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
129
media for the study of the effects of oestrone and vitamin A on mouse
vagina (Lasnitzki, 1961).
Bone rudiments, the classical objects of organ culture, were among
the first to be successfully cultivated in synthetic media (Kieny, 1955;
Biggers, Webb, Parker and Healy, 1957). Biggers and Lucy (1960)
Biggers, Gwatkin and Heyner (1961) and Biggers and Heyner (1961)
have introduced two chemically defined media for the cultivation of
bone rudiments. The first (Medium BL 1) has sixty components and
is a modification of the medium 858 of Healy, Fisher and Parker (1955).
It differs from the prototype in that 5-methyl deoxycytidine and sodium
penicillin G are omitted, Earle's saline is replaced by that of Hanks
and Wallace (1949), and the concentrations of most of the amino acids
are doubled. The most effective concentration of glucose is at least
400 mg/100 ml. The much simpler medium BJGa, with thirty-seven
components, dispenses with the nucleosides (except adenosine), with
the coenzymes, and with many of the vitamins of BL 1. It contains 14
(as compared with 21) amino acids, in concentrations which appear
to be those of medium MB 752/1 (Waymouth, 1959). The modification
BJGb, of thirty-five components, omits glycine and adenosine, and has
a lower glutamine content. An interesting modification introduced
in these media is the replacement of calcium chloride, the usual source
of calcium ions in defined media, by the non-deliquescent compound
calcium lactate (Biggers and Lucy, 1960). Biggers has used these media
for many well-controlled experiments on variations in weight, length
and chemical constitution of chick bone rudiments, and on biosynthetic
pathways (Biggers, 1960; Biggers, 1961a,b; Biggers, Lawson, Lucy and
Webb, 1961; Webb and Biggers, 1961; Lucy, Webb and Biggers, 1961).
That media devised for one purpose may sometimes be adapted to
an entirely different culture system is shown by the work of Wessells
(1961a) who slightly modified the medium MB 752/1, designed for
monolayer cultures of mouse fibroblasts (Waymouth, 1959) for use in
the study of epidermal differentiation in organ cultures of chick skin.
Wessells
5 medium differed from the prototype mainly in containing
a trace element mixture (Kitos, Sinclair and Waymouth, 1962),
increased arginine, and varying amounts of sulphur compounds.
Wessells' studies showed that differentiation of chick skin continues
in such defined medium; that homocysteine can serve as the sole
S-amino acid for tissue younger than the 14 day stage, but that after
that time methionine is needed to permit continued differentiation.
Cornification takes place in the absence of exogenous cystine-cysteine,
but the degree of cornification is dependent upon the presence of a
suitable mixture of S-compounds, including, for maximum effect,
cystine-cysteine. With the same in vitro system, Wessells (1961b, 1962)
129
media for the study of the effects of oestrone and vitamin A on mouse
vagina (Lasnitzki, 1961).
Bone rudiments, the classical objects of organ culture, were among
the first to be successfully cultivated in synthetic media (Kieny, 1955;
Biggers, Webb, Parker and Healy, 1957). Biggers and Lucy (1960)
Biggers, Gwatkin and Heyner (1961) and Biggers and Heyner (1961)
have introduced two chemically defined media for the cultivation of
bone rudiments. The first (Medium BL 1) has sixty components and
is a modification of the medium 858 of Healy, Fisher and Parker (1955).
It differs from the prototype in that 5-methyl deoxycytidine and sodium
penicillin G are omitted, Earle's saline is replaced by that of Hanks
and Wallace (1949), and the concentrations of most of the amino acids
are doubled. The most effective concentration of glucose is at least
400 mg/100 ml. The much simpler medium BJGa, with thirty-seven
components, dispenses with the nucleosides (except adenosine), with
the coenzymes, and with many of the vitamins of BL 1. It contains 14
(as compared with 21) amino acids, in concentrations which appear
to be those of medium MB 752/1 (Waymouth, 1959). The modification
BJGb, of thirty-five components, omits glycine and adenosine, and has
a lower glutamine content. An interesting modification introduced
in these media is the replacement of calcium chloride, the usual source
of calcium ions in defined media, by the non-deliquescent compound
calcium lactate (Biggers and Lucy, 1960). Biggers has used these media
for many well-controlled experiments on variations in weight, length
and chemical constitution of chick bone rudiments, and on biosynthetic
pathways (Biggers, 1960; Biggers, 1961a,b; Biggers, Lawson, Lucy and
Webb, 1961; Webb and Biggers, 1961; Lucy, Webb and Biggers, 1961).
That media devised for one purpose may sometimes be adapted to
an entirely different culture system is shown by the work of Wessells
(1961a) who slightly modified the medium MB 752/1, designed for
monolayer cultures of mouse fibroblasts (Waymouth, 1959) for use in
the study of epidermal differentiation in organ cultures of chick skin.
Wessells
5 medium differed from the prototype mainly in containing
a trace element mixture (Kitos, Sinclair and Waymouth, 1962),
increased arginine, and varying amounts of sulphur compounds.
Wessells' studies showed that differentiation of chick skin continues
in such defined medium; that homocysteine can serve as the sole
S-amino acid for tissue younger than the 14 day stage, but that after
that time methionine is needed to permit continued differentiation.
Cornification takes place in the absence of exogenous cystine-cysteine,
but the degree of cornification is dependent upon the presence of a
suitable mixture of S-compounds, including, for maximum effect,
cystine-cysteine. With the same in vitro system, Wessells (1961b, 1962)