The Nutrition of Animal Cells

CHARITY WAYMOUTH*
Roscoe B. Jackson Memorial Laboratory, B w Harbor, Maine

Page
I. Introduction ................................ 1
11. Biological Media ........................... 5
111. Synthetic Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
IV. Inorganic Substances ...... ..................... 21

VI. Amino Acids and Peptides ....................

VIII. Lipids ............................................
IX. Vitamins ..........................................

X. Hormones ................................. ...... ........... 53

XI. Concluding Remarks ................................................ 57
XII. References ......................................................... 58

I. INTRODUCTION
“Welche Bedeutung vom physiologischen Gesichtpunkt hat es doch z. B., dass
wir den Nahrungsbedarf der verschiedenartigen Gewebszellen zu registrieren
vermogen I” (Albert Fischer, 1941a).
Our present extensive knowledge of animal nutrition has been reached
through investigations with utilitarian and practical economic motives.
The available information about nutritional requirements at the tissue and
cell levels is, by comparison, very limited. There are mechanisms in the
animal body which assure to each tissue its proper nutritive requirements.
Some of these mechanisms may be looked upon as relatively passive, in
that the body fluids which reach the cells contain the appropriate nutrients ;
others are more active, for example the selection of certain nutrients by
particular cells or the accumulation of substances against a concentration
gradient. The passive mechanisms, by definition, depend purely on the

* On leave of absence from the Chester Beatty Research Institute, Royal Cancer
Hospital, London, England. This work has been supported, in London, by grants to
the Royal Cancer Hospital and Chester Beatty Research Institute from the British
Empire Cancer Campaign, the Jane Coffin Childs Memorial Fund for Medical
Research, the Anna Fuller Fund and the National Cancer Institute of the National
Institutes of Health, U.S. Public Health Service; and in Bar Harbor, by the
American Cancer Society, Inc., through a British-American Exchange Fellowship
awarded on the recommendation of the Committee on Growth of the National
Research Council.
1
2 CHARITY WAYMOUTH

environment ; on the other hand, certain specific activities of different cell

types can modify the immediate environment to satisfy special nutritional
demands and can also (by enzymatic activity or by secretion) produce
materials which exert their effects in distant parts of the organism. Be-
cause modification of the environment is an important function of most
cells, the result is an enormously intricate interdependence of every part
of the organism nutritionally, as well as in the general physiological sense.
On account of this tissue interdependence, the nutrition of isolated colonies
of cells from animal tissues has certain complexities which are not met
with in the nutrition of higher animals or of bacteria or free-living animal
microorganisms. Microorganisms exhibit a wider range of powers of
synthesis of complex from simple components than isolated, differentiated
tissues with specialized metabolic functions. The value of protozoa in the
investigation of animd nutrition has been pointed out by Kidder (1952),
who has been a major contributor in this field with his studies on Tetra-
hynzenu. But, as Kidder implies, the nutrition of this independent unicel-
lular organism, in which there is no division of labor, is to be contrasted
on the one hand with multicellular organisms where such a partition of
function exists, and on the other with the individual cells of the metazoan
organism with their strong mutual interdependence. As Carrel (1938) has
said : “the living body is a heterogeneous spatiotemporal continuum of
cells and fluids” in which “neither cells nor tissues are mere cellular aggre-
gates.” Careful adaptation of the environment has been shown (Sanford,
Earle, and Likely, 1948; Likely, Sanford, and EarIe, 1952) to be neces-
sary, to produce conditions in which a single isolated metazoan cell (in
contrast to a fragment of tissue or a population of cells) can survive and
multiply i 7 ~vitro.
It is of particular interest to know whether the nutritional needs of
different tissues for growth and repair differ from those for the mainten-
ance of normal function and to study the possible special nutritive demands
of neoplastic cells in comparison with those of normal cells. Quantitative
and qualitative differences in nutritive pattern between tissues within an
animal are difficult to study in the intact organism, even though it is evident
that such differences exist (cf. Spratt, 1950). One of the aims of
nutritional investigations at the cellular level, as Haddow (1947) has
stressed, is to attempt to disclose these differences by the study of isolated
cell colonies and ultimately to integrate the information for the better
understanding of the whole process of nutrition in maintenance, develop-
ment, and growth, and in neoplasia.
Much unnecessary confusion exists in the field of cell nutrition because
criteria for nutritional adequacy have not been clearly enough defined.
 T H E NUTRITION OF ANIMAL CELLS 3

When cells can proliferate uninterruptedly and be carried through a large

number of subcultures, the medium in which this is possible must be
sufficient in every sense. Dilute fowl serum is nutritionally complete in
this respect for fowl macrophages (Carrel and Ebeling, 1922a ; Jacoby,
1937a ; 1938, 1940, 1941, 1945, 1949). For similar, long-continued cul-
tivation of embryonic fibroblasts in a state of active proliferation, a mixture
of fowl plasma and chick embryo extract is the standard medium, But
dilute fowl serum (or heparinized plasma) alone is not sufficient in the
same sense for fibroblasts (Carrel and Ebeling, 1923b, e, f ) , though it
permits their survival for a year or more (Parker, 1936a). I t is conven-
ient to make a distinction between nutritional conditions sufficient for
the maintenance of cells in a healthy state for more or less prolonged
periods ; and the additional conditions, presupposing the former, required
for proliferative growth. Such a distinction is useful in assessing the
value of individual nutritive substances and of synthetic nutrient mixtures.
The most promising method of designing media of known composition is
first to devise a medium in which the cells can survive. When the medium
is adequate for prolonged survival of cells, the additional conditions which
will permit growth can be recognized. This empirical distinction is impor-
tant because of the seeming paradox that growth stimulation can reduce
survival. I t can do this because, if the cells are stimulated to multiply in a
nutritionally deficient environment, they will the more rapidly exhaust it.
Because the cell and its environment form a changing and a reciprocating
system, the problem of defining nutritional adequacy cannot be simply
solved by making a neat classification of nutrient substances into well-
defined groups according to the needs of a particular tissue for them.
Knight (1945) has stated the position for the nutrition of microorganisms,
and it can equally well be said of the nutrition of animal cells that “a
given substance, required as a compunent of one of the essential metabolic
processes, might appear in three different roles . . . (1) as an ‘essential
nutrient’, when its rate of synthesis by the cell was so slow as to be
insignificant; (2) as a growth stimulant, when its rate of synthesis was
somewhat faster but still slow enough to be a limiting factor; or ( 3 ) as a
substance not required at ail for nutrition, because the cell could synthesize
it so fast that it was not a limiting factor in growth. I t is the metabolic
process which is the essential thing.”
It has been repeatedly pointed out by numerous authors (eg. Hueper
et al., 1933; Cunningham and Kirk, 1942; Fischer, 1946a; Tompkins,
Cunningham, and Kirk, 1947; Waymouth, 1949) that the methods most
commonly used to measure “growth” in cell populations iH vitro are
unsatisfactory. I t is still necessary to emphasize that measurements of
4 CHARITY WAYMOUTH

superficial area of fibroblast cultures can only be used in carefully selected

conditions and with an understanding of the relative contributions to the
total area made by cell migration and by cell division (cf. also Katzenstein
and Knake, 1931). The application of chick embGo extract to fibroblast
cultures, as Willmer and Jacoby (1936) recognized, promotes both activi-
ties. It was still more clearly demonstrated by Doljanski and Goldhaber
(1945) that these two functions are experimentally separable. They irra-
diated fibroblast cultures with a dose of X-rays such that all mitotic
activity was inhibited. Application of embryo extract to these cultures
induced cell migration not significantly less in extent than in unirradiated
cultures in which mitosis was taking place.
B’ecause criteria of growth or nutritional effectiveness are more often
than not unsatisfactory, the reader must treat critically the results reviewed
below. Some of them are based on assessments of survival, some on
measurements of outgrowth, and others on stimulation of mitosis. In
many cases the observations were made over very short periods of time
(e.g., 48 hours). When it is realized that cells can remain morphologically
unaltered and can even undergo mitosis in simple salt solutions during such
periods, it will be clear that little of positive value can be learned about
cell nutrition from these short-term tests. They are useful only in the
negative sense that they demonstrate that the nutrient tested has no toxic
effect and is therefore worthy of more extensive study in longer experi-
ments. The longer the cells are kept in an artificial environment, the
less will they be able to “live on their reserves,” and the more important
does it then become to supply not only the major nutrients but also the
trace substances for which a deficiency may not develop or manifest itself
for several weeks.
The objective for those who would understand the nutrition of individual
tissues or their component cells is to reproduce, in chemically defined
terms, an environment sufficiently close to the normal “passive” environ-
ment for the cells that they can modify it to fulfil completely their peculiar
nutritional requirements. The need to develop nutrient media of known
chemical composition for animal cells has been recognized for a long time
(Lewis and Lewis, 1911a, b). Baker’s (1929) early experiments led her
to hope “that it might be possible to synthetize an artificial medium which
would prove adequate for the maintenance of cell life and multiplication.”
A fully defined nutrient medium was still hardly regarded as a practical
possibility when Willmer and Kendal (1932) wrote : “The future of tissue
culture as an experimental method depends very largely upon whether a
more complete knowledge can be obtained of the requirements of the cells.
The goal at which to aim is a completely synthetic medium whose every
 THE NUTXITION OF ANIMAL CELLS 5

constituent is under control. This is probably an ideal rather than a possi-

bility.” But only a little later Willmer (1935) had apparently conceded the
possibility, though he felt that “there is still . . . a very long road to be
travelled before it will be possible to produce a synthetic environment for
tissue-culture cells.” The ideal has not yet been fully realized, but with the
rapid extension of our knowledge of cellular metabolism, the possibility is
now accepted, the practicability of the project has increased, and the
desirability of its achievement has by no means diminished. Our present
understanding of the nutrition of cells has been derived partly from analysis
of media of biological origin which are nutritionally complete, partly from
the study of the effects of known substances added to more or less com-
plete biological media, and partly from experience with partly or wholly
synthetic media designed on the basis of known biochemical and nutritional
principles. It is the intention, in this review, to assemble what is known
of the general and special nutritive requirements of animal cells, with
particular reference to the nutrition of colonies of cells in tissue culture.
The physiology and nutrition of cells in culture have previously been
dealt with in the following reviews, books or articles : Anon. (1947, 1949,
1950) ; Cameron (1950) ; Carleton (1923) ; Carrel (1924a) ; Fischer
(1930, 1933, 1940, 1941b, 1946a, b, 1947, 1948c) ; Lewis and Lewis
(1925) ; Morgan ( 1950) ; Vogelaar and Erlichman (1933) ; Waymouth
(1950, 1952) ; White (1946, 1947, 1950) ; Willmer (1928, 1935, 1945),
and Winnick (1952).
11. BIOLOGICAL MEDIA
Just as the science of bacteriology was developed by the empirical use
of meat infusions, the techniques for cultivation of cells from the tissues of
higher organisms have been based on the use of biological fluids as culture
media. From the use of lymph by Harrison (1907) and plasma by Burrows
(1910, 1911 ; Carrel and Burrows, 1911a) for relatively short-term studies
of living cells in vitrro, the finding of Carrel (191 1, 1912a, b, c, 1913a, b, c)
that the addition of an extract of chick embryo to the plasma clot wouId
permit continuous proliferation of chick fibroblasts for long periods,
tempted many to use this new tool to study living, growing cells. The fact
that cells, in such a medium, could be forced into a high rate of proliferation
proved so fascinating that it was sometimes forgotten [though not by
Carrel (1938) himself] that cells perform many functions other than repro-
duction and that surviving cells in an artificial environment are a valuable
experimental material. Because of the strong emphasis upon proliferative
growth, the information that has been gathered on nutritional requirements
of cells in tissue culture pertains very largely to actively growing cells.
As has been pointed out elsewhere, in a discussion of stimulation of mitosis
6 CHARITY WAYMOUTH

in relation to cell nutrition, “it appears . . . to be axiomatic that, for cell

division to be possible, adequate nutritional conditions for supplying
energy and materials must first be fulfilled” (Waymouth, 1952). That it
was desirable to attempt to distinguish between substances required for
cell nutrition per se and those for cell multiplication, was recognized by
Baker and Carrel (1926a, b, e), who showed that chick embryo extract
was an excellent source of both kinds of substance. Heaton (1926) and
Hueper et al. (1933), for convenience in studying the properties of
biological media, further emphasized that different conditions are necessary
for survival, for cell multiplication, and for synthesis of protoplasm, and
that each cell type has its special nutritive requirements. One of the
earliest students of cell physiology (Loeb, 1912) had stressed the point
that variations in internal and external environment could separately affect
survival, growth, or movement of the cell.
Many variations have been made on the classic biological medium of
chicken plasma and chick embryo extract, but it remains true that a great
variety of cell types can be grown more or less successfully in this mixture.
Growth-promoting power is not confined to homologous media. Heterolo-
gous plasmas were used by Volpino ( 1910) ; Lambert and Hanes (191 1) ;
Lambert (1912) ; Ingebrigtsen (1912a, b) ; Champy and Coca (1914) ;
Fischer (1924, 1929), and Chlopin (1930), and it is now common
practice to do so. Extracts of chick .embryos were shown to promote
growth of duck (Fischer, 1924 ; Kiaer, 1925), rat (Mottram, 1927),
and rahhit, guinea pig, and human tissues (Fischer, 1941b). Chick
tissues were grown in extracts of rabbit embryos (Carrel and Ebeling,
1923d ; Landsteiner and Parker, 1940). Amphibian tissues have been
grown in media composed wholly of chick plasma and embryo extract
(Hughes and Preston, 1949 ; Danes, 1949). Bovine embryo extracts have
been used for human (Gey and Gey, 1936), rat (Lewis, 1935, 1939), and
chick (Fischer, 1941b) tissues. These early demonstrations of the non-
species-specificity of growth-promoting media established the now general
use of heterologous components in tissue-culture media. The strain L
mouse cells of Sanford, Earle, and Likely (1948) have been maintained
continuously for 11 years in a medium of horse serum and chick embryo
extract.
The plasma or serum component of the medium has usually been derived
from adult animals. The belief became general, after it had been shown to
be SO for the fowl, that serum becomes progressively more and more
inhibitory with the age of the donor (Carrel and Ebeling, 1921a, b ; 1922b;
1923a, c ; Baker and Carrel, 1926a, c, d, 1927), and therefore the serum
of young adult animals has generally been used. There is little more recent
 THE NUTRITION O F ANIMAL CELLS 7
comparative information, but a widespread impression that serum or
plasma from a young animal, or from the fetus, provides a more favorable
medium for cell growth than the corresponding adult serum. There is no
clear evidence for inhibition with increasing age of donor in the case of
horse serum (Earle, personal communication). Fetal plasma or serum,
and especially human umbilical cord serum (Gey and Gey, 1936), is much
used, particularly in media for the growth of tumor cells. Some clues to
the nutritional demands of growing cell colonies may be found by compar-
ing the chemical differences between adult and fetal sera, where the latter
have been shown to be more effective in promoting growth. As examples
(others will be noted later, under the different classes of nutrients), it is
now well known that fructose occurs in large amounts in the fetal blood
of many species (Bacon and Bell, 1948; Barklay et aE., 1949; Hitchcock,
1949, and Goodwin, 1952). The amount of inositol in fetal plasma is
higher than in the adult (Nixon, 1952) ; fetal rabbit plasma has a higher
bicarbonate and a lower chloride content than maternal plasma (Young,
1952). It has long been supposed that human umbilical cord serum had a
higher amino-N content than maternal serum (Morse, 1917), and similar
differences were reported for other species before Christensen (1948) and
Christensen and Streicher (1948), using more reliable methods, demon-
strated a ratio of 1.5 to 2.0 for the fetal : maternal amino acids in human and
rabbit plasmas, and a ratio of about 5 for the guinea pig. The ratio for
human plasma was confirmed by Crumpler, Dent, and Lindan (1950),
who reported a range of 1.03 to 3.00 (fetal :maternal amino acids) in nine
cases, with a mean of 1.60. Schreier and Steig (1950) showed that the
amounts of individual amino acids in human cord serum varied from
23.7% higher (leucine) to 182.1% higher (lysine) than in adult human
serum. The amount of glutamic acid is much increased in fetal blood
(average 9.5 mg./100 ml.) compared with normal adult (average 1.0 ( 9 )
and 1.2 ( b ) ) and maternal (4.3) (White, Beaton, and McHenry, 1952).
The growth-promoting effects produced by tissue extracts vary with
age, even within the embryonic period (Gaillard, 1935, 1942 ; Gaillard
and Varossieau, 1938 ; Miszurski, 1939). Adult tissue extracts have,
however, been used with varying degrees of success, mostly for short-term
experiments (Carrel, 1913a ; Heaton, 1926 ; Hoffman, Goldschmidt, and
Doljanski, 1937; Trowel1 and Willmer, 1939; Doljanski and Hoffman,
1939, 1943; Hoffman and Doljanski, 1939; Hoffman, Tenenbaum, and
Doljanski, 1939a, b, 1940; Hoffman, 1940; Doljanski, Hoffman, and
Tenenbaum, 1939, 1942 ; Hoffman, Dingwall, and Andrus, 1948, 1951;
Margoliash and Doljanski, 1950). Leucocytes are another source of
growth-promoting extracts (Carrel, 1922, 1924b, c, 1927), and these cells
8 CHARITY WAYMOUTH

were believed to be important in the nutrition of other types of cell (Carrel

and Ebeling, 1922c, 1926b, 1928; Fischer, 1925a, b) and to be able to
make and secrete “trephones” (Carrel and Ebeling, 1923g, h ; Carrel,
1924b, c) which could act as intermediaries for the nutrition of more
differentiated cells (des Ligneris, 1931). Other biological fluids which have
been employed at various times in tissue culture media include peritoneal
exudate (Baitsell and Sherwood, 1925), allantoic fluid (Moppett, 1927),
amniotic fluid (Grossfeld, 1949; Enders, 1953) or a mixture of amniotic
and allantoic fluids (Szarski, 1950, 1951), and aqueous humor. (Albrink
and Wallace, 1951). A particularly effective biological fluid is ascitic fluid
from patients with carcinomatosis peritonei (Bergman and Waterman,
1935; Ivers and Pomerat, 1947; Ivers, Pomerat, and Neidhardt, 1948;
Ulloa-Gregori et ab., 1950; Pomerat, Nowinski, and Rose, 1950; de Lustig,
1951 ; Ellis, Nowinski, and Bieri, 1953). Serum ultrafiltrates, introduced
by Simms (1936; Simms and Stillman, 1937) for use with adult tissues,
are often employed. Chemical analyses have been attempted on few of
the medium components except plasma or serum and embryonic extracts.
A general survey has recently been made (Waymouth, 1952) of the
history of the use of embryonic and adult tissue extracts and of leucocyte
extracts as stimulants to cell proliferation. The discussion here will be
confined to the attempts which have been made to analyze the commoner
medium components.
During the period 1920 to 1940, varioys analyses of embryo extracts,
using methods which by present-day standards were somewhat crude, were
made by Carrel, Baker, and Ebeling and by Fischer. The influence of
Carrel in the field of tissue culture at that time led to the general acceptance
of his view (Carrel, 1924a) that the whole of the growth-promoting
activity of his medium for fibroblasts resided in the embryo extract.
Plasma was regarded as an almost inert substratum. This view appeared
to be borne out by the later observations of Carrel and Ebeling (1923b, e) ;
Carrel (1928) ; Fischer and Parker (1929) ; Olivo (1931) and Parker
(1933, 1936b) that, in a medium of diluted serum, fibroblast cultures
could be kept in a state of prolonged survival with little or no increase of
tissue, whereas addition of embryo extract produced an immediate pro-
liferative response. Olivo’s (1931) cultures doubled in mass in a period
of six months ; Carrel (1928) maintained chick embryo heart cultures,
with persistent pulsation, for 104 days in a Ringer-washed plasma clot.
Addition of embryo extract then induced active proliferation. Carrel
(1928) maintained from this experiment that serum has no nutritive
function for fibroblasts and that chick embryo extract is a “complete
food.” At this time he also recorded that “although attempts at frac-
 THE NUTRITION OF AWIMAL CELLS 9

tionation have been made with all possible techniques, no part has been
isolated which was endowed with a greater activating power than the
whole.” However, whereas in 1926 Carrel and Ebeling (1926a) had
stated that “fibroblasts do not feed on plasma, egg albumin, egg yolk,
amino acids or broth. They synthetize protoplasm exclusively from sub-
stances contained in the juice of chick, mouse, guinea pig and rabbit
embryos,” by 1938 Baker had come to the conclusion that embryo juice
alone was unable to furnish the substances necessary for normal fibroblast
growth, but that serum was needed to provide additional nutriment for
continued culture. The result of the twenty-year period of attempts to
isolate active “growth hormones” from embryo extracts was summarized
by Fischer (1941b) in the discouraging words that “it made practically
no difference how an extract was treated, its activity was lowered anyhow.”
While it is lamentably true that analysis of biological media by chemical
methods, and the attempts to attribute special growth-promoting activity
to individual chemical constituents, have not added very greatly to our
knowledge of cell nutrition, a somewhat greater measure of success has
attended the use of physical methods. By these methods (dialysis, ultra-
filtration and ultracentrifugation) it has been possible to eliminate some
parts of the complex biological mixture.
Fractionation of biological media by dialysis or ultrafiltration has a long
history. Wright (1926), by dialyzing embryo extract against a modified
Pannett and Compton saline solution, obtained a protein-f ree dialyzate
(i.e., a dialyzate in the modern, not the original, sense; vide Pirie, 1947)
which, in a medium containing whole plasma, promoted cell division in
cultures of chick heart fibroblasts. On the other hand, Baker and Carrel
(1926e) reported that dialysis of embryo extract against water, using
“very permeable collodion sacks” and adjusting the salt concentration and
p H before use, did not entirely remove the growth-promoting activity of
the extract. They found, however, that an ultrafiltrate only slightly
increased the area of outgrowth of cultures, and permitted survival no
longer than Tyrode solution alone. They concluded that “The growth-
promoting substances which distinguish embryonic juice from other fluids
in its capacity to maintain the life of fibroblasts and epithelial cells indefi-
nitely in vitro are not to be found among its dialyzable components.’’
Jacoby ( 1937b) reached essentially the same conclusion, using dialysis
against Tyrode solution and measuring both areas of migration and
mitoses in the cultures. Tazima (194Oa, b) , who dialyzed both the embryo
extract and the plasma against Tyrode solution, reported that dialyzed
extract gave very poor growth of cultures in their second passage; the
dialyzate, with whole plasma, gave better growth than the plasma diluted
10 CHARITY WAYMOUTH

with Tyrode solution. Outgrowth of fibroblasts was less in whole embryo

extract and dialyzed plasma than in the complete medium, but cultures
could be maintained for up to at least 15 passages. Iris epithelium
could be carried through 24 passages (Kimura, 1938; Tazima, 1940a).
Dialysis of adult chicken heart extract against Tyrode solution was carried
out by Margoliash, Tenenbaum, and Doljanski (1948). When fresh heart
extract was used in the medium, the growth-promoting effect (area meas-
urement) was greatly reduced by dialysis, and negligible growth-promoting
activity was found in the dialyzate. Dialysis of extracts of acetone-dried
heart also reduced the growth-promoting power ; in this case the dialyzate
was not completely without activity, and complete restoration of activity
was achieved by recombination of the dialyzed extract and its dialyzate.
Either fraction taken alone is evidently nutritionally deficient.
Dialysis of plasma and embryo extract against a Ringer-glucose solution
is the basis of Fischer’s approach to the analysis of growth media (Fischer,
1941a, b, 1942a, b ; 1946b, 1947, 19&, b, c, d ; Fischer and Astrup, 1942,
1943; Astrup, Fischer, and Volkert, 1945;Astrup, Fischer and Phlenschla-
ger, 1947; Astrup and Fischer, 1946; Astrup, Ehrensvard, et al,, 1947;
Fischer, Astrup, et d., 1948). The underlying hypothesis is that, by
dialysis, low molecular weight substances are removed from the classic
nutrients, leaving unaffected the high molecular “growth-promoting f ac-
tors” or “Embryonin.” The principle active factor in “embryonin” is
believed to be a labile nucleoprotein or phosphoprotein with catalytic
properties. The deficiency of low molecular “accessory growth factors’’ is
made up by supplementation of the dialyzed medium with known com-
pounds. Fischer has been able, by this method, to demonstrate the im-
portance of amino acids and peptides for the survival of chick fibroblasts.
Supplementation of the medium of dialyzed pIasma and embryo extract
with trypsin-digested serum produced a medium in which growth could
take place: further digestion with acid (i.e., to amino acids) and restora-
tion of tryptophan was sufficient for maintenance only (Fischer, 1941a).
Similarly, supplementation with synthetic mixtures of amino acids main-
tained the cells alive but by no means restored full growth. Boiled kidney
extract (Fischer and Astrup, 1942) was also a satisfactory supplement to
the dialyzed medium. It was, however, shown (Fischer, 1946a) that
cultures which required both an amino acid mixture and a boiled kidney
extract as supplements during the first stages of growth in vitro, could
proliferate at an undiminished, or even a greater, rate from 13 days on-
wards if one or the other of the supplements was omitted. Extracts of
heart tissue were less active supplements to the dialyzed medium than kidney
extracts, when used alone, but were highly active when used with a mixture
 T H E NUTRITION OF ANIMAL CELLS 11

of nine amino acids, or with addition of cystine, lysine, glutamic acid,

tryptophan and arginine (Astrup, Fischer, and Volkert, 1945).
There is some question whether a useful distinction between the “cata-
lytic growth-promoting factors” and the “accessory growth factors”
(Fischer, Astrup, et d.,1948) can be maintained. One of the most out-
standing characteristics of Fischer’s “embryonin” fraction is its great
lability. Apart from the fact that its activity is easily lost, such a fraction
undoubtedly contains many low molecular components held in combina-
tion or adsorption, but nutritionally accessible to the living cells. In other
words, it cannot be said that the small molecules provided as such are
qualitatively or quantitatively the only ones the cells may use. The validity
of the use of dialyzed plasma and embryo extract as a basal medium for
the study of “accessory growth factors” was first disputed by White and
Lasfargues (1949), who were able to restore some of the growth-promot-
ing activity for chick osteoblasts by simple dilution of the dialyzed
medium with Tyrode solution. This effect of dilution has been confirmed by
Barski et al., (1951), who also questioned whether biological components
could ever be treated as inert basal media. They reviewed some of the
earlier work on dialysis, ultrafiltration, and ultracentrifugation of tissue
culture media, and added some valuable new observations. It had already
been shown by Fischer (1941a) that epithelium could survive in an un-
supplemented dialyzed medium ; from Fischer’s laboratory it has now
been demonstrated (Landschutz, 1952) that a dialyzed medium can
permit a significant amount of outgrowth of fibroblasts, provided that
sufficiently large pieces of tissue are used. Cultures which were halved
produced only a minimal amount of outgrowth, whereas undivided cul-
tures in a medium of the same composition increased markedly in area.
In a careful examination of the properties of dialyzed media, Harris
(1951a, b ; 1952a, b) showed tha& an even greater degree of restoration
of ability to support growth could be achieved by adjustment of the
bicarbonate concentration. His dialysis was more thorough than Fischer’s
(eg., his dialyzing fluid was changed daily for eight days), and he
found that media so treated were entirely unable to support any outgrowth
of fibroblasts (Harris, 1952a). Media dialyzed without change of fluid,
as in Fischer’s experiments, could produce a small outgrowth of cells.
Growth-stimulating activity could be restored by adding, to the dialyzed
plasma and dialyzed embryo extract, dialyzate from the chick embryo
extract. Harris made the important observation that there is a marked
fall in p H during dialysis against unbuffered Ringer-glucose solution and
pointed out that this could not be rectified by treating the medium with a
C02-containing gas mixture. Adjustment of the pH to 7.4-7.6 with
12 CHARITY WAYMOUTH

NaHC03 or Na&Os enabled the dialyzed mixture to support sustained

outgrowth. That the effect was not due solely to restoration of the correct
pH, but to compensation for a bicarbonate deficiency, was shown by the
failure of a dialyzed medium neutralized with NaOH to show the same
activity. I t may be recalled that Warburg, Posener, and Negelein (1924)
had demonstrated, for neoplastic cells, that glycolytic activity depends not
only on the presence of a suitable carbohydrate source, but also on the
presence of bicarbonate (optimum concentration 2.5 mM.). Adjustment
of the p H of the dialyzed embryo extract with carbonate has also been
adopted by Landschiitz (1952). This single adjustment seems to restore
conditions in which low molecular materials become available to the
cells. Tissue cultures can obtain enough sugar by the hydrolysis of
polysaccharides to enable them to proliferate in a bicarbonate-adjusted
dialyzed medium for several months (Harris, 1952a) (contrast four to
five days in most of Fischer’s experiments). Sufficient phosphate is also
available in the dialyzed medium ; supplementary phosphate at 1 mM. was
found by Harris to confer no advantage and 3 mM. phosphate were
inhibitory. Besides the inorganic phosphate of the Gey’s fluid (containing
1 mM. phosphate) used as a component of the medium, most of the
phosphate used probably derives from breakdown of phospholipids, which
can take place in the medium at 37” C. (Trowell, 1952). Hass, Schweitzer,
and Boscia (1950, 1951), measuring radial outgrowth of chick lung
fibroblasts in a medium containing guinea pig plasma, reported growth-
promoting activity in the part of embryo extract (dialyzed against water)
which was non-diff usible and water-insoluble. The non-diffusible and
water-soluble fraction was inhibitory. The active water-insoluble fraction,
which could be dissolved in Tyrode solution, could be further fractionated
by precipitation with acetone to give a fraction which was calculated
to induce division of fibroblasts twice in 24 hours. The material was heat
labile (Maganini, Schweitzer, and Hass, 1953) and a great deal of the
protein could be discarded without loss of activity. No active fraction was
obtained entirely free from protein. A strong absorption at 2600 A. and
the presence of phosphorus were characteristics of all fractions retaining
activity. Harris ( 1952b) found a non-protein, partly heat-stable, acid-
labile material, which could correct a deficiency in unsupplemented
dialyzed medium, in a dialyzate from an alcoholic extract of 12-day
chick embryos.
The effect reported by Simms and Stillman (1937), namely that serum
ultrafiltrate overcomes the “dormancy” of adult tissue, in the sense that
outgrowth occurs sooner from adult tissue incubated in serum or serum
ultrafiltrate before planting in a plasma medium than from tissue
 THE NUTRITION OF ANIMAL CELLS 13

previously incubated in Tyrode solution, indicates a protective effect of

the ultrafiltrate compared with the Tyrode solution. Gey and Gey (1936)
have drawn attention to the fact that “. . . prolonged washing in saline
has a definite deleterious effect on fresh tissue.”
Human placental cord serum has been commonly used in tissue culture
media since attention was first drawn to its efficacy by Gey (1929) (cf.
Cey and Gey, 1936) and to that of pregnancy serum by Pybus and Fawns
(1931). Jacquez and Barry (1951) have studied human cord serum by
dialysis against Simms’ X6 balanced saline-glucose solution. This solu-
tion contains bicarbonate and 1.5 mM. phosphate. They found that embryo
extract actively stimulated cell migration in rat fibroblast cultures but
could not alone cause much increase in the volume of tissue, whereas Serum
alone promoted an active increase in culture density, though this was some-
what less than in the complete medium of serum and embryo extract. This
activity of the serum was traced entirely to the non-dialyzable part.
Further fractionation showed that the euglobulin fraction contained most
or all of the activity. Serum albumin had no growth-promoting effect, but
exercised a detoxifying action, probably by adsorption of fatty acids and
other injurious substances (e.g., heavy metal ions). Barski et d . (1951)
also clearly demonstrated by dialysis, ukrafiltration, and ultracentrifuga-
tion that the main factor, the loss of which results in reduction of growth-
promoting and maintenance effects in an embryo extract-plasma medium,
was to be found in the high molecular part of the plasma. Hoffman,
Dingwall, and Andrus (1951) found that the supernatant fluids from
ultracentrifugation of adult sheep heart extracts retained most of their
growth-promoting activity. Wolken (1952) also found activity in the
supernatant fluid after 87% of the total solid material had been brought
down by ultracentrifugation of chick embryo extract; but in his experi-
ments there was no significant difference in effect on the growth of chick
fibroblasts between this supernatant, the remaining pellet, an acetone
supernatant, or an acetone precipitate from a low speed fractionation.
Kutsky and Harris (1952) also found the supernatant active and the
resuspended pellet less active than the whole extract.
Sanford et d. (1952) have made a study by ultrafiltration of the
medium, containing horse serum and chick embryo extract, in which it
has been possible to grow strains of mouse cells in continuous culture for
a period of years. More active fractions of embryo extract and serum
could be obtained by ultrafiltration than by dialysis against water or saline.
The effects of ultrafiltered materials on strain L mouse cells again empha-
sizes the importance of the high molecular fraction of the serum and of
the filtrate of the embryo extract. The protein residue of horse serum
14 CHARITY WAYMOUTH

promoted cell multiplication more than did the ultrafiltrate ; the ultrafiltrate
of chick embryo extract increased the proliferation rate much more than
the non-filtrable part. High molecular complexes corresponding to Fischer’s
“embryonin’” could therefore be eliminated from the medium for the
growth of strain L cells without affecting the growth rate. This is an
important simplification of the classic biological nutrients. Horse serum,
embryo extract and their ultrafiltrates, prepared in Earle’s laboratory,
have also been tested in a similar way on strain 14pf of normal rat fibro-
blasts by Ehrmann and Gey (1953). For these cells, it appeared that
horse serum contains a non-ultrafiltrable inhibitory material. Ehrmann
and Gey confirmed, with their strain of cells, the nutritive effect of chick
embryo extract ultrafiltrate, and found that growth was better in a medium
containing this nutrient and human placental cord serum than in the
embryo extract ultrafiltrate and horse serum. However, the best growth
was still obtained with whole embryo extract and whole human cord serum.
These cells seem to need more from the embryo extract, for maximum
growth, than is provided by the ultrafiltrate. A lyophilized ultrafiltrate
of embryo extract was extracted with 70% ethanol and passed through
an ion exchange column by Rosenberg and Kirk (1953). The d u e n t
contained about 3% of the N of the original ultrafiltrate and was as
active as the untreated ultrafiltrate as a supplement to thoroughly dialyzed
embryo extract, for the growth of chick embryo fibroblasts.
Insofar as any conclusion can be drawn from the analyses of biological
media, it is that we have so.far learned from them little of the nutritional
needs of any tissue for particular metabolites. Many low molecular
components, e.g., coenzymes, vitamins, hormones, and even metallic ions,
are, in nature, bound to proteins ; and cells are endowed with a variety of
enzyme systems capable of acting on substrates of high molecular weight.
It is not, therefore, altogether surprising that the capacity of cells to avail
themselves of high molecular components, or substances associated with
them, is evidently, as Harris (1952a) has shown, very great. On the
other hand, several of the nutrients of low molecular weight are un-
doubtedly equally well utilized in the free state, a fact which encourages
the hope that a complete nutrient medium of known chemical composition
and equal nutritive value with the classic biological media will in due
course be devised.
111. SYNTHETIC
MEDIA
Because of the general belief engendered by Carrel, and referred to
above, that plasma acts largely as a supporting structure and serum as
some kind of buffer against accumulation of toxic products, the early
attempts to devise media of known composition were directed to
 THE NUTRITION OF ANIMAL CELLS 15

the design of synthetic mixtures to replace the embryo extract. Plasma

o r serum were retained. From the supplementation of biological media in
this way it early became clear that “the effect of adding many biologically
important substances . . . such as vitamines, iron salts and oxides,
cysteine, carbohydrates, cholesterol etc.” to a basic medium of plasma,
glycine, nucleic acid and protein digest “had no beneficial action under
the conditions of the experiments” (Baker, 1929). Baker adopted the
view, later expressed by Morgan, Morton, and Parker (1950) in their
work with fully synthetic media that “even though no evidence w a s
obtained of the nutritive value of these substances, the negative results
must not be accepted as final, for it is conceivable that any one of them
might contribute to the functional requirements of the cells, but that its
effect could not be observed in experiments on growth when some other
substance necessary for complete nutrition was absent” (Baker, 1929).
I n 1933, Vogelaar and Erlichman designed a feeding solution (Table I)
for use with serum in growing human fibroblasts. This medium contained
Witte’s peptone, which had already been found by Baker and Carrel
( 1926a) ; Carrel, Baker, and Ebeling (1927), and by Fischer and Demuth
(1927-1928) to have stimulatory effects on tissue cultures. Later (Voge-
laar and Erlichman, 1938) a more fully defined feeding solution was de-
scribed containing glycine in place of the peptone (Table I). Baker ( 193541,
1936), following closely the composition of Vogelaar’s medium, but
adding ascorbic acid, cysteine, glutathione and vitamin A, introduced
two serum-containing nutrient solutions (Table I), one for fibroblasts and
epithelial cells and the other for monocytes. Chick fibroblasts in a horse
plasma coagulum proliferated two or three times as rapidly in Baker’s
medium as in Vogelaar’s first (1933) medium and, whereas in the latter
solution the survival time was only 12 to 14 days, in Baker’s medium the
cultures were maintained in active proliferation for over six weeks. The
medium for monocytes differed chiefly from that for fibroblasts in the
concentrations of the components, containing smaller amounts of peptone
and salts, more glucose, and additional B vitamins. Monocytes proliferated
in this medium for 80 days. Various elaborations on these mixtures were
made, and one solution (Table I) was devised by Baker and Ebeling
(1939) for use as a maintenance medium, without serum. This was not
fully defined, as it contained peptone and a digest of whole blood. Fibro-
blast cultures were kept in this medium for 30 to 43 days in the absence
of serum. A slight modification of Baker’s (1936) medium was used by
Wilson, Jackson, and Brues (1942) (Table I) for the growth of chick
embryo tissues in a thin plasma clot but without added serum. A com-
parison was made in this solution of the growth and nitrogen utilization
16 CHARITY WAYMOUTH
Is I
h
g$g
000
I I I I I?
000000
0s
0s
OS
0's

0 0 0
09
0'21

Id Id u+u+u+Gl6~
M
a 21
PI
pr)
2 II
I I I I I I I I I I I I I I I I ~ 1 1 1 1 1
121 I I I S
121 I I I2 8
"I1 ~

I I I I I I
4
I IXI I I l 3 Ih
ZI I I I I I I I
1121 I I I 2 I g I I I I I I I I
CI
131 l N2
W Y I I I I I I ;I I I I I
I I I 1181 I Z I 1 I I I I I I I
 L-Leucine 15.6 15.6 -
m-Isoleucine 10.4 10.4 4.0
DL-Aspartic acid - - 6.0
as as par tic acid - 6.0 -
DL-Glutamic acid - - 15.0
L-Glutamic acid - 14.0 -
L-Arginine 7.8 7.8 7.0
L-Histidine 2.6 2.6 2.0
DL-Methionhe 13.0 13.0 3 .O
DL-Phenylalanine 5.0 5.0 5.0
L-Cystine - 1.5 2.0
m-Tryptophan 4.0 - 2.0
L-Tryptophan - 4.0 -
L-Tyrosine - - 4.0
L-Proline - 5.0 4.0
L-Hydroxyproline - 1.o
- -
I

L-Glutamine 10.0
Cysteine-HC1 9.0 1.125 0.10 0.10 0.01
Glutathione 1.0 0.34 0.10 0.10 0.005
Ascorbic acid 0.25 0.085 0.05 0.05 0.005
Carotene - 0.01 0.01 -
Vitamin A 900 to 18oou. 0.01 0.01 0.01
Calciferol (vitamin D) - - - 0.01
Menadione (vitamin K) - - 0.001
a-Tocopherd phosphate - - 0.001
Thiamine 0.0053U. o.Ooo1 0.01 0.01 0.001
Riboflavin 0.0001u. 0.0034 0.01 0.01 0.001
hrridoxine 0.05 0.05 0.0025
- - 0.0025
0.05 0.05 0.0025
- - 0.0025
Calcium pantothenate 0.01 0.01 0.001
Biotin 0.01 0.04 0.001
Folic acid 0.0001 0.005 0.001
Inositol 0.05 0.05 0.005
p-Alanine - 0.05 -
Choline 0.5 0.1 0.05
 Feeding Solution for
Human Fibroblasts.
ooi I I 1 I I I 1 1 I I I I 1 1 I 1 1 12 Vogelaar and
Erlichman (1933).

+
c Glycine-Containing
ggr I I I I I I I I I I 1 I I I I I I 121 p&saqt;tion.
.PI-
00 Erlichman (1938).

Serum-Containing
881 I I I I I I I I I I I I I I I I I l o51 E:$;s:osr:::Wthof
Epithelium.
aW
88 Baker (1936).

Serum-Containing
991 I I II I I II I II I I II I I I0I g:z$teiSs' Growth Of
Baker (1936).
mcl
1 -
Modified Baker's
I I I II I I I I I I I IIII I I I I g
lo1 ~F::,oafYE:,;
(1942).

(1939).

upplemented Dialysed
I l l I I I I I I I I I I I I I I I I I @I Plasma for Growth of
Chick Fibroblasts.
Fischer (19484.
 THE NUTRITION OF ANIMAL CELLS 19
I I I I 1 1 1 1 1
I I I I "1111
5
20 CHARITY WAYMOUTH

of chick embryo muscle tissue in the presence of (1) Witte’s peptone and
(2) peptone which had been hydrolyzed with acid to amino acids, and
supplemented with tryptophan. There were some morphological differences
between the cells in the two variants. Nitrogen was well utilized from
the amino acid-containing medium and mitotic rates over a period of eight
days were similar to those in the unhydrolyzed peptone medium.
Undeterred by the tenacity with which tissue culturists retained an
unquestioning faith in the Components of the classic nutrients and in high
molecular “growth-promoting factors” of the “trephone” or “embryonin”
type, and encouraged by notable success in devising a fully synthetic
nutrient for plant tissues, White (1946) tackled the problem of designing,
from current knowledge of the certain and probable requirements of
animal cells, a synthetic nutrient medium. This medium (Table I) con-
tained a variant of the mixture of amino acids found by Rose (1938) 10
be able to maintain nitrogen equilibrium in the rat; a high glucose con-
centration, a salt mixture similar to the conventional balanced salt solutions
but containing a ferric salt, and a number of vitamins. Not all of the
individual components were shown to be essential, and indeed to check the
absolute and relative concentrations of all the components would be a
very formidable task. The results with this mixture were so promising that
it warranted much further study and attempts to improve upon it. With
the original mixture, chick embryo tissue cultivated in roller tubes, directly
on the glass without clot or other source of high molecular or unknown
material, could be maintained for about 50 to 60 days. The addition of
five more amino acids, and minor changes in some of the other components,
resulted in a mixture (White, 1949) (Table I ) which could maintain
chick embryo cells for up to 80 days, with several changes of the nutrient
fluid. By contrast, cells of the same kinds in a balanced salt-glucose solu-
tion will survive about eight to ten days. The original medium of White
(1945) was not able, alone, to support the life and growth of fowl
niacrophages (Jacoby and Darke, 1948), which can proliferate in-
definitely in fowl serun1,diluted with Tyrode solution (1 : l ) . However,
10 to 2O:h of serum in 90 or 80% of the synthetic medium formed a
mixture in which these cells could multiply actively and be iriaintained ior
up to two months. Stuermer and Stein (1950) reported that when stromal
cells of the human decidua were cultivated in a biological medium (human
placental cord serum, fowl plasma, and chick embryo extract), fibroblasts
and epithelial cells grew out. In White’s (1946) synthetic medium, only
fibroblasts migrated; in seven to ten days the explants, originally 1 mni.,
had produced 3- to 4-mm. wide collars of outgrowth.
The second fully synthetic medium was described by Morgan, Morton,
 THE NUTRITION OF A N I M A L CELLS 21

and Parker (1950) (Table I). In their earlier experiments, these authors
started their cultures in a biological medium €or several days before subject-
ing them to the synthetic mixture. Later (Morton, Morgan, and Parker,
1951), this practice was discontinued. The medium no. 199 contains a
much larger number of components than White’s media, designedly, as
it “was felt that the preliminary media should include as many known
nutritional factors as possible, even though many of them had no apparent
effect.” (Morgan, Morton, and Parker, 1950). Under the best conditions
(renewal of medium at weekly intervals after an initial starting period of
three days in a biological medium of serum and embryo extract), cultures
of chick embryo muscle survived for an average period of fifty days, and
a maximum of seventy days. Without the initiaI starting period in a
non-synthetic medium, but with a seven-day starting period in a minimal
amount of the synthetic medium, the average survival time was thirty-
seven days.
The synthetic medium of White (1949), and that of Morgan, Morton,
and Parker (1950), were tested by Evans et ul. (1953) on replicate
cultures of mouse strain L cells. Whereas in the best biological media the
cell population increased twelve to sixteen fold in six days, and in Earle’s
salt-glucose solution the population fell to 1.5% or less of the initial value,
in White’s medium, renewed every two or three days, the final cell number
was 21 to 30% of the initial value. In Morgan, Morton, and Parker’s
(1950) medium the final cell number was 41 to 67% of the number of
cells inoculated, slightly higher counts being obtained with media freshly
prepared than with stored media. Both types of synthetic medium are
clearly unsatisfactory, as they stand, for this strain of cells and under this
regime. Though Evans et ul. discuss the possible role of adaptation of
the cells to media, no attempts were made in this investigation to permit
gradual adaptation of the strain L cells to the synthetic media.
In the sections which follow, evaluation of the nutritional role of the
various components of biological and synthetic media and their effects on
different cell types, will be attempted.
SUBSTANCES
IV. INORGANIC
The first attempts to maintain physiological function in isolated tissues
and organs were made at a time when the composition of the cell was
regarded as much more stable and constant than it is now known to be.
It was then thought that the provision of the necessary inorganic salts
in the proper proportions and in osmotic equivalence with the interstitial
fluids was all that was required. From these studies emerged the well-
known “physiological salt solutions,” with or without glucose, of Ringer
22 CHARITY WAYMOUTH

(1886), Locke (1895, 1901), Tyrode (1910) and others for homoeo-
and poikilotherm tissues (Table 11). The first studies aiming to establish
the optimum concentrations of various ions in fluids for tissue cultures
TABLE 11
SALTSOLUTIONS
BALANCED USEDIN TISSUECULTURE.
MEDIA
mg./100 ml.

u6
a
rd

b
u
NaCl 900 900 800 900 480 900 700 800 800 800 680 800 700
KCl 42 42 20 42 60 42 37 38 20 20 40 40 37.5
CaCll 25 24 20 20 15 12 17 13 20 11.1 20 20
_ - - - - - -
Ca(NO8),.HaO - - - - - - A.
LI
MgCla.6HsO - - 21.4 - 50 - 21 21 10 20.3 - - -
- - - - - - 70 - - - 20 20
MgSO4.7HsO
MgHPO4 - - - - - - - - - - -_ _ - - - - - 56.2
CaHs(P04)I - - - 10 10 - - -
NalHPOl - - - - - - 12 12 - 21.3 - 10 5.75
NaHIP04 - - - - - - - - 4.3% 10.8 - -
KHsPOI - - - - - - 3.0 2.5 - -
Fe(NOs)a.9H10 - - - - - - - - - - - -
10 - 2.6
0.13
NaHCO, - 30 100 20 - 53 227 25 - 101 220 34 55.0
Glucose -2100 100 - - -
100 100 100 100 100 200 850

were made by Lewis and Lewis (1911a). I n spite of their careful in-
vestigations, and the subsequent publication of descriptions of balanced
salt solutions specifically for use in tissue culture media by Drew (1922,
1923), Pannett and Compton (1924), Roffo (1925), Gey and Gey
(1936), Parker (1938), Simms and Sanders (1942), Earle (1943),
Hanks (1948, 1949), White (1949), and Osgood et d. (1951) (Table
II), the most generally used salt-glucose solution has, until quite recently,
been the formula of Tyrode (1910) (with or without modification, e.g.
by Willmer and Kendal, 1932), designed for quite another purpose.
As the pioneer experiments of Lewis and Lewis (1911a, b ; 1912)
demonstrated, survival of tissue cultures in a salt-glucose mixture alone
is very short. The inorganic requirements of different types of tissue may
be different. For example, Willmer (1927), using various salt-glucose
solutions as diluents in biological media, showed that when only NaCl, KCl,
CaC12, NaHC03, and glucose were used, outgrowth of chick fibroblasts
was optimum in 0.8% NaCl and 0.8% glucose; slightly hypotonic
 T H E NUTRITION OF ANIMAL CELLS 23

NaCl stimulated fibroblast migration; 0.9% NaCl was optimum for

chick epithelium. This contrasts with the fact that Parshley and Simms’
(1950) 216 solution for adult epithelium contains a lower salt concentra-
tion (0.68% NaCl) than their X6 solution for adult fibroblasts (0.8%
NaCl). The 216 solution moreover contains lower concentrations of
KCI and MgCI2 than the X6 solution, and no calcium or bicarbonate.
These investigators found that the growth of adult epithelium is stimulated
by a high phosphate concentration (Parshley and Simms, 1946, 1950) and
that, in contrast to adult fibroblasts, epithelium does not require a diluent
containing bicarbonate.
The concentrations of phosphate (usually in the form of a buffer) and
of bicarbonate provided in various solutions used in tissue culture media
have varied over a wide range. The amounts of phosphorus in the usual
salt solutions vary from Tyrode (0.36 mM.), through Pannett and
Compton’s (1924) solution (0.57 mM.), White’s (0.58 mM.), Earle’s
(0.9 mM.), Gey and Gey’s (1936) (1.0 mM.), Hanks’ (0.86 or
1.44 mM.), Drew’s (1.26 mM.), to Simms’ X6 solution for fibroblasts
(1.5 mM.). W r t h (1948) has used for virus culture a modification of
Tyrode’s solution containing six times the usual amount of phosphate,
i.e., 2.16 mM. Human pIasma inorganic phosphate (as P) is 2.6 to
5.4 mg./100 ml. (average, 3.2 mg./100 ml., i.e. 1 mM.) and the total
phosphate (as P ) is 10.0 to 14.1 mg./100 ml. (average 12.1 mg./100 ml.,
i.e. 3.9 mM.) (Stearns and Warweg, 1933, quoted by Krebs, 1950). For
monkey plasma, McKee et d. (1946) give an average phosphate of 2.1
mM. The inorganic P of fowl plasma is 2.45 to 4.25 mg./100 ml. (0.8
to 1.4 mM.), and forms most of the acid-soluble phosphorus fraction
(Davidson and Waymouth, 1946). Harris (1951a, b, 1952a) showed that,
in a dialyzed medium containing sources of bound phosphate, supplemen-
tary inorganic phosphate was not essential and that the addition of 3 mM.
phosphate was inhibitory. Phospholipid P is present in amounts of the
same order as inorganic P, i.e., 2.2 to 4.0 mg./100 ml. (Davidson and
Waymouth, 1946) in fowl plasma, and it is known that phospholipids can
break down on incubation at 37”C . and release inorganic P. The phos-
pholipid P of embryo extract, prepared by extraction of embryo pulp with
Tyrode solution, is 1.3 to 2.5 mg. P/100 ml. I t seems, therefore, that salt
solutions such as Tyrode’s or Pannett and Compton’s, low in phosphate,
should be satisfactory diluents for biological media rich in organic phos-
phate. As a basis for fully synthetic nutrient media, phosphate to a total
concentration of at least 2 mM. should be supplied, either entirely as in-
organic phosphate or partly as organic phosphorus compounds.
In short-term experiments, W. H. Lewis (1929) demonstrated that
24 CHARITY WAYMOUTH

NaHC03 is essential to the maintenance of heart beat in three-day chick

embryo heart in Locke’s solution (without sugar or phosphate, neither of
which is necessary to the maintenance of this function). KCl and CaC12
were also shown to be essential to maintain heartbeat. Lewis and Lewis
(1912), in experimental culture media with varied NaC1, KCl, CaC12, and
NaHC03 concentrations, commonly used NaHC03 at 20 mg./100 ml.
(2.38 mM.), and in no case did they employ more than 100 mg./100 ml.
The original Tyrode (1910) solution contained NaHC03 at 100 mg./100
ml. (11.9 mM.). A later modification of this solution for use in tissue
culture (Willmer and Kendal, 1932) contained half this amount of
bicarbonate. The solution of Adler (1909, quoted by Tyrode, 1910)
contained more phosphate (12.6 rng./loO ml. NaHZP04, 1.05 mM.)
and much more bicarbonate (351.0 mg./100 ml. NaHC09, 42 mM.)
than any of the other early salt solutions designed to imitate the ionic
composition of blood plasma. This solution does not seem to have been
much used in tissue culture work. Roffo (1925) obtained good growth
of cultures with 30 to 53 mg./100 ml. (3.37 to 6.32 mM.) NaHC03.
The original formula of Gey and Gey (1936) for a saline solution for
use in roller tubes contains 227 mg./100 ml. (27 mM.) NaHC03, but a
solution containing one-ninth of this was recommended for use with
slide cultures with their smaller gas phase. The lower concentration has
become generally adopted for both purposes (Gey, personal communica-
tion, 1953 ; Cameron, 1950; Parker, 1950). Simms and Sanders’ (1942)
solution X6 for adult fibroblasts contains 101 mg./100 ml. (12 mM.)
NaHC03, and Harris’ (1952b) F6 solution contains 140 mg.Jl00 ml.
(16.7 mM.). The salt solutions in common use that contain a high
bicarbonate concentration, near to that in human plasma, are Earle’s
(1943) saline, with 220 mg./100 ml. (26.2 mM.) and Dubin and Yen’s
( 1950) modified Krebs-Ringer-bicarbonatesolution for macrophages, with
210 mg./100 ml. (25 mM.). The average bicarbonate content of human
plasma or serum, as NaHC03 (Hald, 1933, 1947, quoted by Krebs,
1950) is 226 mg.Jl00 ml. (range 205 to 280 mg./100 mi.) and that for
monkey plasma (McKee et al., 1946) falls within the same range.
Bicarbonate or carbonate is essential for the outgrowth of fibroblasts, and
Harris (1952a) obtained in a medium with 200 mg./100 ml. an outgrowth
twice that with 100 mg./lOO ml., and standardized on 210 mg./100 ml.
(25 mM.).
The balanced salt solutions are generally used as diluents for biological
media such as plasma, serum, and tissue extracts, and they may form
up to 90% of the medium in some cases. Gey and Gey (1936) emphasized
the deleterious effect of prolonged contact with saline upon freshly
 THE NUTRITION OF ANIMAL CELLS 25

isolated tissue. Roffo (1925) made a study of the effects of varying the
proportions of the different cations, especially K and Ca, on the develop
ment of normal chick heart and of two tumors (a sarcoma and a carcinoma
of rat) in tissue cultures. In agreement with Carrel and Burrows (191Ib),
Lamhert (1914), Ebeling (1914), and Pannett and Compton (1924), he
found that hypotonic media, (e.g., plasma diluted to contain 0.51% NaCl)
are favorable €or the initial outgrowth of cells. H e prepared a series of
modified Ringer solutions (Table 111) and tested these as diluents for

TABLE 111
ROFFO’S (1925) MODIFIEDRINGERSOLUTIONS
mg./100 ml.

NaCl 900 900 900 900 900

KCI 42 21 42 0 58
CaCL 24 24 12 86 0
NaHCOl 30 53 53 30 30

fowl plasma in his media. Normal chick heart grew well in (3) and (5),
poorly in (2) and very poorly in the K-free solution (4). The fuso-
cellular sarcoma grew best in the low-Ca medium (5), well in (3) which
was better than normal Ringer ( I ) , and gave little or no growth in (2)
or (4). The rat carcinoma grew well in ( 3 ) and (5). These experiments
demonstrated the necessity for K and the relatively smaller importance
of Ca. According to Jazimirska-Krontowska (1930), the outgrowth and
sugar consumption of normal tissue were reduced by high concentrations
of Ca. A high concentration of K greatly reduced the area of the cultures,
but sugar consumption remained high. Brues et al. (1940) found that
tissue cultures could tolerate high concentrations of K, up to 400 mg./100
ml. Lymphocytes seem able to withstand similarly high concentrations of
K (greater than 300 mg./100 ml., Trowell, 1953). Parshley and Simms
(1950) indicate that 1 mM. Mg and a very low Ca are optimal for adult
epithelial cultures. After depleting the medium of free cations, Shooter and
Gey (1952) found, by supplementation, that Ca, Mg and K were all
essential for the growth of a strain of normal rat fibroblasts. Either
Ca or Mg alone, in a Na- and K-containing medium, restored outgrowth
to some extent; both were necessary for even 24 hours’ continued growth.
Restoration of cations was made by means of solutions of Ca(NOa)a,
K H 2 P 0 4 , MgS04, and CuSO4. A trace-metal solution containing Fe,
Zn, Mn, and Co (as sulfates) and ammonium molybdate, improved
survival in Shooter and Gey’s experiments over the use of the Ca-, Mg-,
K-, and Cu-containing supplement alone.
26 CHARITY WAYMOUTH

The use of trace metals in media for tissue cultures has been studied
only sporadically, and the effects of anions have been even more thoroughly
neglected. Lewis and Lewis (1912) incorporated traces of ferric oxide
into a medium for the cultivation of sympathetic nerve. The tolerance
of chick connective tissue towards CuS04 and NaAsOs were studied by
Wilson (1922). H e found that 1.G mg. Cu,/lOO ml. were toxic, while
0.8 mg. Cu/loO ml. was tolerated. These concentrations are much
greater than the range used by Roffo and Calcagno (1928) ; they found
0.005 mg. Cu/lOO ml. to be toxic and 0.002 mg.Jl00 ml. to give good
growth. For human fibroblasts, Vogelaar and Erlichman (1934) found an
optimum Cu concentration (0.75 mg./lOo ml.) in the range indicated by
Wilson. Concentrations of 0.37 and 0.56 mg. Cu/100 ml. were less favor-
able; 1.12 mg. Cu/lOO ml. was distinctly toxic. Shooter and Gey (1952)
include 0.1 mg. Cu/lOo ml. in their medium. Uei (1926) reported that
small amounts of Fe and Mg increased the growth of a rat sarcoma in
vitro: Zn and Cu were inhibitory. Vogelaar and Erlichman (1933), and
following them Baker (1936), included hemin in their media. As a source
of iron, the amounts they used (0.oooO55 mM.) would probably not add
significantly to that present in other components of their media. Ehrmann
and Gey (1953) found that hemoglobin had a distinct stimulatory effect
on the outgrowth of rat fibroblasts in a 25% serum medium; they used
200 mg./100 ml., which would provide about 0.12 mM. of iron. Iron
was supplied as Fe(NOs)s by White (1946, 1949) in his synthetic
media, at 0.003 mM. The effects of ferric, cupric, and manganese chlorides
on the survival times of chick heart cultures in biological media were studied
by Hetherington and Shipp (1935). Ferric chloride (FeCl&H20) at
100 mg.Jl00 ml. (3.7 mM.) prolonged the life of the cultures for eight
days longer than the controls (33 days) ; MnC12.4Hz0 at 1.0 mg./lOO ml.
prolonged survival by two days, and CuC12.2H20 at 1.0 mg.Jl00 ml.
deferred the peak death rate by five days. Small amounts (of the order
of 0.005 mM. or less) of the chlorides of Fe, Mn, Cu, Zn, and Co were
included by Fischer st al. (1948) in their synthetic supplementary medium
V-605. Morgan, Morton, and Parker (1951) found that small quantities
of Co were toxic in biological media but that larger amounts could be
tolerated in synthetic media. Histidine and purines form complexes
with Co, and histidine and some other components of the synthetic
medium no. 199 (Morgan, Morton, and Parker, 1950), probably the
purines, exert a protective effect. The amounts of Zn, Pb, Cu, Fe, Al,
Co, and Mn in various biological and synthetic media have been studied by
Healy, Morgan, and Parker (1952) as a “basis for further studies on
the mineral requirements of animal cells in tissue culture.” Several trace
 THE NUTRITION OF A N I M A L CELLS 27

metals are found in human and chicken plasma in amounts greater than
0.1 mg.Jl00 ml., e.g., Zn (Vikbladh, 1950, 1951; Healy, Morgan, and
Parker, 1952) and F e (Healy, Morgan, and Parker, 1952). These and
others which are present in smaller amounts deserve closer study to
determine their effects in tissue nutrition.
Most of the balanced salt solutions contain as anions only chloride,
phosphate, bicarbonate, and sometimes sulfate. Vogelaar and Erlichman
(1939) found that 80% of the chloride in the medium for human thyroid
fibroblasts could be replaced by iodide. Nitrate is included by White
(1949) in his synthetic medium. As the nutritional needs of cells
become more clearly defined, the role of the various anions will have to
be investigated.

V. CARBOHYDRATES AND OXYGEN

The majority of the balanced salt solutions used in preparing and dilu-
ting tissue culture media have been fortified with glucose. The principal
exceptions are Ringer’s (1886) solution, which was not designed for tissue
culture but has often been used as a diluent ; and Drew’s (1922, 1923) and
Pannett and Compton’s (1924) solutions, which were developed for use
in tissue culture media. By far the most commonly used formula has been
that of Tyrode (1910). This solution was designed for use in pharma-
cological experiments on surviving mammalian intestine and contains 100
mg./100 ml. glucose, which is close to the average normal mammalian
blood sugar level. Other salt-glucose solutions (Locke, 1901; Simms and
Sanders, 1942; Gey and Gey, 1936; and Earle, 1943) also contain this
amount, though a modification of Gey’s solution (Cameron, 1950) con-
tains 200 mg./100 ml. Hanks’ solution (1948, 1949) originally described
with 200 mg./100 ml. has sometimes been used with 100 mg./100 ml.
(e.g., Weller and Enders, 1948) and, for prolonged maintenance, with
400 mg./100 ml. (Hanks, 1948). Willmer (1942) drew attention to the
higher glucose content of bird compared with mammalian blood (cf. also
Wright, 1928; Hill, Corkill, and Parkes, 1934, and Erlenbach, 1938), and
modified the formula for Tyrode solution used in studying carbohydrate
metabolism in chick tissue cultures to include 200 mg./100 ml. glucose.
Studies on the effects of various concentrations of glucose on cultures
of connective tissue were made by M. R. Lewis (1921, 1922). She found,
as did Wilson, Jackson, and Brues (1942), that in the complete absence
of glucose, cultures of embryonic connective tissue degenerated and died
within two to three days. I n 0.25% glucose, the formation of vacuoles
within the cells was delayed, but survival was increased by only a few
days. Still higher concentrations (0.5, 0.75, and 1.0%) permitted two to
28 CHARITY WAYMOUTH

four weeks’ survival. In 2.0 to 5.0% the cells degenerated rapidly on ac-
count of acid formation. Lens epithelium in tissue culture was found by
Kirby, Estey, and Wiener (1933) to tolerate 0.478% but to be inhibited
by 0.578% glucose. Carrel (quoted by Ebeling, 1936) found “no inter-
ference with tissue growth” at 0.3% glucose. According to Willmer
(1927) and Demuth (1931), there is a direct relation between the amount
of fibroblast migration and glucose concentration up to 1.0% Ebeling
(1936) tested the effect of several concentrations on cultures of fibro-
blasts, leucocytes and iris epithelium. For all these tissues, the optimum
results were obtained within the range 0.39 to 1.15% glucose. Concentra-
tions above 2.0% were in general inhibitory to growth or productive of
cell granularity. Latta and Bucholz (1939) reported that 1 to 2%
glucose inhibited, and 5% stopped, fibroblast growth in vitro without
affecting embryonic muscle migration. Friedheim and Roukhelman ( 1930)
claimed that fibroblasts could grow in glucose up to 7.5%.
It is evident that cells in tissue culture can tolerate, at least for a short
time, concentrations of glucose much higher than physiological. However,
high concentrations of the order found optimum by Ebeling (1936) have
not often been used. Lewis and Nettleship (1932-1933) found a concentra-
tion of 500 mg./100 ml. glucose beneficial in a medium (Kendall’s medium)
composed of an extract of hog intestine in a buffered saline solution.
Kirk (e.g., Signorotti, Hull, and Kirk, 1950; Boyer and Kirk, 1952; and
Stewart and Kirk, 1952) has recently adopted a Tyrode solution contain-
ing 400 mg./100 ml. glucose (and NaCl reduced to 770 mg./100 ml.)
as a component of biological media for the study of the quantitative aspects
of growth in tissue culture, and Burt’s (1943b) medium for spinal ganglia
also contains this amount. Wilson, Jackson, and Brues (1942) found
that a high mitotic rate was slightly longer maintained with 500 mg./lOO
ml. than with 100 mg./100 ml. glucose, in cultures of mixed chick embryo
tissue. There was, however, no increase in the mitotic rate, and cultures
in which all mitoses were blocked by colchicine continued to use glucose
at an unaltered rate. The relationship of carbohydrate concentration to
the onset of mitosis in adult epithelium, in Vivo and in Vitro, has been
examined by Medawar (1947, 1948a, b) and by Bullough (1949, 1950,
1952; Bullough and Johnson, 1951a, b, c), Medawar’s (1948a) medium
for adult skin contained 500 mg./100 ml. glucose. H e used, in some cases,
a Krebs-Ringer-bicarbonate extract of adult tissue, supplemented with
glucose to this relatively high level. B’ullough and Johnson’s (1951b)
solution incorporated 400 mg./100 ml. glucose.
The requirements of different tissues for carbohydrate may differ in
amount and also in kind. Baker’s (1936) serum-containing feeding solu-
 THE NUTRITION OF ANIMAL CELLS 29

tion for fibroblasts and epithelium contained 100 mg./100 ml. glucose;
the corresponding medium for monocytes contained twice this amount.
A later formula (Baker and Ebeling, 1939) for fibroblasts contained,
however, 300 mg./100 ml. Fischer’s (1918a) “basic nutrient” and me-
dium V-605 (Fischer et al., 1948) (Table IV) contain a total of 100
mg./100 ml. sugars, made up of 80 mg. glucose, 10 mg. mannose and 10
mg. galactose. The simpler formulas V-612 and V-614 (Table V ) contain
200 mg./100 ml. glucose only. Astrup, Fischer, and $%lenschlager (1947)
found, in agreement with Lewis (1921, 1922) that cells were unable to
TABLE I V
MIXTURESOF ACCESSORY
FISCHER’S GROWTHSUBSTANCES
(1 Medium V-605 (Fischer el aL, 1948)
(2{ “Basic nutrient” (Fischer, 1948a)
~

rng./100ml. mg./100 ml.

NaCl 750.0 Cozymase 0.5
KCI 20.0 Thiamine 0.3
CaCL 20.0 Riboflavin 0.02
MgCL 10.0 Pyridoxine 0.03
Na,HPOI 5.0 Pantothenate 0.007
NaHCO, 100.0 Biotin 0.0007
FeCl, 0.06 p-Aminobenzoic acid 0.1
CUCll 0.02 Choline-HC1 1.0
MnCL 0.03 Nicotinic acid 0.03
ZnCL 0.10 Creatine 1.0
CoClr 0.001 Hypoxanthine 10.0
Glucose 80.0 Glutathione 0.5
Mannose 10.0 Ascorbic acid 0.2
Galactose 10.0 Methyl naphthohydro-
Tnositol 2.0 quinone sulfate 0.0005
Sodium succinate 1.0 Adenosine triphosphate 20.0
Sodium fumarate 1.o Fructose diphosphate 10.0
Sodium malate 1.o 8-Glyccrophosphate 10.0
Sodium oxaloacetate 1.o Inosinic acid 3.0
(1) (2)
Medium V-605 “Basic nutrient”
mg./100 ml. mg./100 rnl.
DbThreonine 1.2 -
DL-Valine 1.4 -
>Leucine 0.9 -
DL-Isoleucine 1.0 -
L-Aspartic acid - 0.59
L-Glutamic acid - 1.41
~-Lysine-2HCI 1.5 1.51
L-Arginine-HC1 0.2 0.77
L-Histidine-HCI 0.5 0.31
DL-Methionine 0.6 0.86
DL-Phenylalanine 0.7 -
L-Cystine 0.5 0.15
L-Tryutophan 0.2 0.50
t-P<oiind - -
Glutamine 25.0
30 CHARITY WAYMOUTH

TABLE V
MEDIAV-612
FISCHER’S AND V-614
mg./100 rnl.
NaCl 750.0
KCI 20.0
CaCI, z0.0
MgClt 10.0
NatHP04 5.0
NaHCO, 100.0
Glucose 200.0
DL-Threonine 2.4
m-Valine 2.8
DL-Phenylalanine 1.4
L-Leucine i .8
DL-Isoleucine 2.0
L-Lysine-2HC1 3.0
L-Arginine-HC1 0.4
~-Histidine-HCI i .o
L-Cystine 1.o
L-Tryptophan 0.4
L-Glutamine 25.0
Glutathione 1.0
Fructose diphosphate 20.0
p-Glycerophosphate 20.0 Omitted in
Inosinic acid 6.0) V-614
- ~~ - ~-
(Fischer et al., 1948)

survive in sugar-free media, but they reported that fructose or mannose

could replace glucose ; that galactose and maltose showed some activity ;
but that sucrose, lactose, xylose, arabinose, liver glycogen, soluble starch,
p-glycerophosphate, pyruvate, lactate, and glucosaniine were ineffective
substitutes. Lewis and Lawler (1931) had, under different conditions
from those of Astrup ef aE., examined the effect of starch on tissue cultures.
In their experiments, chick embryo skin cells multiplied and grew abun-
dantly for two to three weeks in a Locke-bouillon-glucose medium contain-
ing 0.5% glucose. Without glucose, the cells died in three to four days.
When starch was substituted for glucose, the cells grew at first and
survived for seven to ten days. Maltose had already (Lewis and Lewis,
1911a) been shown to be usable as a substitute for glucose in a simple
medium containing only saits, amino acids, and polypeptides.
In experiments which are relevant to the strictly tissue-culture studies,
Warburg, Posener, and Negelein (1924) showed that glucose and man-
nose were readily, and fructose and galactose less readily, used as carbo-
hydrate sources for glycolysis by tumor cells. Spratt (1949, 1950) com-
pared a number of carbohydrates for their effects in permitting develop-
ment of early chick embryos in vitro on a basal medium of salts and agar.
Mannose and glucose were equally effective ; fructose, galactose and mal-
 THE NUTRITION OF ANIMAL CELLS 31

tose were utilizable, but progressively greater concentrations were required.

On a molar basis, the relative efficiencies were (glucose 100) : mannose
100; fructose 40; galactose 10, and maltose 10. Pyruvate and lactate
(110 to 440 mg./lOO ml.) could also be used (Spratt, 1950). It is inter-
esting to compare, also, these studies with those of Elman and Weichsel-
baum (1952) on the utilization of fructose as a source of energy for
protein synthesis during intravenous alimentation. Both glucose and
fructose are effective as sources of calories, but fructose enters the cell
more rapidly than does glucose. T o quote Elman and Weichselbaum:
“fructose is better than glucose for protein synthesis from infused amino
acids, which are often given . . . as a source of protein food. The reason
is that after intravenous infusion amino acids enter the cell just as readily
as fructose, both being metabolized together, the one for energy, the other
for protein synthesis. By contrast, amino acids given with glucose tend to
be used for caIories rather than protein synthesis, because energy needs
are given first priority and the infused glucose at this time is still outside
the cell.” Worzniak (1952) briefly reported the effects of various carbo-
hydrates on explanted cultures of chick heart, muscle, gut, and liver in a
medium composed otherwise only of Tyrode solution. H e found that, of
the three hexoses tested, mannose was superior to glucose or fructose for
maintaining the life of the cultures, and that proliferation of both fibro-
blasts and epithelium took place. Glucose, and still more so fructose,
favored epithelium rather than fibroblasts. Glycogen, lactate, dihydroxy-
acetone, pyruvic aldehyde, and glucosamine failed to support proliferation,
though heart beat persisted for many days in the presence of lactate.
Phosphorylated hexoses were not superior to mannose, but were utilized.
The triose intermediates, with the exception of dihydroxyacetone, and the
Krebs’ cycle group of substances, supported growth almost as well as
glucose. Pyruvate was able to support fibroblast proliferation, but there
was little or no epithelial growth, and viability was diminished. Snellman
(1937) substituted fructose or galactose for glucose in media for cultures
of the Jensen rat sarcoma and found a decrease of 40 to 70% in lactic
acid production, but normal growth. Hanging-drop cultures of normal
chick tissues, in a Tyrode solution containing sodium lactate in place of
glucose, ‘‘showed cellular activity comparable to that shown by cultures
in ordinary Tyrode solution and far in excess of that shown by cultures
in Tyrode solution without either glucose or lactate” (Pomerat and
Willmer, 1939). This was confirmed by Wilson, Jackson, and Brues
(1942). The medium of Thomas and Borderioux (1948) for the culture
of organs of adult Urodeles contains calcium gluconate in place of glucose.
The number of alternative carbohydrate sources which have been men-
32 CHARITY WAYMOUTH

tioned makes it appear not particularly surprising that chick fibroblasts can
proliferate, in a medium composed of plasma and embryo extract, both of
which have been dialyzed against a sugar-free salt solution, without added
sugar (Harris, 1951b). Outgrowth can be increased not only by glucose
but by several other hexoses or by maltose o r glycogen. In all cases the
carbohydrates were depleted in the medium and lactic acid produced.
Salisbury (1947), from his experiments with tissue cultures of normal
and malignant cells, concluded that tumor cells were permeable to sucrose,
but that the normal cells examined were not. Heart beat can be maintained
in tissue in a state of reduced metabolic activity in a wholly synthetic
medium containing sucrose (optimum concentration 1.7%) instead of
glucose (White, 1946). The high glucose, concentration (0.8%) found
by Willmer (1927) to be best for outgrowth of fibroblasts in a medium
containing only salts (NaCl, KCI, CaClz and NaHC03) and glucose, and
the similar high concentration (0.85%) arrived at by White (1946) for
maximum survival in the first (and therefore not complete) synthetic
medium, support the view that the essential structure and functions of the
cells can be better maintained in nutritionally deficient media when high
concentrations of carbohydrate are available. In fully adequate nutrient
milieux, lower concentrations of carbohydrate (at, or slightly above,
“physiological” levels) are sufficient. It is of interest that, in the nutrition
of the protozoon Tetrahymenu, Kidder (1952) has found glucose to exert
a sparing action on amino acids, though no carbohydrate source is essential
for this organism (Manners and Ryley, 1952). It remains to be seen, in
tissue culture nutrition, how far amino acids can spare carbohydrate as
an energy source, and vice versa. It has been shown (Fischer, Fischer,
et al., 1953) that C14-labeledglucose, added to the classic biological medium
of plasma and embryo extract, is incorporated into the amino acids of the
protein of the growing embryo chick heart tissue.
There is, as Willmer (1941, 1942) showed, in general no correlation
between high growth rate and high glucose consumption or lactic acid
production. H e suggested that glucose consumption and lactic acid pro-
duction are associated with cell movement, which in turn is related to the
incidence of cell division. In tEe early stages of cultivation of chick tissues
in vitro, glucose utilization is high (Wilson, Jackson, and Brues, 1942),
starting at 2.6 mg./100 mg. wet tissue per day and falling, as the glucose
is depleted, to about 0.5 mg./100 mg./day on the third and fourth days.
Cultures on a schedule of daily renewal of medium, or replenishment of
glucose only, used a total of 22 mg. glucose per 100 mg. tissue in 11 days,
i.e., an average of 1.6 mg./day. Of this glucose, 60 to 70% appears as
lactic acid. The results of Willmer (1942) on the carbohydrate utilization
 T H E NUTRITION OF ANIMAL CELLS 33

of chick osteoblasts in media with or without embryo extract are in

general agreement with Wilson, Jackson, and Brues ( 1942). Glucose
consumption was high initially when embryo extract was provided. Both
Willmer and Wilson, Jackson and Brues concluded from the results on
their culture systems that some lactate may be formed from a source other
than glucose.
Many of the tissues which grow most satisfactorily in tissue culture
have a high capacity for anaerobic glycolysis. Early chick embryos can
metabolize glucose or mannose to lactic acid anaerobically (Needham and
Nowinski, 1937). Glycogen, disaccharides, and phosphorylated hexoses
are not attacked. Glycogcnolytic activity in embryo muscle develops after
the fifteenth day of incubation. There is, however, a wide variation in
the requirements of different tissues for oxygen. Medawar (1947) and
Bullough (1952) have shown that fragments of adult mammalian epider-
mis can survive in complete absence of oxygen for many days. For this
type of tissue, mitotic activity increases with increasing oxygen tension
over quite a wide range (Bullough and Johnson, 195la). This is a general,
but not a universal, characteristic of adult tissues (Parker, 1936a). Krebs’
cycle intermediates (glutamate, fumarate, or citrate), added to a saline
medium with an oxygen gas phase, increase the rate of oxygen consump-
tion and the rate of mitosis in adult epidermis by about 25 to 30% (Bul-
lough, 1952). Some of the literature on the oxygen requirements of
tissue cultures is reviewed by Hudspeth, Swann, and Pomerat (19.50).
Very young embryonic cells can withstand lower oxygen tensions than
older embryonic cells. Heart tissue from four- to five-day chick embryos
survived many hours in “pure” nitrogen (Burrows, 1921) and, after a
latent period of 10 to 24 hours, during a period of a few hours produced
a small amount of outgrowth. Mitosis was possible in tumor cells at
lower oxygen tensions than were required by chick embryo myoblasts
(Wright, 1928). Heart tissue from the ten-day chick embryo requires
1.8% oxygen (Burrows, loc. cit.) or 1.7% (Wright, loc. cit.) ; the fifteen-
day heart tissue requires 5.4% (Burrows). In accordance with this is
the finding of Danes and Leinfelder (1951), that the oxygen consumption
of seven-day chick heart tissue cultures could be reduced 16% without
affecting cellular activity. Lowering the oxygen consumption further
suppressed cell activity progressively, to the point of complete inhibition
when the oxygen consumption was reduced by 85%. Other studies of the
oxygen requirements of tissue cultures and of the relation between tissue
growth and oxygen tension have been made by Ephrussi *etaE. (1929) and
by Paulmann (1940). I n general, above the minimum requirements, mi-
gration of fibroblasts is favored by low and inhibited by high oxygen ten-
34 CHARITY WAYMOUTH

sion. The reverse is true for nerve fibers ; the character of the outgrowth
from explants of chick spinal cord varies with the oxygen concentration.
A high oxygen content (%% Oz, 4% COz) stimulated the outgrowth of
nerve fibers and suppressed connective tissue. Connective tissue cell
migration is stimulated by very low oxygen tensions (e.g. 2%), though
the total absence of oxygen prevents all outgrowth (Hudspeth, Swann, and
Pomerat, 1950). Very high concentrations of glucose (1 to 2%) sup-
pressed respiration and inhibited outgrowth. At physiological concentra-
tions and upward (e.g., 5.5 to 37 mM.), glucose inhibited respiration in the
Ehrlich mouse carcinoma (Brin, 1953 ; McKee and Lonberg-Holm, 1953) ;
only at low concentrations is oxygen consumption stimulated.
Leucocytes have a high oxygen consumption in vitro, though the meta-
bolic intensity is markedly influenced by the composition of the surrounding
medium (Hartman, 1952; Delaunay and Pag+s, 1946; Macleod and
Rhoads, 1939). Oxygen deficiency causes giant cell formation in tissue
cultures of lymph nodes (Barta, 1925, 1926). Trowel1 (1952) has dem-
onstrated the need for an abundant oxygen supply for the survival of
lymph nodes, which consume rather more than their own volume of oxy-
gen per hour. His cultures were maintained in an atmosphere of 100%
oxygen. Bone marrow also needs a high oxygen tension. Rosin and Rach-
milewitz (1948) found that 12% or less of oxygen was injurious to rabbit
bone marrow in vitro; the cells were kept in excellent condition by 50%
oxygen, diluted with nitrogen (no carbon dioxide). Parker ( 1936a),
keeping the COz uniform at 8% (in a medium containing a high bicar-
bonate content and 400 mg./100 ml. glucose), varied the 0 2 and Nz in
the gas phase for cultures of adult rabbit spleen in a fluid medium. The
cells were much better preserved in 80% oxygen than in 21% or 40%.
After four days in 2176 oxygen, there was marked necrosis and degenera-
tion.
VI. AMINOACIDSAND PEPTIDES
Supplementation of a simple saline medium with amino acids and pep-
tides was one of the early steps taken by Lewis and Lewis (1911a) towards
the understanding in chemical terms of tissue culture nutrition. With the
same aim of simplification, Smyth (1914) described a tissue culture
medium of agar and trypsinized peptone. The objective of Burrows and
Neymann (1917, 1918) was also explicitly to work toward a “synthetic
medium suitable for the growth of tissue cells outside of the animal
organism,” and they expressed the opinion, which is still valid, that “since
the preparation of such a medium would lead directly to a better under-
standing of cellular metabolism this problem has stood forth as one of the
most important of those presented by the tissue culture method.” It is
 T H E NUTRITION O F ANIMAL CELLS 35

unfortunate, therefore, that their categorical report that amino acids and
peptides were toxic probably had its influence in driving investigators
interested in the nutrition of cells back from the synthetic approach to
the analysis of biological media. Carrel, Baker, and Ebeling included in
their long series of studies of the effects of biological media and their com-
ponents on tissue growth, investigations of amino acids, peptides, and
protein digests. It was already apparent to Carrel in 1924 that “amino
acids under the same concentration as in the blood have no poisonous
effect on fibroblasts and epithelial cells in pure cultures, and that some of
them increase the rate of cell migration and multiplication” (Carrel,
1924a). Burrows and Neymann had used excessively high concentrations,
and their (1918) statement that “low dilutions” of amino acids stimulate
contraction of heart fragments seems to have been overlooked. Both Car-
rel (1924a) and Ebeling (1924) were of the opinion that the amino
acids were not used by the cells as a source of nitrogen, and that they
were effective only in promoting cell migration, but not cell multiplication.
Likewise, the addition of a mixture of sixteen amino acids to a dialyzed
embryo extract produced an increase in area of fibroblast cultures, but no
increase in the mass of tissue (Baker and Carrel, 1926e). Gerarde, Jones,
and Winnick (1952a) found that a mixture of nineteen amino acids in the
proportion found in bovine serum albumin, added at 10.0, 50.0, 100.0, or
500.0 mg./100 ml. to Tyrode solution, was not able to prevent autolysis in
cultures of chick lung, heart, o r intestine. In this case, however, the me-
dium was not designed to be nutritionally complete.
There have been many reports of the effectiveness of peptones (especial-
ly Witte’s peptone) in stimulating growth in tissue cultures. Baker
(1933), Baker and Carrel (1926a, 1928a), and Carrel and Baker (1926a,
b, c, 1927) found that a proteose prepared from fibrin actively promoted
cell proliferation and Guillery ( 1930) made similar claims for preparations
made in the same way from embryo extract. Fischer and Demuth (1927)
separated an active proteose from Witte’s peptone. So did Kuczinski,
Tenenbaum, and Werthemann (1925), who mention that their preparation
was rich in vitamin B. By this, as in so many studies on partially purified
biological materials, is admitted the possibility that the effects observed are
not only, or perhaps sometimes even mainly, attributable to the quantita-
tively preponderant components.
Carrel, Baker, and Ebeling (1927) compared the effects, on rat sarcoma
cells, of various supplements to Tyrode solution. Digests of egg albumin
and of casein gave poor “growth.” Addition of glycine increased the rate
of growth by about 70%, and addition of “nucleic acid” [amount and
source unspecified, but perhaps the thymus nucleic acid (Levene) referred
36 CHARITY WAYMOUTH

to by Baker and Ebeling (1938) ] plus glycine to a digest of egg albumin

increased the area 91%. “Nucleic acid” plus glycine and casein digest
gave an even greater increase in area (193%) over the controls. The
sarcoma cells responded much better than normal cells to peptic digests
(Baker and Carrel, 1928a, b, c). Willmer and Kendal (1932), who used
normai chick fibroblasts, prepared a thermostable heteroproteose from
Witte’s peptone, which greatly stimulated the cells to migratory activity
and cell division. Glycine was examined by Vogelaar and Erlichman
(1936b) as a supplement to media for human fibroblasts. At this time, a
very high concentration (525 mg./100 ml.) was used, and this was not
capable of replacing the Witte’s peptone in the feeding solution. Later,
Vogelaar and Erlichman (1938) used a medium (Table I) containing
1/10 of this concentration (52.5 mg./100 ml.), which gave good results
in the absence of Witte’s peptone.
Glycine was not one of the mixture of nine amino acids (prepared after
the analysis of fibrin made by Bergmann and Niemann (1936) ) used by
Fischer (1941a) to supplement dialyzed biological media. Later he
(Fischer, 1948a) found that a supplementary medium containing no amino
acids except cystine, glycine, and glutamine was able to maintain cultures in
a condition comparable to that in a supplement containing the nine Berg-
mann and Niemann amino acids. Omitting single amino acids from the
mixture of nine [reported as Rose’s (1938), but apparently actually Berg-
mann and Niemann’s (1936) mixture], Fischer (1948b) found that
lysine could be dispensed with over a four-day growth period without
significant reduction in outgrowth. Omission of cystine caused severe
reduction, and of arginine, tryptophan, or glutamine a moderate reduction,
of the total area at the fourth day. Stimulating effects on tissue cultures
have been observed with cystine, with glutathione (Hueper and Russell
1933; B’etker and Wormiak, 1952), and with arginine (Hueper and
Russell, 1933; Bach and Lasnitzki, 1947). Cystine is regarded by Fischer
(1941a, 1948a, d) as possessing peculiar importance for the cells. The
absence of cystine, even when the other amino acids were provided, led
to complete inhibition of growth in Fischer’s dialyzed system. A compari-
son of the effects of amino acid mixtures based on the composition of two
proteins (lactoglobulin and bovine serum albumin) with tryptic or peptic
digests of the same proteins was made by Fischer (1948a). The digests
(it is not clear whether both enzymes were equally effective) increased
the area of outgrowth far beyond that obtained in the artificial amino acid
mixtures. Thus, With a lactoglobulin digest, the area at fourteen days was
about twice that in the amino acid mixture simulating lactoglobulin, and
was still increasing, while in the mixture of twenty amino acids the area
 THE NUTRITION OF ANIMAL CELLS 37
had reached a maximum at seven days. The persistent idea that peptides
have an important stimulating effect was put forward by EhrensvLrd,
Fischer, and Stjernholm (1949) in their conclusion “that we have to seek
for the components possessing maximal activity in respect to the induction
of cell proliferation between some special definite limits of molecular
weight, namely the upper limit of trichloroacetic acid precipitability and
the lower limit of non-dialyzability.” While it seems to be established
that peptides stimulate fibroblast migration, the evidence for the actual
utilization of peptides by the cells is very meager and at best circumstantial.
It is possible that, for tissue cells, as has been shown (Kihara and Snell,
1952; Kihara, Klatt, and Snell, 1952) for bacteria, small peptides may
have advantages over their component-free amino acids. For example,
Kihara, Klatt, and SneIl showed that leucyltyrosine and glycyltyrosine
are far superior to tyrosine in growth-promoting activity for Streptococcus
fueculis, because free tyrosine, but not the peptides, is attacked by a
tyrosine decarboxylase present in the cells. This is an example
of a principle which may have far-reaching implications in the
search for the optimum nutritional requirements of different cell types.
The fact that the Ehrlich mouse ascites tumor cells can take up a-glutmyl-
glutamic acid, glycylglycine and triglycine (Christensen and Rafn, 1952)
(though the uptake of these peptides by these cells is less than that of the
free amino acids), nevertheless indicates that such small peptides can be
assimilated. Whether larger peptides, some of which are known to have
important physiological functions, e.g., strepogenin as a growth factor for
certain bacteria and the leukotaxinlike peptides (eight to fourteen residues
in length) in inflammation and capillary permeability (Duthie and Chain,
1939; Spector, 1951), are taken up by the cells or utilized in their nutri-
tion, remains doubtful. According to Winnick (1952), amino acids or
peptides can be used by the cells to build new cell protein and “it is even
conceivable that whole protein molecules may be assimilated with only
minor structural modifications”. Some evidence has been brought forward
in support of this hypothesis (Francis and Winnick, 1953). It has been
claimed (Bohus Jensen, quoted by Fischer, 1950) that the “presence of
large peptides in the medium causes a remarkable increase in the fre-
quency of cell divisions.” Fischer (1942a, 1947) attempted to demonstrate
that the peptides from homologous plasma, which could be expected to
conform to the pattern of the species-specific protein in the tissues, were
more readily used for the growth of fibroblasts than peptides from hetero-
logous plasma. Only one type of tissue (chick embryo fibroblasts) was,
however, used, and there was a wide variation in the effects with digests
from plasmas from various species. The evidence was hardly sufficient to
38 CHARITY WAYMOUTH

justify the generalization that homologous peptides are nutritionally su-

perior. I n experiments of the same sort, Fischer (1950) tested peptic
digests of normal and tumor tissue for their growth-promoting power on
normal myoblasts. The increase in area of the cultures treated with the
tumor digest (from a methylcholanthrene-induced fowl sarcoma) was in
one case slightly, in another markedly less than in controls treated with
normal tissue digests. Again the evidence is too slight to warrant generali-
zation. And, as has been pointed out elsewhere in this review, increases
in area alone are an uncertain and often entirely misleading guide to the
true growth of tissue. The ability of cells to use heterologous or homolo-
gous peptides still remains an open question.
A great deal of information is now available on two basic phenomena
of cell behavior and biochemistry, highly relevant to the mechanism of
amino acid utilization in tissue nutrition. These are: (1) the capacity of
cells actively and selectively to concentrate amino acids from their environ-
ment, and (2) the mechanisms and scope of transamination with its
implications for the ability of the cells to modify the amino acid population
presented to them. The application of this knowledge to the understanding
of tissue nutrition and the design of nutrient media has hardly begun. T o
these two should perhaps be added the information which is accumulating,
but is still relatively fragmentary, on the incorporation of amino acids into
other cell constituents, e.g., glycine and aspartic acid into nucleic acid
purines and pyrimidines.
The concentrative uptake of amino acids has been studied over a number
of years by Christensen and his collaborators. The interest of this phenom-
enon for tissue growth was commented on by Christensen and Streicher
(1948), who speculated on their findings that the concentrations of amino
acids in fetal tissues, and in actively regenerating liver, are several times
those in the corresponding normal adult tissues. They suggested that
“increased protein synthesis and growth may be initiated or promoted by
increased concentrations of amino acids”’ and that therefore the “concen-
trating function of cells for amino acids represents a possible point for the
control of growth.” Fetal guinea pig muscle, for example, contains an
amount of a-amino acids three times that in the muscle of the maternal
guinea pig. The concentrative ability for amino acids of Ehrlich’s mouse
carcinoma (ascites form) cells is very great (Christensen and Henderson,
1952; Christensen and Riggs, 1952). Glycine gradients of 60 mM./1.
water between cells and suspending fluid can be achieved in glycine-
enriched media at the end of 2 hours’ incubation. The ratio of concentra-
tions of glycine in cells to ascitic fluid without added glycine is 12.4.
The uptake is dependent on the presence of oxygen and is temperature
 THE NUTRITION OF ANIMAL CELLS 39

sensitive, the ratio cells-fluid being maximum near the physiological

temperature. Concentration of amino acids is closely associated with the
maintenance of ion balance, and glycine uptake can be inhibited by 40
meq./l. of potassium, During glycine concentration (Christensen and
Riggs, 1952) or tryptophan concentration (Riggs, Coyne, and Christensen,
1953) the cells lose K and take up Na. These are additional instances of
the principle of the control of the ionic environment by amino acids, first
studied by Krebs and Eggleston (1949) in respiring brain slices in relation
to the effects of glutamic acid and glucose on potassium exchange. An
appreciation of the implications of these studies is essential to the under-
standing of the possible ways in which materials are utilized in the
system cells plus medium which constitutes the tissue culture. According
to Christensen et al. (1952), the cells of higher animals show a character-
istic responsiveness to extracellular levels of amino acids. The amino acid
content of the cell is adjusted in relation to the environment, the intracel-
M a r level always remaining higher than the external level. The concentra-
tive activity Of cells for amino acids decreases during embryonic develop-
ment and in general as cells become specialized. C1*-labeled glycine at 1.33
mM., and DL-alanine and DL-phenyhlanine at 2.66 mM., are taken up by
the proteins of embryonic chick lung, intestine and heart cells. The labeled
atom of glycine-l-CIJ appears in the serine as well as in the glycine of
the heart and lung proteins (Gerarde, Jones, and Winnick, 1952a).
The ability of mouse heart in tissue culture to effect transaminations
was demonstrated by Jacquez, Barclay, and Stock (1952). Of eleven
mouse and four rat tumors examined, nine had negligible transaminating
ability. Bach and Lasnitzki (1947) found that arginine (100 mg./100 ml.)
significantly increased the growth of tissue cultures of carcinoma 63 ;
chick heart fibroblasts and mouse embryo lung were unaffected. Trans-
aminase activity is, however, fairly widespread among different tissues, and
the capacity of the cells thus to modify their nutritional environment
should be borne in mind in assessing their nutritional needs. The fact
that CI4 from labeled glucose is incorporated into aspartic acid and alanine
(Fischer, Fischer, et d., 1953) indicates the intervention of a trans-
aminase in embryo chick tissue, able to convert the pyruvate and oxaloace-
tate formed from the CI4 glucose into these amino acids. Some CI4 is also
found in the tissue serine, glycine, glutamic acid, and proline. It ,is signifi-
cant that the synthetic medium of White (1946), containing a mixture of
ten amino acids corresponding to those originally reported by Rose (1938)
to be essential for the maintenance of N equilibrium in the rat, was much
improved as a maintenance medium for normal chick tissues by the
incorporation of glycine, glutamic acid, aspartic acid, proline, and cystine
40 CHARITY WAYMOUTH

(White, 1949). Parallel with this finding in tissue culture, Rose, Oester-
ling, and Womack (1948) have also shown that rats fed a diet
containing nineteen amino acids gain 25% more weight in a 28 day period
than those on the original 10 amino acids. Glutamic acid was particularly
important. Parshley and Simms (1950) added aspartic acid to their
special salt solution designed for use in media for adult epithelium, after
finding that this amino acid stimulated the cells of thyroid and skin in tissue
culture. Morgan, Morton, and Parker’s (1950) medium no. 199 contains
a mixture of nineteen amino acids based on analyses of tissue protein
(Block and Bolling, 1945). It is by no means certain that a mixture of
amino acids in the proportions found by analysis in a given protein is the
best material for the normal synthesis of the same or similar protein in a
biological system. There is much to suggest that extensive interconversion
can take place, and that therefore smaller numbers of amino acids are
sufficient. Much remains to be learned about the interrelations of the amino
acids and about the design of the most effective mixtures for tissue nutri-
tion.
The total concentrations of amino acids provided must also be suitable,
though surprisingly high concentrations of certain individual amino acids
(glycine, 1,ooO; phenylalanine, tyrosine, aspartic acid, SO0 ; tryptophan,
histidine, 400; arginine, lysine, 200 mg./100 ml.) were found by Brues
et al. (1940) to be non-inhibitory to fibroblasts. White (1946) tested a
wide range of concentrations of his ten-amino acid mixture (0.15 to 150
mg./100 ml.) and found a regular increase in survival with concentration
up to 45.0 mg./100 nil., and a decrease at 150.0 mg./lOO mi. A concentra-
tion of 100 mg./100 ml. was taken as optimum. With the addition of the
further five amino acids (White, 1949), the total concentration was
raised to 136.5 mg./lCQ nil. Morgan, Morton, and Parker (1950) tested
a narrower range (50 to 500 mg./100 ml.) of dilutions and found 100
mg.Jl00 ml. of their nineteen-amino acid mixture to be most favorable.
These concentrations are of the same order as the amounts of free amino
acids in rabbit fetal plasma, rather more than in human cord serum, some-
what less than the concentration in guinea pig serum (Christensen and
Streicher, 1948) and about three times the amount in adult human blood
(Krebs, 1950). It is remarkable that Bullough and Johnson ( 1 9 5 1 ~ )
found that a relatively very high concentration (340 mg./100 ml. ;0.02 M.)
of a single amino acid (glutamic acid) increased the rate of mitosis in
fragments of adult mouse ear epidermis in vitro. This is, however, of the
same order as the concentration of aspartic acid (300 mg./lOO mi.) used
by Parshley and Simms (1950) in their 216 solution for adult epithelium.
The concentration of glutathione in chick embryo extract was reported
 T H E NUTRITION OF ANIMAL CELLS 41

to be 40 mg.Jl00 ml. (Hueper and Russell, 1933). As their standard

medium contained one-fourth embryo extract, this would contribute about
10 mg./lOO nil. glutathione to the medium. The various synthetic and semi-
synthetic media in which glutathione has been included have usually con-
tained less than this. It has mostly been added with the aim of stabilizing
ascorbic acid, rather than for its possible contribution as a peptide or
source of amino acids to the nutrition of the cells. Glutathione has lately
been shown to be the prosthetic group of a glyceraldehyde-3-phosphate
dehydrogenase (Racker and Krimsky, 1952). Baker’s media for fibro-
blasts and monocytes ( 1936) contained respectively 0.34 and 1.O mg./100
ml., and Baker and Ebeling’s (1939) medium 1.2 mg./lOO ml. glutathione.
White’s ( 1946, 1949) media contained 1.0 mg./100 ml. ; Morgan, Morton,
and Parker (1950) had only 0.005 rng./100 ml. in mixture no. 199.
Fischer’s (Fischer et al., 1948) media V-605 and V-612 contain respective-
ly 0.5 and 0.1 mg./lOO ml. and these media are designed as supplements
to dialyzed media, so the final concentrations would be lower. However,
considerably more (8 to 10 mg./100 ml.) seems to have had a beneficial
effect (Astrup and Fischer, 1946) and Fischer (1948b) states that gluta-
thione plus a dialyzed medium caused “large areal spreading of the cells”;
here the concentration appears to have been 200 mg./lOO ml. He (Fischer,
1948c) devised a medium (Table I ) , in which heart fibroblasts could be
maintained for ten passages, containing a final concentration of 14.8
mg./100 ml. glutathione.
Cysteine was used at 9.0 mg./lOO ml. by Vogelaar and Erlichman
(1933) and Erlichman (1935) in their feeding solution for human fibro-
blasts and at 14.2 mg./100 ml. in their modification (Vogelaar and Erlich-
man, 1938) containing glycine. Baker’s (1936) media contained 1.125
mg./lM ml. (for monocytes) and 9.0 mg./100 ml. (for fibroblasts), as
did Baker and Ebeling’s (1939) medium and Wilson, Jackson, and Brues’
(1942) medium. White (1946, 1949) used 0.10 mg./100 ml. Morgan,
Morton, and Parker (1950) included 0.01 mg./lOO ml. cysteine in medium
no. 199. Pires Soares (1947), using guinea pig testis and embryonic
chick heart, prepared tissue cultures in biological media (60% plasma, 20%
embryo extract plus 20% saline with or without added cysteine). I n the
range 0.125 to 1.875 mg./100 ml. in the final medium, the highest and
lowest concentrations had little effect compared with the optimum of 0.75
mg.Jl00 ml. in increasing the area of the cultures of both types of cells
in a forty-eight-hour period. Parallel with the increase in area, he observed
an increase in mitotic index and prolongation of the life of the (hanging-
drop) cultures, without transfer to fresh medium, from ten days in the
controls to fifteen days in the medium containing 0.75 mg./100 ml. cys-
42 CHARITY WAYMOUTH

teine. All phases of mitosis were increased, but most markedly the number
of telophases.
Glutatnine is a major contributor to the free =-amino N of animal tissue
and plasma (Hamilton, 1945). The normal glutamine concentrations in
dog and human plasmas are 6 to 12 mg./100 ml. Heart tissue contains 225
mg./100 ml. which comprises 50 to 60% of the total free a-amino N of
the tissue. Fischer’s (1948a, c) media contained 25.0 and 18.5 mg./100
ml. respectively ; in Ehrensvard, Fischer, and Stjernholm’s ( 1949) me-
dium, the final concentration of glutamine was also about 25 mg./100 ml.
Morgan, Morton, and Parker’s ( 1950) reported that glutamine increased
the life span of their fibroblast cultures, at 10 mg./100 ml.
VII. PURINES, ACIDS
AND NUCLEIC
PYRIMIDINES,
From some of their early fractionation experiments on embryo extracts,
Baker and Carrel (1926a) reported that the proteins therein were “a
mixture of nucleoprotein and glycoprotein with mucin-like properties’’ ;
but they could not attribute to the isolated fractions, or to other nucleo-
proteins, to sodium nucleate prepared from embryo pulp, or to nucleic
acid from thymus, any growth-stimulating effects. However, Baker and
Ebeling (1938, 1939) included 20 mg./100 ml. thymus nucleic acid in
their synthetic maintenance medium. Fischer (1939) on the other hand
isolated from beef embryo extract a “nucleoprotein” fraction which he
found to accelerate the growth of his cultures. Both ribonucleic acid and
deoxyribonucleic acid were present, but he maintained that the “growth-
promoting activity seems in the meantime to follow the fractions contain-
ing the ribose nucleotides,” i.e., the fraction precipitated by glacial acetic
acid. Later he reported (Fischer, 1940, 1941b) that reprecipitation
always caused reduction of activity. Repeated precipitation with dilute
HCl at 0” C. gave “more and more a distinct maximum of absorption in
ultra-violet at 2600 A characteristic of nucleic acid” but less and less
growth-promoting activity. Fischer was therefore forced to consider the
possibility that some substance other than the nucleoprotein itself was
responsible for the activity of the “nucleoprotein” fraction. The fraction
contained more P than could be accounted for as nucleic acid, it con-
tained 2% s, and probably polysaccharides of the chondroitin sulfate type
(Fischer, 1940, 1941b). There is no evidence for the utilization of nucleo-
proteins or nucleic acids as such in cell nutrition, although Tennant,
Liebow, and Stern (1941) and Tennant, Stern, and Liebow (1942)
suggested that nucleates stimulated migratory activity in mouse fibroblast
cultures.
Studies on changes in nucleic acids in growing tissues have been made
 T H E NUTRITION OF ANIMAL CELLS 43

by Willmer (1941, 1942), Davidson and Waymouth (1943, 1944a, b,

1945, 1946), Davidson, Leslie, and Waymouth (1949), Davidson and
Leslie ( 1951), Leslie and Davidson (1951a, b), Hull and Kirk (195Oa, b,
c), Boyer and Kirk ( 1952), and Gerarde, Jones, and Winnick (195Zb).
The finding that the growth-promoting effect of chick embryo extract was
not reduced by treatment with the enzyme ribonuclease (Davidson and
Waymouth, 1943) strengthened the probability that free polynucleotides
are not important nutrients.
Caution in generalizing from one organism to another about metabolic
pathways is always desirable but is particularly necessary in the case of
the precursors of the nucleic acids. It has been shown (Abrams and
Goldinger, 1952) that, in rabbit bone marrow, hypoxanthine can act as a
precursor of both adenine and guanine in the nucleic acids. Adenine and
guanine themselves can be used by the rabbit cells (Abrams and Goldinger,
1951), but there is not in the rabbit, as in the rat, any extensive intercon-
version of the two purines. Moreover, the rat cannot use hypoxanthine as a
precursor of the polynucleotide purines (Gebler et ad., 1949). There are
now many instances to show that an exogenous source of preformed
purine is not essential. With labeled molecules, it has been demonstrated
that in the rat (Abrams and Goldinger, 1952) and the pigeon (Greenberg,
1948; Valentine, Gurin, and Wilson, 1949; Sonne and Lin, 1952, 1953)
the following can act as precursors of various parts of the hypoxanthine
molecule : formate, glycine, glutamate, aspartate, and glutamine. Labeled
COa is incorporated into both purine and pyrimidine rings, in rat tissues
in vivo (Heinrich and Wilson, 1950). Reichard and B'ergstrom (1951)
have shown that, in the rat, there is considerable synthesis of both ribonu-
cleic acid and deoxyribonucleic acid purines from glycine, and of pyrimi-
dines from orotic acid. Ribonucleic acid pyrimidines can also incorporate
aspartic acid (Lagerkvist, Reichard, and Ehrensvard, 1951). The biosyn-
thetic incorporation of formate and bicarbonate into nucleic acids in mice
and rats is dependent upon folic acid (Skipper, Mitchell, and Bennett,
1950; Drysdale, Plaut, and Lardy, 1951) ; that of bicarbonate into ribonu-
cleic acid in the rat is dependent upon biotin (MacLeod and Lardy, 1949).
There is therefore no reason to assume that purines or pyrimidines as
such, or in the form of nucleosides or nucleotides, are needed as nutrients.
There exists a small amount of evidence upon the effects of some of these
substances on cells growing in tissue culture.
Hopkins and Simon-Reuss (1944) tested the effects of hypoxanthine
in 15% embryo extract on the growth of periosteal fibroblasts. The areas
were increased over the controls, at 5 mg.Jl00 ml. ; 10 mg./100 ml. was
less effective. In similar cultures in Carrel flasks, analyzed by the photo-
44 CHARITY WAYMOUTH

graphic method of Willmer and Jacoby (1936), 5 mg.JlO0 ml. hypoxan-

thine caused, over a period of 30 to 40 hours, a marked stimulation of
mitosis. No similar effect was obtained with adenine. Ehrensvard,
Fischer, and Stjernholm (1949) included hypoxanthine in a simple sup-
plement to a dialyzed medium. They found the optimum concentration in
the fluid phase in Carrel flasks to be about 1 mg./100 ml. Adenine and
guanine were found to be inactive. Rerabek and Rerabek (1952) treated
Maximow cultures of fibroblasts with purine and pyrimidine bases. These
authors found that adenine at 1.69 mg./lOO ml. caused area and mitotic
increases, greater than the increases produced by the four bases (adenine,
guanine, cytosine, and uracil) at the same (0.125 mM.) molarity. Cytosine
alone gave some effect, guanine alone was inhibitory, and uracil had no
distinct effect. Trowel1 (1953) found adenine and adenosine at 2 mM.
toxic to lymphocytes ; he quotes evidence of SchiZler that leukemic lympho-
cytes in vitro rapidly convert purines to allantoin. Concentrations as low
as 0.6 mM. of adenosine, adenylic acids, and adenosine triphosphate can
cause pre-prophase inhibition of mitosis in chick fibroblasts (Hughes,
1952), and concentrations in this range or a little higher can cause nucleolar
fragmentation, though they do no apparent damage to interphase cells.
The synthetic medium no. 199 of Morgan, Morton, and Parker (1950)
included a number of nucleic acid components, namely adenine at 1.0
mg.JI00 ml., guanine, xanthine, hypoxanthine, thymine, and uracil each
at 0.03 mg./100 ml., ribose and deoxyribose each at 0.05 mg.Jl00 ml. This
medium also contained muscle adenylic acid at 0.02 mg./100 ml. and ade-
nosine triphosphate at 1.0 mg./100 ml. Fischer’s (19484 supplements
V-605 and basic nutrient contained 10 mg./100 ml. hypoxanthine and 3.0
mg./100 ml. inosinic acid. Also using a dialyzed medium, Harris (1952a)
found no effect on the increase in area in ten days of his fibroblast cultures
with 10 mg./100 ml. ribonucleic acid. The nucleotides and nucleosides ( 1
to 100 mg./100 ml.) were inactive or inhibitory, and so were the seven
purine and pyrimidine bases (adenine, guanine, cytosine, uracil, thymine,
xanthine, and hypoxanthine) at 1 or 10 mg./100 ml. and the cofactors
adenosine triphosphate ( 1 mg./100 ml.) and diphosphopyridine nucleotide
(0.1 mM. or 6.6 mg./100 ml.).
VIII. LIPIDS
Little has been added to our knowledge of lipid metabolism in tissue
culture systems since the chemical events in such systems were reviewed
by Fischer in 1933. Carminati (1933) reported that a synthetic (distearyl)
lecithin stimulated chick fibroblasts. Davidson and Waymouth ( 1944b,
1945, 1946) showed that fibroblast cultures do not grow well in a medium
 THE NUTRITION O F ANIMAL CELLS 45

consisting of defatted plasma and defatted embryo extract, but that

defatted sheep embryo extract promoted greater increase in nucleoprotein
phosphorus than the untreated extract, in the presence of whole plasma.
The enzyme lecithinase A (which dissociates Iecithin to Iysolecithin with
release of an unsaturated fatty acid), and lysolecithin itself (50 mg./100
ml.), produced morphological and chemical changes in chick heart cells
which suggested that this treatment may favorably improve the capacity
of the cells to take up nutrient materials. Leslie and Davidson (1951a)
showed that there is an early increase in phospholipid (during the first
24 to 48 hours) in chick heart explants, at the time when carbohydrate
metabolism is particularly intense (Willmer, 1942). Insulin increased
the phospholipid content of the cultures, but reduced the amount per cell
(Leslie and Davidson, 1951b) .
I t is now very well established that lipogenesis is intimately linked with
active carbohydrate metabolism, and that carbohydrate and acetate, as
well as pyruvate, oxaloacetate, lactate, etc., provide carbon sources for
fatty acid synthesis. Free fatty acids do not normally accumulate in
lipogenetic tissues, but are immediately esterified to glycerides. Cholesterol
incorporates acetate or acetoacetate ; Popjik and Beeckmans (1950) have
shown that the rabbit fetus is independent of the mother for lipid synthesis
and can incorporate C14 acetate into cholesterol and into both glyceride
and phospholipid fatty acids. Popjik and Muir (1950) discuss the
probability that a- and P-glycerophosphates may be precursors of phospho-
lipid P.
Lipid synthesis and metabolism are believed to be affected at various
points by almost all of the B vitamins (thiamine, riboflavin, pyridoxine,
pantothenate, nicotinic acid, folic acid, biotin, choline, and inositol) (cf.
Frazer, 1952; Bloch, 1952). I n vivo, but not with any certainty in vitro,
insulin and the hormones of the adrenal and pituitary glands are concerned
in fat metabolism.
Fischer et aE. (1948) included in their medium V-605 fumarate, malate,
oxaloacetate, and succinate, most of the B vitamins and several carbohy-
drates. It is improbable that fatty acids as such are nutritionally useful.
Morton, Morgan, and Parker (1950) studied the effects of a series of
“Tweens” (polyoxyethylene sorbitan esters of fatty acids) on myoblast
cultures in synthetic media. The monolaurate, monopalmitate, mono-
stearate, monooleate (Tween So), and trioleate esters, and free oleic acid
were tested at 0.05 to 500 mg./100 ml. Concentrations of 5 mg./100 ml.
and less were not inhibitory. Tween 80, because it contained an unsatu-
rated fatty acid, was incorporated into medium no. 199 at 2.0 mg./lOO ml.,
partly for its possible intrinsic value, and partly as a vehicle for non-water-
46 CHARITY WAYMOUTH

soluble components (Morgan, Morton, and Parker, 1950). Jacquez and

Barry (1951) showed that oleic acid at 1 mg./100 ml. was toxic to rat
fibroblasts in a medium containing embryo extract and the globulins of
human placental cord serum. Higher concentrations could be tolerated
in the presence of serum albumin, which binds the fatty acid.
IX. VITAMINS
1. The Fat-Soluble Vitamins
a. Vitmin A . The survival and growth of several types of tissue is
Vitro appear to be affected by vitamin A. The life of liver and spleen
cultures was prolonged by addition of the vitamin to the medium
(Bisceglie, 1926). Baker (1935a) increased the vitamin A content of
chicken serum two-hundredfold by allowing the serum to stand overnight
in the presence of purified vitamin A. Fibroblast cultures in a horse
plasma clot and a semisynthetic nutrient containing chicken serum so
fortified formed colonies up to three times as large as control cultures
without added vitamin. Large amounts of the hypervitaminotic serum
were toxic; the optimum effect was found when it formed 1% of the
medium. This would mean slightly more than doubling the normal
concentration, allowing for a proportion of normal serum in the medium.
The incorporation of vitamin A into the medium prevented fat accumula-
tion and increased the survival time. The semisynthetic media subse-
quently described by Baker (1936) contained 900 to 1,800 units/100 ml.
for fibroblasts and 50 to 100 units/100 ml. for monocytes. Normal fowl
plasma contains 200 to 400 units/100 ml. vitamin A and about 300
units/100 ml. carotene; mouse plasma contains only 20 to 60 units/100
ml. vitamin A (Fell and Mellanby, 1952). Gordonoff and Ludwig (1935,
1936) also found that an increase in vitamin A stimulated the growth of
chick fibroblasts, and of a mouse carcinoma in culture. Proliferation
of both these types of cells was greatly inhibited by the absence of vitamins
( A B1,BP, C, D, and E) from the plasma used in the medium, or by
the absence of vitamins A or BI alone. Small amounts of vitamin A Were
found by Vollmar (1939) to stimulate the growth of both normal and
tumor cultures ; larger amounts were inhibitory, and more strongly so to
the tumor cells than to the normal cells. Fell and Mellanby (1952)
produced hypervitaminosis A in plasma, both “artificial” (by adding
vitamin A alcohol or acetate to normal fowl plasma) and “natural” (by
feeding large amounts of the vitamin to birds). Plasma from these birds
contained three to four times the normal amount of vitamin A and about
half the normal amount of carotene. The vitamin in the “artificial”
hypervitaminotic plasma remains solubie in fat solvents ; in the “natural”
 THE NUTRITION OF ANIMAL CELLS 47

hypervitaminotic plasma, it is not extractable with petroleum ether unless

the proteins have first been denatured. Both types of vitamin-rich plasma
produce profound changes in limb bud rudiments of the chick and mouse
in &fro, the effects with the free vitamin being more drastic than those
of the combined form. Another remarkable effect was produced by Fell
and Meflanby (1953) by treating ectoderm from six- to seven-day chick
embryos with vitamin A at about 1,ooO to 2,000 units/100 ml. Amounts
of vitamin A of this order prevented keratinization and caused the
epithelium to differentiate into an actively secreting mucous membrane,
often ciliated and histologically similar to normal chick nasal mucosa. On
transfer to normal medium, after seven to fourteen days in the medium
rich in vitamin A, there was a t first continued rapid development of the
ciliated and secretory epithelium for four to five days. Thereafter the
basal cell layer multiplied and formed a squamous epithelium similar to
that produced in cultures carried throughout in normal medium.
One international unit of vitamin A is equivalent to 0.344 pg. vitamin
A acetate or 0.6 pg. p-carotene (W.H.O. Tech. Rept. Series, 1950, no. 3).
Human plasma contains about 0.025 mg. vitamin A per 100 ml. and has
a total carotenoid content of 0.09 mg./100 ml. (Krebs, 1950). The total
vitamin A plus carotene (0.01 mg./100 ml. of each) used by White
(1946, 1949) and by Morgan, Morton, and Parker (1950) in their
synthetic media are therefore of the same order as the amounts in human
or mouse plasma and considerably lower than in fowl plasma.
b. Vitamin D. Hosono and Narisawa (1931) added vitamin D to
the culture medium for chick heart and also used plasma from birds fed
vitamin D orally. They found that the “heart tissue grew more than twice
as well as in control plasma.’” On the contrary, Gordonoff and Ludwig
(1936) found that, in contrast to the unfavorable effects produced by
deficiencies of vitamins A and BI, neither avitaminosis D nor addition of
vitamin D to normal plasma had any effect on normal or tumor cultures.
Vollmar (1939) also obtained no clear effect. Baker’s (1936) media for
fibroblasts and monocytes contained, respectively, 15 to 30 and 1 to 4
units per 100 ml. vitamin D. The international unit of vitamin D is
equivalent to 0.025 pg. crystalline vitamin D3 (cholecalciferol). The
synthetic medium of Morgan, Morton, and Parker (1950) contains a
concentration of 0.01 mg./100 ml. calciferol (ergocalciferol) , which is
about the amount found in egg yolk.
c. Yitartin E. While Gordonoff and Ludwig (1936) found that
plasma deficient in vitamin E, or supplemented with an excess, had no
effects on their cultures, JuhAsz-Schaffer (1931) and Rossi (1935, 1936)
(quoted by Vogelaar and Erlichman, 1937) reported stimulation of
48 CHARITY WAYMOUTH

cultures of chick embryo tissues. According to Vollmar (1939), normal

tissues were stimulated by vitamin E at 1 g./100 ml. and were not inhibited
even by a concentration as high as 10 g./lOO ml. One gram per 100 ml.,
however, inhibited the growth of tumor cultures. In their medium no.
199, Morgan, Morton, and Parker ( 1950) included a-tocopherol phosphate
at 0.001 mg./100 ml. The total tocopherol concentration in human pIasma
is about 1.2 mg./lOO ml. (Krebs, 1950).
d. Vitamin K . This vitamin is included in Morgan, Morton, and
Parker’s (1950) medium no. 199 at 0.001 mg./100 ml. Nothing is known
of its possible influence on the nutrition of cells.
2. The Water-Soluble Vitamins
a. Vitom’fi C. Vogelaar and Erlichman (1937) reported that 0.5
mg./iOO ml. ascorbic acid favored the growth of the Crocker mouse
sarcoma 180 in tissue culture. On the other hand, Vollmar (1939) found
that 1 mg./lOo ml. stimulated normal tissue but had no effect on the
tumor tissues tested. High concentrations (200 to 500 mg./100 ml.)
were found by Hengstmann (1938) to inhibit, and low concentrations to
have no effect on the growth of human and chick embryonic tissues.
Epithelial cells from embryonic guinea pig kidney and parotid remained
healthy in a washed coagulum in the presence of a buffered saline contain-
ing ascorbic acid, but not without it (Chambers and Cameron, 1943).
Plasma from scorbutic guinea pigs also caused deterioration of these
tissues. Here, however, the effect might be an indirect or a multiple one.
For example, the amounts of the important amino acids glycine and
glutamic acid are greatly reduced in scorbutic guinea pig tissues (one-
third of normal in liver and muscle, Christensen and Lynch, 1948), and
may well also be reduced below the nutritional optimum in the plasma.
Messina and Verga (1937) and Nungester and Ames (1948) found that
the phagocytic activity of leucocytes was increased by ascorbic acid.
Within the range of 0.10 to 0.60 mg./lOO ml., which is well within physio-
logical limits, the phagocytic activity and the fragility of leucocytes in peri-
toneal exudates of the guinea pig varied with ascorbic acid content. Above
0.60 mg./100 ml., there was no increase in phagocytic activity (Nungester
and Ames, loc. cit.). These observations suggest that, apart from its prob-
able metabolic role, ascorbic acid may have an influence on the physical state
of the cell surface and so on the exchange of substances between the cell
and its environment. A curious effect was produced by a rather high
concentration (20 mg./100 ml.) of ascorbic acid on cultures of the Ehrlich
adenocarcinoma which had been maintained through 680 passages in a
rat and chicken plasma and chick embryo extract medium (Gaillard, 1942).
 T H E NUTRITION O F ANIMAL CELLS 49

Three to four days after addition of the vitamin, a thickening of the

center of the explant took place, which proved to be a core of cartilage.
,4 concentration of 10 mg./lOO ml. was able to activate cell proliferation
and growth in cultures of kidney tubules (Chambers and Cameron, 1944).
Concentrations within the physiological range were used by Baker (1936)
(0.25 mg./100 ml. for fibroblasts and epithelium, 0.085 mg./100 ml. for
monocytes) . B’aker and Ebeling’s (1939) maintenance medium contains
0.3 mg./100 ml. ascorbic acid. Fischer’s (19484 supplement V-605
contains 0.2 mg./lOO ml. The synthetic media of White (1946, 1949) and
of Morgan, Morton, and Parker (1950) contain respectively 0.05 and
0.005 mg./100 ml.
b. Vitamins of the B Group.
( 1 ) THIAMINE. From the experiments of Gordonoff and Ludwig
(1935) and of Rossi (1935, 1936) it appeared that some part of the B
complex was important as a component of nutrients for tissue cultures.
Plasma from avitaminotic animals was unfavorable and completely in-
hibited the growth of chick embryo fibroblasts and of mouse tumor cells.
Plasma from hypervitaminotic birds stimulated cultures and favored the
survival of tissue in Vitm (Gordonoff and Ludwig, 1936). Very high
concentrations of a brewers’ yeast preparation of “B vitamin” were
inhibitory, but more dilute preparations were stimulatory (Rossi, 1935).
Gordonoff and Ludwig (1935, 1936) concluded that thiamine (vitamin
B1) was essential to the proliferation of normal chick and of mouse
carcinoma cells. Paterson and Thompson ( 1943) also studied avitaminotic
plasma as a tissue culture medium. Conditions favorable to growth could
be restored by adding a brewers’ yeast extract low in thiamine and in
biotin. It was concluded that this supplement provided a source of some
essential and unidentified member of the B complex. Added thiamine
was not effective. I n contrast to this, and in agreement with Gordonoff
and Ludwig, Hengstmann (1938) claimed a stimulating effect of thiamine
on chick embryo heart, human embryonic and chicken leucocyte cultures,
at optimum concentrations in the range 5 to 10 mg./100 ml. Higher con-
centrations were inhibitory. The confusion is increased by the report
of Vollmar (1939) that 500 mg./lOO ml. thiamine will stimulate chick
heart and inhibit mouse tumor cultures. Some of the differences in the
ranges of concentrations found effective by different workers may be due
to an underestimation of the lability of free thiamine. Thiamine in the
natural, protein-bound form is stable to ordinary conditions of storage,
but free thiamine, in addition to being labile to heat and alkali, is liable
to inactivation by oxidation (Kandutsch and Baumann, 1953). However,
50 CHARITY WAYMOUTH

in short-term hanging-drop cultures, Hetherington ( 1946) found that

thiamine at 6.25 to 200 mg./100 ml. had no detectable effect on brain,
skin, or heart cultures. Concentrations of thiamine below (1 to 5 mg./100
ml.) and within (8 to 24 mg./100 ml.) the physiological range had no effect
on spinal ganglia in tissue culture (Burt, 1943b). Very high concentra-
tions were inhibitory. Plasma from thiamine-deficient birds (Burt, 1943c)
inhibits axon growth, but this inhibition, like that found by Paterson and
Thompson ( 1943) was not reversed by addition of thiamine to the medium.
Avitaminosis-B probably produces indirect as well as direct effects on
the composition of the plasma, For example, it is known that severe
thiamine deficiency in rats or pigeons results in a marked reduction in
the enzymes concerned in transamination (Kritzmann, 1940, 1943, quoted
by Braunstein, 1947). Administration of thiamine in Vim rapidly restores
the enzyme activity; but addition of thiamine in vtcro to minced tissues
from thiamine-deficient animals does not result in reactivation of the
transaminase system (Barron et al., 1941). Baker and Ebeling’s (1939)
maintenance medium (medium IV) contains O.ooO1 mg./100 ml., which
is less than the average normal level in human plasma (0.005 mg./100 ml.)
(Krebs, 1950). Fischer’s (19484 medium contains 0.3 mg./100 ml.
The synthetic media of White (1946, 1949) and Morgan, Morton, and
Parker (1950) contain respectively 0.01 and 0.001 mg./100 ml. thiamine.
(2) RIBOFLAVIN.In short-term cultures, riboflavin has little
(Gordonoff and Ludwig, 1936) or no effect (Hengstmann, 1938; Hether-
ington, 1946). Baker and Ebeling ( 1939) incorporated 0.0034
mg./100 ml. into their medium; Fischer (1948a) had 0.02 mg./100 ml.;
White (1946, 1949) had 0.01 and Morgan, Morton, and Parker (1950)
0.001 mg./100 ml. Human plasma contains 0.0032 mg./100 ml. total
riboflavin (free riboflavin plus flavin nucleotides) (Suvarnakich, Mann.
and Stare, 1952).
(3) PYRIDOXINE. Hetherington ( 1946) included pyridoxine in the
series of B vitamins which he tested on hanging-drop cultures, and he
found no effects attributable to this vitamin under the conditions of his
experiments. Fischer (1948a) had 0.03 mg./100 ml. in his medium
V-605 ; White (1946, 1949) used 0.05 mg./100 ml. in his synthetic media.
Morgan, Morton, and Parker (1950) included both pyridoxine (0.0025
mg./100 ml.) and pyridoxal (0.0025 mg./100 ml.) in their medium no.
199. In view of the part played by pyridoxal phosphate as a co-
decarboxylase, and in transamination reactions (Gunsalus, 1950), it is
possible that pyridoxal alone would be a sufficient addition. Transamina-
tion reactions are depressed by pyridoxine deficiency, though not so
drastically as by thiamine deficiency. Both pyridoxal phosphate and
 THE NUTRITION O F A N I M A L CELLS 51

pyridoxamine phosphate are effective coenzymes of transaminase (Cohen,

1951 ; Meister, Sober, and Peterson, 1952) ; pyridoxine is not able, in etitro,
to activate transamination in deficient tissues (Schlenk and Fisher, 1947).
Pyridoxal phosphate is also a coenzyme of histaminase (Sinclair, 1952)
and of desulf urase and transsuliurase (Tarver, 1952).
(4) BIOTINAND FOLIC -4~1~ Of. all the €3 vitamins examined, biotin
and folic acid are those on which the best evidence exists for positive
effects on the growth and survival of tissue cultures. Hamilton and Plotz
(1942) recorded stimulation of the growth of mouse and chick epithelium,
fibroblasts, muscle, and nerve cultures by biotin at about 0.03 mg./100 ml.,
though Burt (1943a) did not find that biotin at this concentration had
any effect on chick spinal ganglia, or on other types of cells, in tissue
culture. Hetherington (1946) reported that the survival time of chick
fibroblasts in hanging-drop cultures was doubled by biotin (0.15 to 0.5
mg./100 ml.) and fdic acid (0.015 to 0.5 mg./lOO ml.). Degeneration of
nerve cell cultures was prevented by 0.15 mg./100 ml. folk acid; 18.5
mg./100 ml. biotin (saturation) was not toxic to these cells (Painter,
Pomerat, and Ezell, 1949). Folk acid at 1.0 to 5.0 mg./100 ml. promoted
maturation of megaloblasts from pernicious anemia bone marrow, in a
medium containing pernicious anemia plasma. In a medium containing
bovine serum ultrafiltrate (not beef plasma, as reported), maturation
could be achieved with 0.1 mg./100 ml. folk acid (Thompson, 1952).
The synthetic media of White (1946, 1949) contained 0.01 and 0.04
mg./100 ml. biotin and O.OOO1 and 0.005 mg./100 mi. folk acid, and
that of Morgan, Morton, and Parker (1950) O.OOO1 mg./100 ml of each
vitamin. Fischer’s ( 1948a) medium contained 0.0007 mg./lOO ml. biotin,
and no folic acid. The biotin and folk acid contents of human plasma are
given by Krebs (1950) as 0.00127 and 0.00175 mg./100 ml., respectively.
(5) NICOTINIC ACIDA N D NICOTINAMIDE.Up to 200 mg./100 ml. of
nicotinamide (Brues et d.,1940) and 250 mg./100 ml. of nicotinic acid
(Hull, Perrone, and Kirk, 1950) could be tolerated by fibroblast cultures
without toxic effect. Nicotinamide at 12.5 mg./100 ml. was found to be
optimum for chick embryo heart and brain, and 25.0 mg./100 ml. for
chick embryo skin (Hetherington, 1946). “AS little as” 300 mg./100 ml.
nicotinamide damaged nerve fibers (Painter, Pomerat, and Ezell, 1949) ;
2,500 mg./100 ml. were totally inhibitory. Morgan, Morton, and Parker
(1950) included both nicotinic acid and nicotinamide in their mixture
no. 199, each at 0.0025 mg./100 ml. White’s (1946, 1949) media had
0.05 mg./100 ml. nicotinic acid, which is of the same order as the amount
used by Fischer (1948a) (0.03 mg./100 ml.), and corresponds to the
amount of nicotinic acid plus amide in an average human plasma (Krebs,
52 CHARITY WAYMOUTH

1950). The chick embryo synthesizes nicotinic acid, so that the amount
in the embryo is finally twenty times that in the unfertilized egg (Snell
and Quarles, 1941).
(6) PANTOTHENATE. The amount of pantothenate in chicken blood
varies significantly with the amount in the diet, but falls within the range
0.02 to 0.05 mg./100 ml. (Pearson, Melass, and Sherwood, 1946) ; this
is higher than the average for human plasma given by Krebs (1950), i.e.,
0.012 mg./100 ml. Calcium pantothenate was included in the media of
Morgan, Morton, and Parker (1950) at 0.001, Fischer (194th) at 0.007,
and White (1949) at 0.01 mg./100 ml.
(7) P-ALANINE. Fischer (1941a), treating p-alanine as an amino acid
inter aliu, but also having in mind its possible use as a pantothenic acid
precursor, found that it had no effect, alone, on his fibroblast cultures
in dialyzed media, but that it enhanced the effect of cystine when the two
together were supplied at 1.7 mg./100 ml. amino-N. Vogelaar (1953)
reports that cystine can be dispensed with in a feeding solution for human
fibroblasts, but attests the importance of p-alanine. The synthetic medium
of White (1949) contained 0.05 mg./100 ml. 8-alanine.
(8) INOSITOL.Fetal plasmas contain more inositol than adult (Nixon,
1952). The amounts in human fetal and adult plasmas are 8.9 and
0.68 mg./100 ml. respectively, and in sheep, 29.0 and 1.4 mg./100 ml.
The media of White (1946, 1949) contained 0.05 mg./l00 ml. and of
Morgan, Morton, and Parker (1950) 0.005 mg./100 ml.
(9) ~AMINOBENZOIC ACID. A semisynthetic medium for malaria
parasites, containing proteose peptone, could be made fully synthetic by
replacing the peptone (150 mg./100 ml.) by p-aminobenzoic acid (0.01
mg./100 ml.). This was the optimum concentration; 1.0 mg./100 ml.
was inhibitory to the growth of the parasites (Anfinsen et al., 1946). At
1,500 mg./100 ml. (saturation), p-aminobenzoic acid was not toxic to
nerve fibers in vitro (Painter, Pomerat, and Ezell, 1949). The medium of
Fischer (1948a) for embryonic fibroblasts contained 0.1, and Morgan,
Morton, and Parker’s (1950) medium no. 199 contained 0.005 mg./100
ml. p-aminobenzoic acid.
(10) CHOLINE. The free choline in plasma is rather constant at 0.1
to 0.2 mg./100 ml. (Bligh, 1952). Amounts of 500 mg./100 ml. could be
tolerated by fibroblast cultures (Brues et d.,1940) without inhibitory
effect. Choline was used by Fischer (194th) at 1.0 mg./100 rnl. in his
mixture V-605, by White at 0.5 mg./100 ml. (1946) on 0.1 mg./lOO ml.
(1949), and by Morgan, Morton, and Parker (1950) at 0.05 mg./100 nil.
One of the methods by which fixed tissue cells can be transformed into
macrophages is the addition of choline to the culture medium (Thomas,
 THE KUTRITION O F A N I M A L CELLS 53

1937). Chivremont (1943, 1945, 1948; Bacq and Chhremont, 1944)

has studied this transformation very thoroughly and he found (1943) that
the optimum concentration for niuscle cells in hanging-drop cultures was
3 to 4 mM. (40 to SO mg./100 ml.) and in Carrel flasks 1.3 mM. (16
mg./100 ml.). Acetylcholine at similar molarities was also effective.
Hepatic cells are morphologically altered in a similar way (Frederic,
1951), but the optimum concentration of choline was found to be higher
(10 mM., i.e., 121 mg./100 ml.). Among many amines examined, LettrC
and Albrecht (1943) found that choline chloride at 16 to 160 mg./100 ml.
did not produce vacuolisation of chick fibroblasts.
(1 1 ) VITAMINBiz. Human serum contains an average of 20 mpg./lOO
ml. (range 8 to 42) of vitamin BIZ (Rosenthal and Sarett, 1952). Vitamin
B12 at 0.001 to 0.01 mg./100 ml. had no effect on the maturation in zritro
of megaloblasts from bone marrow of pernicious anemia patients (Thomp-
son, 1952). Evidence for its nutritional value to other cells is not yet
available. Chick fibroblasts have been shown to be able to tolerate exces-
sively high concentrations of vitamin I312 (up to 0.5 mg./lOO ml.)
(Waymouth, unpublished), but this and very much lower concentrations
have no apparent effect on growth in short-term experiments.
X. HORMONES
With the exception of insulin, thyroxin, and some of the steroid
hormones, which have received a good deal of attention, there have been
few systematic studies on the effects of hormones on tissue cultures or of
their possible roles in cell nutrition.
Insulin in a very wide range of concentrations has been incorporated
into tissue culture media. Gey and Thalhimer (1924), at a time when
insulin had not been very highly purified, used the preparation then availa-
ble at 1 to 2 g./100 ml., and reported larger and heavier growth of
fibroblasts. This was probably something over 20,000 units per 100 ml.
( 1935 International units). Kuczinski, Tenenbaum, and Werthemann
(1925) used 0.66 unit/100 ml. to counteract the deleterious effect of a
high (0.25%) glucose concentration on guinea pig liver cultures. Roffo
(1928a, b ; 1930) studied the effects of insulin on normal and neoplastic
tissues in Vitm and Roffo and Ferramola (1930) measured glycolysis in
cultures of normal and tumor cells at 0 and 48 hours and found glycolysis
greater when insulin was added. Gomes da Costa (1935) found that small
concentrations of insulin increased respiration and depressed glycolysis ;
high concentrations diminished respiration and stimulated glycolysis.
Insulin, at an optimum concentration of slightly less than 0.2 unit/100 ml.,
was considered by Vogelaar and Erlichman (1933) to be a cause of
54 CHARITY WAYMOUTH

marked improvement in any medium containing glucose. B’aker ( 1936),

in her modification of the Vogelaar medium, used 0.09 unit/100 ml. for
fibroblasts and 0.012 to 0.024 unit/100 ml. for monocytes. Baker and
Ebeling’s (1939) medium, and Vogelaar and Erlichman’s (1938) medium,
contain 0.1 unit/100 ml. For leukocyte cultures, Wallbach (1938)
employed 1 unit/100 ml. Latta and Bucholz (1939) found that migra-
tion and proliferation of chick heart fibroblasts were not affected, in the
first and second passages in d r o , by the addition of insulin up to 100
units/lO nil. to the standard medium of heparin-plasma and embryo
extract. 333.3 units/100 ml. caused a slight effect and a-marked increase
in fat deposition. Fetal heart fibroblasts were stimulated to proliferation
and increase in area by 100 units/100 ml. (von Haam and Cappel, 194Ob),
and slightly larger amounts (200 to 300 units/100 nil., i.e., approximately
10 to 15 mg./100 ml.) were found by Leslie and Davidson (1951b) to
promote proliferation in chick heart explants in a “fully adequate growth-
promoting medium.” A rise in ribonucleic acid phosphorus per cell was
obtained. In conjunction with cortisone and/or pituitary growth hormone
(Leslie, 1952), insulin caused an increase in lipid phosphorus also.
Crystalline or protamine zinc insulin at 1,ooO units/100 ml. (45 mg./100
tnl.) did not at first inhibit nerve fiber outgrowth, though it did so at
48 hours (Painter and Pomerat, 1948). Current evidence points to the
importance of the function of insulin in reversing thiamine dephosphoryla-
tion (Foa et d., 1952) and in increasing cell membrane permeability to
glucose and other biologically important sugars and so accelerating their
uptake (Ross, 1953). The extent to which insulin exerts any true
hormonal effect in tissue cultures is uncertain. It is possible that such
effects as are observed are largely unspecific. Fischer (1941a) demon-
strated that insulin (when denatured) was among the sulfur-containing
compounds that could in part replace cystine in media rendered deficient
by dialysis.
S m u r a (1931) studied the effects of thyroxin on chick fibroblasts in
plasma and embryo extract. Ten milligrams per 100 ml. were inhibitory ;
UP to 1 mg./100 ml., growth increased with concentration. Cultures in
plasma (without embryo extract) were stimulated by 0.1 or 1.0 mg./100
ml., and degeneration was retarded. Vogelaar and Erlichman (1936a),
on the other hand, found that thyroxin over a wide range of concentrations
(0.01 to 10 mg./100 ml.) had no obvious effect on the growth of human
fibroblasts. In fetal mouse heart cultures, von Haam and Cappel (194Ob)
showed that the addition of O.ooO1 to 0.01 mg./loO ml. thyroxin produced
a more rapid gain in area and more mitoses, compared with normal
controls. Baker (1936) used 0.009 or 0.000113 mg./100 ml. thyroxin
 THE NUTRITION O F AXIM A L CELLS 55

respectively in her media for fibroblasts or monocytes. Carrel (quoted by

Parker, 1938) was able to produce profound morphological changes in
normal fowl leucocytes in vitro by 2.5 mg./100 ml. thyroxin.
Adrenaline at 0.01 to 10 mg./100 ml. was inhibitory to mouse heart
fibroblasts (von Haam and Cappel, 1940b). This may be due, according
to the theory of LettrC and Albrecht (1941), to the effect of its oxida-
tion product, adrenochrome. LettrC believes that tumor cells, in contrast
to normal cells, have lost the capacity to oxidize adrenaline and that tumor
celIs are therefore not subject to mitotic inhibition in vitro in the presence
of adrenaline. The uninhibited growth of tumor cells in viva is likewise
ascribed to the failure of this regulatory mechanism. Gaillard and Veer
(1948) found that adrenochrome could increase the radial migration
of fibroblasts, but it also reduced the number of cells entering mitosis and,
at certain concentrations, caused profongation of the metaphase without
giving rise to any morphological abnormalities. Baker and Ebeling’s
(1939) medium contained adrenaline, adrenal cortical hormone, antuitrin
and pitressin.
Antuitrin was found by Semura (1931) to be inhibitory to chick
fibroblasts at 10 to 1000 mg./100 ml. Trowel1 and Willmer (1939) and
Davidson and Waymouth (1943) found that anterior pituitary extracts
had no effect on the growth or nucleoprotein content of chick fibroblasts.
From ovarian explants derived from rabbits previously treated for three
to four days with 100 to 200 units of prolan, or with 15 t o 20 ml. of
pregnancy urine, there was a notable growth of fibroblasts, compared with
explants from untreated animals (Vercesi and Guercio, 1935). Lactogenic
hormone was found not to stimulate the growth of chick connective tissue,
epidermis or esophageal epithefium (SalIe and Shechmeister, 1936).
Estrone, at an optimum concentration of 0.01 mg./100 ml., stimulated
fetal mouse heart fibroblasts to greater outgrowth and slightly increased
mitotic activity (von Haam and Cappel, 194Oa). Human malignant
ascites cells were not affected by a medium saturated with estrone (666
mg./100 ml.) (Ivers, Pomerat, and Neidhardt, 1948). A medium con-
taining 1.25 mg./100 ml. estrone sulfate had, in one case, a remarkable
stimulatory effect on the epithelium of human malignant ovarian tissue
(Rose, Townsend, and Pomerat, 1951). Among other steroids, von
Mollendorff (1941) found that mitotic disturbances were caused by
estrone, estradiol, testosterone, methyltestosterone and diethylstilbestrol at
0.10 to 0.25 mg./100 ml. According to Bullough (1952) the mitotic
rate in mouse epidermis is increased by glycogen and androgens, the
duration of mitosis (about 2% hours) remaining unaltered. With estrone,
56 CHARITY WAYMOUTH

the number of mitoses is increased and the duration of each mitosis is

reduced to less than one hour.
Cortisone is said to stimulate the secretory activity of the renal proximal
tubules of the chick in tissue culture (Chambers and Cameron, 1944) and
to reduce the number of cells which migrate from lymph node cultures
(Heilman, 1945). There is, however, as Barski and de Brim (1952) and
Trowell (1953) have noted, some conflict among the results of different
workers with adrenal cortical hormones on cells in vitro. Ruskin, Pomerat,
and Ruskin (1951) found that the toxicity of various cortisone prepara-
tions (acetate, sulfate, etc.) varied with the preparation and mode of
solubilizing it, but that the toxicity was in no case high. In cultures of
adult rabbit subcutaneous tissue, spleen, and kidney, and of embryonic
mouse liver and lung, which were carried on for two to three weeks,
Barslci and de Brion (1952) observed the effects of various concentra-
tions of cortisone or deoxycorticosterone. At 5 to 10 mg./lOO ml.,
cortisone had no effect on the growth of various fibroblasts or on renal
epithelium, nor did it visibly affect collagen formation in vitro. Fifty
milligrams per 100 ml. caused degeneration at fifteen to seventeen days
in adult rabbit spleen, kidney, and subcutaneous tissues grown in a medium
containing, probably in addition to an embryonic extract and a balanced
salt solution, 30% homologous serum, 10% horse serum and 5% Ringer
solution containing 0.05% Tween 20 (polyoxyalkylene sorbitan mono-
laurate) rfl the steroid. After about seventeen days in 50 mg./lOO ml.
cortisone, all epithelium in the kidney cultures disappeared, leaving healthy
histiocytes. Serum from cortisone-treated rabbits stimulated cell migra-
tion. Deoxycorticosterone acetate at 50 mg./100 ml. in spleen cultures
completely (but reversibly up to six days) inhibited outgrowth. Cornman
( 1950) showed that deoxycorticosterone reversibly inhibited heart beat
in tissue culture, and that this inhibition was more effective in the absence
of potassium. It has been suggested (Elliott and Yrarrazaval, 1952) that
the adrenal hormones (especially cortisone) govern the permeability of
cell membranes. Trowell (1953) has reviewed previous work on the
effects of cortisone (and C-ll-oxygenated adrenal steroids in general) on
lymphocytes in vitro. Several groups of workers, e.g., Baldridge et ad.
(1951) and Delaunay, Delaunay, and Lebrun (1949) had found no
effects even with rather high concentrations (e.g., 100 mg./100 rnl. corti-
sone acetate). Using his method (Trowell, 1952) for the maintenance of
whole lymph nodes in culture for relatively short periods, Trowell (1953)
estimated lymphocyte viability by counts of pyknotic (dead) cells in fixed
and stained films after five hours’ treatment with cortisone. Five hours
had been found (Trowell, 1953) to be the time of maximum toxic effect
 THE NUTRITION OF ANIMAL CELLS 57

with agents such as cyanide, mercury, and X-rays. I n the range 0.01 to 1.0
mg./100 ml., a statistically significantly toxic effect was noted even with
0.01 mg./lOO ml. Increasing the concentration one-hundredfold increased
the effect only slightly. When it was found that the maximal effect was
obtained with cortisone, not after five but after forty-six hours, a greater
spread of effect with concentration appeared, and at the highest concentra-
tion (1.0 mg./lOO ml.), 50% of the lymphocytes were pyknotic after
forty-six hours. Under the same conditions, other steroids (ll-deoxy-
corticosterone, 11-deoxycorticosterone acetate, testosterone, estradiol, and
progesterone) had little or no effect compared with that of cortisone.
XI. CONCLUDING
REMARKS
It cannot be said that a clear picture of the general and special nutritional
needs of metazoan cells emerges from the present stock of biochemical
information. There are, however, indications that the design may be
drawn in bold strokes. The ionic environment has important effects on
the balance between cells and nutrient medium. The p H and the oxida-
tion-reduction potential, not perhaps strictly nutritional factors, are never-
theless highly relevant to the maintenance of proper physiological con-
ditions. So is the gas exchange between cells and environment. Sources
of energy and of all the materials for the renewal or synthesis of proto-
plasm must be available to the cells. The needs of the cells for prolonged
maintenance may prove fewer in number and simpler in structure than has
often been assumed. Of the special requirements of growing cells there is
still little precise knowledge.
Much of the information which has been reviewed here needs to be
rescrutinized and systematically retested. Because embryonic cells have
proved such convenient experimental material, our knowledge of cell
nutrition relates preponderantly to these cells of high growth potential.
Comparative investigations are needed of the nutritional requirements of
many tissues, embryonic, adult, and neoplastic, in relation to maintenance,
growth, and (where appropriate) function. The development of media
of exactly known chemical composition in which cells can survive and
function for long periods is most important for controlled physiological and
pharmacological studies at the cellular level. The study of growth in rela-
tion to cell nutrition has long been handicapped by insufficiently precise
quantitative methods for assessing growth. Uniform strains of cells, derived
from single cells (Sanford, Earle, and Likely, 1948; Likely, Sanford, and
Earle, 1952) are now available, and cell nuclei can be enumerated (Sanford
et al., 1951) as a means of determining changes in cell population. These
must prove powerful aids to the study of nutrition and growth. The key
58 CHARITY WAYMOUTH

questions to open up the next advance are: (1) How far can a given
environment be modified by any particular cell type, so that the cell can
maintain full functional and metabolic activity? and (2) With how few
and how simple components can this environment be prepared ex-
perimentally ? The application and amplification of information from the
whole field of biochemistry will be needed to supply the answers.
ACKNOWLEDGMENT
The author acknowledges gratefully the helpful and constructive criticisms of
Dr. Philip R. White and Dr. Wilton R. Earle, who read the manuscript.
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