116
C H A R I T Y W A Y M O U T H
promote the growth of strain-L, clone 929, mouse cells (Evans et al.,
1956b; McQuilkin, Evans and Earle, 1957; Waymouth, 1959; Perlman,
1962); of monkey kidney cells (Melnick et al., 1957) and of HeLa cells
(Allison and Arnstein, 1961). Melnick et al. (1957) were among the
first to point out that deficiencies of the vitamins do not generally
produce immediate effects, and that at least 3 weeks may elapse before
evidence of a deficiency is clearly seen in cultures. The omission of
coenzymes from medium N C T C 109, which also contains a full
complement of vitamins, was demonstrated by Evans, Fioramonti and
Earle (1959) to have no deleterious effect upon N C T C strain 2071 cells,
over a test period of 4 months. The capacities of different cell types to
synthesize inositol vary widely, from which it follows that requirements
for this compound span a wide range. Chang (1958) showed differences
in the requirements for this vitamin between strains of similar cells with
different nutritional histories. Cultures of cells with high synthetic
ability for inositol can supply the needs of cultures of highly inositoldependent cells grown in "parabiosis" with them, i.e. separated by a
semi-permeable Cellophane membrane (Eagle, Agranoff and Snell,
1960). Haff and Swim (1957b) demonstrated requirements for folic
acid, nicotinamide,, pantothenate, pyridoxal, riboflavin and thiamine
for strain RM3-73 rabbit fibroblasts. For a strain of human uterine
fibroblasts, Swim and Parker (1958d) showed the need for choline and
inositol, as well as for the four B vitamins required by the rabbit
fibroblasts. In the presence of pyridoxal, the rabbit cells can dispense
with eight amino acids which this strain requires in its absence. The
uterine cells also require six additional amino acids if pyridoxal is
omitted. These observations again point very cogently to the unsoundness of categorizing components of a medium as "essential" or "nonessential", and to the importance of considering every system as a whole.
In spite of their frequent inclusion in media, e.g. in those of the 199
through 1066 series of Parker et al., some satisfactory synthetic media
do not contain any of the fat-soluble vitamins. For general purposes
of maintaining life and growth, they seem to be unnecessary though,
again, they may be beneficial. White (1946, 1949) used vitamin A
and carotene. The media 199 and 858 of Parker contain vitamins
A, D , E and K, though the later formula C M R L 1066 omits them
(Parker, 1961). The importance of these vitamins, and of many hormones, is much more evident in organ cultures than in cell culture
systems (see below and Chapter 17). There is a large literature on the
application of various hormones to cultures of cells (see Chapter 16),
though not many of these studies have been made in completely
defined media. One hormone which has appeared in tissue culture
media since its first isolation (Gey and Thalhimer, 1924), and may now
C H A R I T Y W A Y M O U T H
promote the growth of strain-L, clone 929, mouse cells (Evans et al.,
1956b; McQuilkin, Evans and Earle, 1957; Waymouth, 1959; Perlman,
1962); of monkey kidney cells (Melnick et al., 1957) and of HeLa cells
(Allison and Arnstein, 1961). Melnick et al. (1957) were among the
first to point out that deficiencies of the vitamins do not generally
produce immediate effects, and that at least 3 weeks may elapse before
evidence of a deficiency is clearly seen in cultures. The omission of
coenzymes from medium N C T C 109, which also contains a full
complement of vitamins, was demonstrated by Evans, Fioramonti and
Earle (1959) to have no deleterious effect upon N C T C strain 2071 cells,
over a test period of 4 months. The capacities of different cell types to
synthesize inositol vary widely, from which it follows that requirements
for this compound span a wide range. Chang (1958) showed differences
in the requirements for this vitamin between strains of similar cells with
different nutritional histories. Cultures of cells with high synthetic
ability for inositol can supply the needs of cultures of highly inositoldependent cells grown in "parabiosis" with them, i.e. separated by a
semi-permeable Cellophane membrane (Eagle, Agranoff and Snell,
1960). Haff and Swim (1957b) demonstrated requirements for folic
acid, nicotinamide,, pantothenate, pyridoxal, riboflavin and thiamine
for strain RM3-73 rabbit fibroblasts. For a strain of human uterine
fibroblasts, Swim and Parker (1958d) showed the need for choline and
inositol, as well as for the four B vitamins required by the rabbit
fibroblasts. In the presence of pyridoxal, the rabbit cells can dispense
with eight amino acids which this strain requires in its absence. The
uterine cells also require six additional amino acids if pyridoxal is
omitted. These observations again point very cogently to the unsoundness of categorizing components of a medium as "essential" or "nonessential", and to the importance of considering every system as a whole.
In spite of their frequent inclusion in media, e.g. in those of the 199
through 1066 series of Parker et al., some satisfactory synthetic media
do not contain any of the fat-soluble vitamins. For general purposes
of maintaining life and growth, they seem to be unnecessary though,
again, they may be beneficial. White (1946, 1949) used vitamin A
and carotene. The media 199 and 858 of Parker contain vitamins
A, D , E and K, though the later formula C M R L 1066 omits them
(Parker, 1961). The importance of these vitamins, and of many hormones, is much more evident in organ cultures than in cell culture
systems (see below and Chapter 17). There is a large literature on the
application of various hormones to cultures of cells (see Chapter 16),
though not many of these studies have been made in completely
defined media. One hormone which has appeared in tissue culture
media since its first isolation (Gey and Thalhimer, 1924), and may now