3. C O N S T R U C T I O N A N D USE OF S Y N T H E T I C M E D I A
115
twelve (i.e. including threonine), plus glutamine, can support avian
lung cells (Neuman and Tytell, 1960). Haff and Swim's (1957a) rabbit
fibroblasts RM3-56 require serine as well as glycine for optimal growth,
though these cells can synthesize serine slowly. The Jensen sarcoma
(McCoy et al., 1959a) can grow, but not well, in a twelve amino-acid
mixture. Glutamine and asparagine are stimulatory, and glucose
carbon can act as a satisfactory precursor for alanine, aspartic acid
and glutamic acid, proline, serine and glycine, though the addition
of the last two improves growth. Maxwell, Orr and M c C o y (1959)
have isolated an asparagine-dependent variant of the Jensen sarcoma
cell. The Novikoff hepatoma also requires, in addition to the twelve
"essential" amino acids, glycine or serine for growth (McCoy et al.>
1959b). In the absence of arginine, these cells undergo a morphological
change from round to fibroblast-like cells.
As the vitamins were identified, and their empirical importance in
nutrition recognized, they were incorporated into media for cells. In
each case, their mode of action was at first obscure, but effects on cell
nutrition were demonstrable. Baker's early semi-synthetic media
(1936, 1938, 1939) contained the then recognized vitamins C, A
(containing D ) , and vitamins B x and B 2 .
Most synthetic media today contain thiamine, riboflavin, members
of the folic acid, pyridoxine and nicotinic acid groups, pantothenate,
choline and inositol. The requirement for biotin is equivocal. Eagle
(1955d) reported that HeLa and strain-L cells did not require it, but
his media were supplemented with serum, which would supply biotin.
Recent studies on the function of biotin (e.g. Whitaker and Umbreit,
1961), indicating for it a role in the exchange of inorganic phosphate
with A T P , suggests that the presence of biotin in tissue culture media
might raise the efficiency of phosphate exchange and perhaps diminish
the need for the high concentrations of inorganic phosphate which
have been found beneficial (Waymouth, 1954b, 1956, 1959). Several
studies have been made of the relative effects of the cofactors and their
parent vitamins. Eagle (1956) studied the relative activities of some of
them, for strain-L and HeLa cells. In most cases, the vitamins are
equivalent or, as in the case of pantothenate, more active (cf. also
Mouchette, 1957). Folic acid is less active than the natural citrovorum
factor (leucovorin). Fischer and Welch (1957) also found, for their
L-5178 mouse leukaemic cells, that leucovorin was more effective, as
did Swim and Parker (1958d) for uterine fibroblasts and Schindler,
Day and Fischer (1959) for a strain of neoplastic mast cells. In Eagle's
system, /?-aminobenzoic acid was inactive, though this precursor is
used in Trowell's (1955, 1959) media for lymph nodes and other organ
cultures. Several media contain vitamin B 1 2 , and it has been shown to
115
twelve (i.e. including threonine), plus glutamine, can support avian
lung cells (Neuman and Tytell, 1960). Haff and Swim's (1957a) rabbit
fibroblasts RM3-56 require serine as well as glycine for optimal growth,
though these cells can synthesize serine slowly. The Jensen sarcoma
(McCoy et al., 1959a) can grow, but not well, in a twelve amino-acid
mixture. Glutamine and asparagine are stimulatory, and glucose
carbon can act as a satisfactory precursor for alanine, aspartic acid
and glutamic acid, proline, serine and glycine, though the addition
of the last two improves growth. Maxwell, Orr and M c C o y (1959)
have isolated an asparagine-dependent variant of the Jensen sarcoma
cell. The Novikoff hepatoma also requires, in addition to the twelve
"essential" amino acids, glycine or serine for growth (McCoy et al.>
1959b). In the absence of arginine, these cells undergo a morphological
change from round to fibroblast-like cells.
As the vitamins were identified, and their empirical importance in
nutrition recognized, they were incorporated into media for cells. In
each case, their mode of action was at first obscure, but effects on cell
nutrition were demonstrable. Baker's early semi-synthetic media
(1936, 1938, 1939) contained the then recognized vitamins C, A
(containing D ) , and vitamins B x and B 2 .
Most synthetic media today contain thiamine, riboflavin, members
of the folic acid, pyridoxine and nicotinic acid groups, pantothenate,
choline and inositol. The requirement for biotin is equivocal. Eagle
(1955d) reported that HeLa and strain-L cells did not require it, but
his media were supplemented with serum, which would supply biotin.
Recent studies on the function of biotin (e.g. Whitaker and Umbreit,
1961), indicating for it a role in the exchange of inorganic phosphate
with A T P , suggests that the presence of biotin in tissue culture media
might raise the efficiency of phosphate exchange and perhaps diminish
the need for the high concentrations of inorganic phosphate which
have been found beneficial (Waymouth, 1954b, 1956, 1959). Several
studies have been made of the relative effects of the cofactors and their
parent vitamins. Eagle (1956) studied the relative activities of some of
them, for strain-L and HeLa cells. In most cases, the vitamins are
equivalent or, as in the case of pantothenate, more active (cf. also
Mouchette, 1957). Folic acid is less active than the natural citrovorum
factor (leucovorin). Fischer and Welch (1957) also found, for their
L-5178 mouse leukaemic cells, that leucovorin was more effective, as
did Swim and Parker (1958d) for uterine fibroblasts and Schindler,
Day and Fischer (1959) for a strain of neoplastic mast cells. In Eagle's
system, /?-aminobenzoic acid was inactive, though this precursor is
used in Trowell's (1955, 1959) media for lymph nodes and other organ
cultures. Several media contain vitamin B 1 2 , and it has been shown to