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
125
Sanford, Earle and Likely, 1948; Sanford, Covalesky, Dupree and
Earle, 1961), or use of an agar substrate (Puck, Marcus and Cieciura,
1956) has been invoked to explain the success of cloning methods.
Deficiency of single nutrient components may make the difference
between success and failure, and supplementation with compounds
which the cells can synthesize only in sub-optimal amounts may provide
just this difference (Lockart and Eagle, 1959; Eagle and Piez, 1962)
by modifying the environment favourably in the critical initial stages
(see Chapter 8). Neuman and McCoy (1958) found that the cloning
efficiency for cells of the Walker 256 carcinosarcoma of the rat could
be increased by inclusion of a-keto acids in the medium. Similarly
we (Waymouth, 1960a) have shown that N C T C 929 cells in certain
synthetic media can be grown from smaller inocula when a-ketoglutarate is included in the medium, although this is a metabolite
which these and other cells produce in fairly substantial amounts in
cultures of larger cell populations (Leslie and Paul, 1954; Westfall,
Peppers and Earle, 1955, 1960).
We look forward to the time when we shall have synthetic media
especially designed to permit primary explants of cells or tissues to
retain most of the properties they have in vivo. We are not yet equipped
either to recognize all the needs of each cell type, or to meet them in
terms of specialized media. Meanwhile, cells do alter in response to the
imperfect media which we supply, and we may suppose that we are, at
least sometimes, selecting cells which are viable under the particular
conditions which we provide. Many criteria of stability or alteration
are needed, and indeed are actively being used (Ross, Treadwell and
Syverton, 1962) to characterize cultivated cells. One which has been
much studied is the chromosome pattern (see Chapter 12). With few
exceptions, those cells which have been successfully grown serially
exhibit marked deviations from the normal diploid chromosome complement of the species (Hsu and Moorhead, 1957). Generally, the
predominant number of chromosomes is large (e.g. the hypotetraploid
number which prevails in many sublines of N C T C 929) and is accompanied by structural changes in the chromosomes to new forms not
characteristic of the stem cell (Hsu and Klatt, 1958; Hsu, 1959). This
is reminiscent of the condition in transplanted tumours after long periods
of repeated serial passage (Levan, 1956). It has even been suggested
that aneuploidy is a necessary condition for long-term cultivation
in vitro (Hsu and Moorhead, 1957; Hayflick and Moorhead, 1961).
Diploid strains of human cells have been maintained for periods of
at least several months (Tjio and Puck, 1958; Chu and Giles, 1959),
with up to or over fifty serial passages (Hayflick and Moorhead, 1961),
but diploid lines of longer duration appear not to have been established.
125
Sanford, Earle and Likely, 1948; Sanford, Covalesky, Dupree and
Earle, 1961), or use of an agar substrate (Puck, Marcus and Cieciura,
1956) has been invoked to explain the success of cloning methods.
Deficiency of single nutrient components may make the difference
between success and failure, and supplementation with compounds
which the cells can synthesize only in sub-optimal amounts may provide
just this difference (Lockart and Eagle, 1959; Eagle and Piez, 1962)
by modifying the environment favourably in the critical initial stages
(see Chapter 8). Neuman and McCoy (1958) found that the cloning
efficiency for cells of the Walker 256 carcinosarcoma of the rat could
be increased by inclusion of a-keto acids in the medium. Similarly
we (Waymouth, 1960a) have shown that N C T C 929 cells in certain
synthetic media can be grown from smaller inocula when a-ketoglutarate is included in the medium, although this is a metabolite
which these and other cells produce in fairly substantial amounts in
cultures of larger cell populations (Leslie and Paul, 1954; Westfall,
Peppers and Earle, 1955, 1960).
We look forward to the time when we shall have synthetic media
especially designed to permit primary explants of cells or tissues to
retain most of the properties they have in vivo. We are not yet equipped
either to recognize all the needs of each cell type, or to meet them in
terms of specialized media. Meanwhile, cells do alter in response to the
imperfect media which we supply, and we may suppose that we are, at
least sometimes, selecting cells which are viable under the particular
conditions which we provide. Many criteria of stability or alteration
are needed, and indeed are actively being used (Ross, Treadwell and
Syverton, 1962) to characterize cultivated cells. One which has been
much studied is the chromosome pattern (see Chapter 12). With few
exceptions, those cells which have been successfully grown serially
exhibit marked deviations from the normal diploid chromosome complement of the species (Hsu and Moorhead, 1957). Generally, the
predominant number of chromosomes is large (e.g. the hypotetraploid
number which prevails in many sublines of N C T C 929) and is accompanied by structural changes in the chromosomes to new forms not
characteristic of the stem cell (Hsu and Klatt, 1958; Hsu, 1959). This
is reminiscent of the condition in transplanted tumours after long periods
of repeated serial passage (Levan, 1956). It has even been suggested
that aneuploidy is a necessary condition for long-term cultivation
in vitro (Hsu and Moorhead, 1957; Hayflick and Moorhead, 1961).
Diploid strains of human cells have been maintained for periods of
at least several months (Tjio and Puck, 1958; Chu and Giles, 1959),
with up to or over fifty serial passages (Hayflick and Moorhead, 1961),
but diploid lines of longer duration appear not to have been established.