60 80 100120140160
Time, days
19-2
288
J. Litwin
combinations of other amino acids permitted sustained HEDLF growth (Litwin,
1974), it must be assumed that the combinations given above satisfy some minimum
necessary requirement of these cells. Eagle, Piez & Levy (1961) have shown that cells
in vitro have the ability to concentrate many fold the amino acids present in the
medium at low concentrations. Possibly glutathione or glutamine + cysteine are
absolutely necessary for sustained growth because of their role in the •y-glutamyl
transferase mechanism for amino acid transport across the cell membrane (Meister &
Tate, 1976). However, this possibility does not explain the activity of cystathionine or
serine + homocysteine.
Table 5. The growth of MRC$ cells in medium containing 1 mM glutamine, 0-2 niM
cysteine, 2 fig/ml folic acid, modified Earle's salts, IOTM Napyruvate and 10% calf serum
{GCF medium) plus one additional amino acid
Media
P.D.
Eagle's MEM§
360
GCF
576
GC +Eagle's vitamins
GCF:
609
+ i-o mM tyrosine
+ i'O mM histidine
+ o-2 mM arginine
+ 0-2 mM lysine
+ O-2 mM tryptophan
+ o-2 mM methionine
+ o-2 mM threonine
+ o-2 mM valine
+ o-2 mM leucine
+ o-2 mM isoleucine
+ o-2 mM proline
+ 0-2 mM hydroxyproline
+ o-2 mM phenylalanine
+ 0-2 mM alanine
+ 0-2 mM taurine
+ o-2 mM serine
+ o-2 mM glutamic acid
+ o-2 mM aspartic acid
D.C.
G.R.
%A.
75
048
79
187
0-41
81
188
0-48
78
273
382
139
188
o-33
0-23
58
49
55"°
182
0'45
55-1
187
160
181
159
036
82
89
393
51-5
487
608
57-4
54-6
55'i
202
181
181
622
187
187
50-1
637
181
202
54' 2
649
62-4
597
159
187
181
181
036
o-
43
o-43
o-35
o-37
o-39
o-37
0-41
o-35
0-40
0-44
0-41
0-42
0-47
86
85
84
79
79
79
90
84
86
82
87
86
85
74
Abbreviations are the same as in Table 1.
§ Culture became contaminated after time indicated in Table.
Folic acid was the only vitamin required in media containing MEM amino acids
with glutamine + cysteine or serine + homocysteine. The addition of other vitamins
had no stimulating effect although nicotinamide increased cell longevity. Presumably
the quantity of other vitamins in the serum was sufficient for sustained cell growth once
folic acid was supplied.
Although lesser growth was obtained with serine + homocysteine than with glutamine 4- cysteine, the former is of interest because significant growth was obtained in the
absence of added glutamine. Cysteine could not replace homocysteine nor did homo-
Amino acids and vitamins for diploid cell growth
289
serine + cysteine support growth. According to Eagle, Washington & Friedman (1966),
HEDLF cells can split cystathionine to serine + homocysteine but lack the ability to
synthesize cystathionine from homoserine + cysteine. Further investigations with
this medium were not pursued.
No amino acid added to GCF medium was found to stimulate growth or longevity of
MRC5 or Wl-38 cells, although tyrosine and histidine were stimulatory to S cells
growing in glutathione medium (Table 1, see also Litwin, 1974), It remains to be
determined what combinations of amino acids will increase the growth rate of these
cells.
Table 6. The growth of W/-38 cells in medium containing 1 mMglutamine (G), o-2mM
cysteine (C), 2 fig/ml folk acid (F), modified Earle's salts, 1 mM Na pyruvate and
10% calf serum {GCF medium) plus one additional amino acid
P.D.
Media
D.C.
G.R.
%A.
Eagle's MEM
332
116
0-44
62
GCF
42-2
162
0-34
57
312
131
0
0
029
—
126
036
i39
O'34
0
—
44
—
63
65
—
3O-4
119
320
420
131
i55
131
134
o-33
0-25
GCF:
+ i-o mM tryosine
+ i-o
+ C2
+ 0-2
+ 02
+ 0-2
+ o-2
+ 02
+ 0-2
+ 0-2
+ o-2
+ 0-2
+ o-2
+ o-2
+ o-2
+ 0-2
+ 0-2
+ o-2
mM
mM
mM
mM
mM
mM
mM
mM
mM
mM
mM
mM
mM
mM
mM
mM
mM
histidine
arginine
lysine
trytophan
methionine
threonine
valine
leucine
isoleucine
proline
hydroxyproline
phenylalanine
glutamic acid
aspartic acid
alanine
glycine
serine
44-0
27-1
0
355
42-4
409
031
o-34
o-37
164
183
167
029
134
126
o-33
417
139
126
n-6
76
o-34
o-35
o-33
44-8
31-9
39'3
46-9
376
O'33
028
0-40
55
48
59
59
64
58
67
5i
61
60
57
63
70
Abbreviations are the same as in Table 1.
A simultaneous growth comparison of the S, MRC5 and WI-38 strains in different
media but with the same batch of calf serum showed that all 3 grew equally well in
MEM and glutamine + cysteine media. In this experiment neither WI-38 nor the S
cells grew in cystathionine medium although they grew in serine + homocysteine. In
earlier experiments the S cells grew well in cystathionine medium and the growth in
glutathione medium was greater. It is possible that the inconsistencies in the growth
results from experiment-to-experiment may be dependent upon the quality of the
serum used and some diploid cell strains may be more sensitive to variations in serum
quality than others. In a preliminary experiment with 6 independent serum batches on
the growth of WI-38 cells in MEM and GCF media (data not presented here) there
290
J. Litwin
occurred considerable variation in the growth rate and longevity in both of these
media.
Although the importance of cysteine (or cystine) and glutamine for human diploid
fibroblasts growth is well documented (Cornfield & Hay, 1978; Ham, Hammond &
Miller, 1977; Griffiths, 1970) the concentration of cysteine in the medium may be a
possible source of growth variation. Ham et al. (1977) claimed that 0-09 mM cysteine
was optimal for WI-38 cell growth, 0-2 mM marginal and 0-3 mM markedly inhibitory. Our results showed that a concentration of o-oi to 0-2 mM cysteine gave comparable growth results whereas no growth occurred at 0-4 mM. Thus, the 0-2 mM cysteine concentration used in GCF medium may be on the border of being inhibitory.
Although the average cell attachment of the S cells tended to be slightly lower in the
test media than in MEM, the MRC5 and WI-38 cells showed no difference. Thus, the
amino acids and vitamins which were absent from the various media had no effect on
cell attachment. During the serial passage of these cells the attachment varied from
100 to 20-30%. In general, attachment decreased with age.
The in vitro life span (population division level) of cells growing in glutamine +
cysteine medium was frequently as great as the same cell strain growing in MEM,
although the growth rate tended to be less. Previous experience with HEDLF cells
indicated that conditions which reduced the growth rate also reduced longevity. The
present data contradict this impression and suggest that the optimum growth rate may
not be directly related to optimum longevity.
The author wishes to thank Mr3 Ann BjOrk and Mrs Annette Litwin for their excellent technical assistance.
REFERENCES
CORNFIELD, V. A. & HAY, R. J. (1978). Effects of cystine or glutamine restriction on human
diploid fibroblasts in culture. In Vitro 14, 787-794.
EAGLE, H., PIEZ, K. A. & LEVY, M. (1961). The intracellular amino acid concentrations required for protein synthesis in cultured human cells. J. biol. Chem. 236, 2039-2042.
EAGLE, H., WASHINGTON, C. & FRIEDMAN, S. M. (1966). The synthesis of homocystine,
cystathionine and cystine by cultured diploid and heteroploid human cells. Proc. natn. Acad.
Set. U.S.A. 56, 156-163.
GRIFFITHS, J. B. (1970). The quantitative utilization of amino acids and glucose and contact
inhibition of growth in cultures of human diploid cell WI-38. J. Cell Sci. 6, 739—749.
HAM, R. G., HAMMOND, S. L. & MILLER, L. L. (1977). Critical adjustment of cysteine and
glutamine concentrations for improved clonal growth of WI-38 cells. In Vitro 13, 1-10.
HAYFLICK, L. (1965). T h e limited in vitro lifetime of human diploid cell strains. Expl Cell Res.
37, 614-636.
HAYFLICK, L. & MOORHEAD, P. S. (1961). The serial cultivation of human diploid strains. Expl
Cell Res. 25, 585-621.
JACOBS, J. P. (1966). A simple medium for the propagation and maintenance of human diploid
cell strains. Nature, Lond. 210, 100-101.
JACOBS, J. P., JONES, C. M. & BAILLIE, J. P. (1970). Characteristics of a human diploid cell
designated MRC5. Nature, Lond. 227, 168-170.
JACOBS, J. P. (1976). The status of human diploid cell strain MRC5 as an approved substrate
for the production of viral vaccines. J. tiol. Stand. 4, 97—99.
LITWIN, J. (1970). Standardization of human diploidcell cultivation. Appl. Microbiol. 20,899—906.
Amino acids and vitamins for diploid cell growth
zgi
J. (1972). Human diploid cell response to variations in relative amino acid concentrations in Eagle's medium. Expl Cell Res. 72, 566-568.
LITWIN, J. (1974). Growth of human diploid fibroblasts in media with different amino acid
composition. J. Cell Sci. 14, 671—680.
MEISTER, A. & TATE, S. S. (1976). Glutathione and related y-glutamyl compounds: Biosynthesis and utilization. A. Rev. Biochem. 45, 559-604.
LITWIN,
(Received 9 April 1979)