470
C L I F F O R D G R O B S T E I N
discussed below. For the moment the important point is that general
culture environments which preserve the integrity of relatively complex
populations of cells have favored differentiation, whereas environments
and factors which disperse and simplify the relations within such populations have tended to inhibit differentiation.
Certainly this phenomenon is not independent of the character of the
nutrient medium and other cultural factors, and it is to be assumed that
as media more effective for differentiation are found, dispersal of cells
into them will be less anti-differentiative in effect. In some measure this
already is occurring. T o date, however, complex media—incorporating
uncharacterized materials found in sera and embryonic tissue extracts—
have been more widely used in studying differentiation in organ cultures
than the simpler, defined media which support growth in many strains
of cultured cells. With few, but recently increasing, exceptions (Wolff,
Haffen, Kieny and Wolff, 1953; Lasfargues and Murray, 1959;
Wessells, 1961a, b ; Lasnitzki, 1961), organ culturists continue to use
mixtures of animal serum and embryonic extracts, at least as supplements to defined media. In some instances differentiative behavior has
been shown to be altered by sera and extracts from different sources or
in different ratios. A number of early workers found that deletion of
embryo juice from complex media favors differentiation, an effect
ascribed to removal of the growth-promoting activity of the embryo
juice. Gaillard (1935) reported that extracts from embryos of different
ages had differing differentiation-promoting and maintaining properties, and that optimal effects could be obtained by employing extracts
from embryos of ascending age. Endo (1960) found that varying the
ratio of horse serum and chick-embryo juice alters the relative amount
of chondrogenesis and osteogenesis of chick-embryo femora in roller
tubes. Wolff and associates (see Borghese, 1958,, for references; Sengel,
1960) have examined the role of various amino acids and other components of standard media, and have produced variations in a number
of differentiating systems. Considerable effort has been devoted to
finding a defined medium for culture of embryonic chick long bones
(Biggers, Gwatkin and Heyner, 1961). A generally satisfactory medium
to support differentiation in all systems, however, has not yet been
defined. Recently, Wessells (1961b) found that chick-embryo shank skin
survives and continues differentiation in vitro on a protein-free, defined
medium modified from Waymouth's MB 752/1. Methionine appeared
to be essential for full keratinization, and by varying its availability the
differentiation process could be controlled in part. Cysteine and cystine
were not essential to early cornification, but supplying them increased
the amount of cornified material formed. It is clear that these and
related results hold promise of understanding the overall nutritional
C L I F F O R D G R O B S T E I N
discussed below. For the moment the important point is that general
culture environments which preserve the integrity of relatively complex
populations of cells have favored differentiation, whereas environments
and factors which disperse and simplify the relations within such populations have tended to inhibit differentiation.
Certainly this phenomenon is not independent of the character of the
nutrient medium and other cultural factors, and it is to be assumed that
as media more effective for differentiation are found, dispersal of cells
into them will be less anti-differentiative in effect. In some measure this
already is occurring. T o date, however, complex media—incorporating
uncharacterized materials found in sera and embryonic tissue extracts—
have been more widely used in studying differentiation in organ cultures
than the simpler, defined media which support growth in many strains
of cultured cells. With few, but recently increasing, exceptions (Wolff,
Haffen, Kieny and Wolff, 1953; Lasfargues and Murray, 1959;
Wessells, 1961a, b ; Lasnitzki, 1961), organ culturists continue to use
mixtures of animal serum and embryonic extracts, at least as supplements to defined media. In some instances differentiative behavior has
been shown to be altered by sera and extracts from different sources or
in different ratios. A number of early workers found that deletion of
embryo juice from complex media favors differentiation, an effect
ascribed to removal of the growth-promoting activity of the embryo
juice. Gaillard (1935) reported that extracts from embryos of different
ages had differing differentiation-promoting and maintaining properties, and that optimal effects could be obtained by employing extracts
from embryos of ascending age. Endo (1960) found that varying the
ratio of horse serum and chick-embryo juice alters the relative amount
of chondrogenesis and osteogenesis of chick-embryo femora in roller
tubes. Wolff and associates (see Borghese, 1958,, for references; Sengel,
1960) have examined the role of various amino acids and other components of standard media, and have produced variations in a number
of differentiating systems. Considerable effort has been devoted to
finding a defined medium for culture of embryonic chick long bones
(Biggers, Gwatkin and Heyner, 1961). A generally satisfactory medium
to support differentiation in all systems, however, has not yet been
defined. Recently, Wessells (1961b) found that chick-embryo shank skin
survives and continues differentiation in vitro on a protein-free, defined
medium modified from Waymouth's MB 752/1. Methionine appeared
to be essential for full keratinization, and by varying its availability the
differentiation process could be controlled in part. Cysteine and cystine
were not essential to early cornification, but supplying them increased
the amount of cornified material formed. It is clear that these and
related results hold promise of understanding the overall nutritional