2. M E T H O D S
69
working with hanging drops and test-tube cultures, cells can now be
grown when actually kept in suspension (Earle, Schilling, Bryant and
Evans, 1954; Earle et al., 1956), either in some form of "shaker-flask"
system, e.g. Erlenmeyer flasks placed on a platform oscillating horizontally in two dimensions, or in special bottles which can be filled with
known gas mixtures and stirred magnetically, or in "tumbler tubes"
(Fig. 5b), which are essendally roller tubes mounted on a wheel in
such a way as to turn "head-over-heels" about twice per minute
(Owens, Gey and Gey, 1953). For most of these cultures serum must
be present in the medium (Bryant, Schilling, Earle and Evans, 1960),
though increasing the viscosity of the fluid by the addition of hyaluronic
acid or methyl cellulose has also been found to give satisfactory results
and to keep the cells in suspension (Earle et al., 1954; Kuchler, Marlowe
and Merchant, 1960). Care has naturally to be taken about the speed
of rotation and the character of the shaking in order to prevent
frothing of the medium, and mechanical damage to the cells. For many
purposes, such as the study of growth or enzyme production, where
uniformity of the cell population is important, it is desirable also to
prevent the cells from clumping into large masses, though this, as
will be seen in the next paragraph, is not necessarily so. The cells
can in some cases be prevented from clumping by the addition of
"Darvan" (0-03%) to the medium (Merchant, Kuchler and Munyon,
1960). "Darvan" is a polymerized organic salt of sulphonic acid and
probably coats the cells and gives them a negative charge. Again the
ugly question arises, how "normal" are these cells? Such dispersed
suspension cultures can have many advantages (see Merchant and
Eidam, 1961) for the study of the growth process and related metabolic
events, and cells can now certainly be cultured in the mass. Population
densities can sometimes reach very high levels. Cherry and Hull (1960)
record an optimum density of 2 X 10
6 cells/ml in their experiments, but
maxima of 3 X 10
7
/ml have been recorded by others.
Fourth, when cell differentiation rather than growth is the chief aim,
the shaker-flask and its movement may be so designed as to encourage
the meeting, clumping and re-aggregation of the suspended cells. Such
aggregates have given important information in studies of cell-differentiation, cell-contact and cell-cohesion in embryonic tissues (seep. 495).
Once again the nature of the medium, the manner of shaking and the
temperature are very important (Moscona, 1961a). Some test of the
viability of different types of cells after isolation can be obtained by such
methods, since if the cells differentiate in the aggregated masses,
they can then be identified with some certainty, though even here
there are loop-holes in the interpretation for metaplasia and alternative
pathways of differentiation.
69
working with hanging drops and test-tube cultures, cells can now be
grown when actually kept in suspension (Earle, Schilling, Bryant and
Evans, 1954; Earle et al., 1956), either in some form of "shaker-flask"
system, e.g. Erlenmeyer flasks placed on a platform oscillating horizontally in two dimensions, or in special bottles which can be filled with
known gas mixtures and stirred magnetically, or in "tumbler tubes"
(Fig. 5b), which are essendally roller tubes mounted on a wheel in
such a way as to turn "head-over-heels" about twice per minute
(Owens, Gey and Gey, 1953). For most of these cultures serum must
be present in the medium (Bryant, Schilling, Earle and Evans, 1960),
though increasing the viscosity of the fluid by the addition of hyaluronic
acid or methyl cellulose has also been found to give satisfactory results
and to keep the cells in suspension (Earle et al., 1954; Kuchler, Marlowe
and Merchant, 1960). Care has naturally to be taken about the speed
of rotation and the character of the shaking in order to prevent
frothing of the medium, and mechanical damage to the cells. For many
purposes, such as the study of growth or enzyme production, where
uniformity of the cell population is important, it is desirable also to
prevent the cells from clumping into large masses, though this, as
will be seen in the next paragraph, is not necessarily so. The cells
can in some cases be prevented from clumping by the addition of
"Darvan" (0-03%) to the medium (Merchant, Kuchler and Munyon,
1960). "Darvan" is a polymerized organic salt of sulphonic acid and
probably coats the cells and gives them a negative charge. Again the
ugly question arises, how "normal" are these cells? Such dispersed
suspension cultures can have many advantages (see Merchant and
Eidam, 1961) for the study of the growth process and related metabolic
events, and cells can now certainly be cultured in the mass. Population
densities can sometimes reach very high levels. Cherry and Hull (1960)
record an optimum density of 2 X 10
6 cells/ml in their experiments, but
maxima of 3 X 10
7
/ml have been recorded by others.
Fourth, when cell differentiation rather than growth is the chief aim,
the shaker-flask and its movement may be so designed as to encourage
the meeting, clumping and re-aggregation of the suspended cells. Such
aggregates have given important information in studies of cell-differentiation, cell-contact and cell-cohesion in embryonic tissues (seep. 495).
Once again the nature of the medium, the manner of shaking and the
temperature are very important (Moscona, 1961a). Some test of the
viability of different types of cells after isolation can be obtained by such
methods, since if the cells differentiate in the aggregated masses,
they can then be identified with some certainty, though even here
there are loop-holes in the interpretation for metaplasia and alternative
pathways of differentiation.