498
A. A. MOSCONA
to cell aggregation phenomena is not clear, and their implications might
be misleading.
D. EXTRINSIC FACTORS IN
ROTATION-MEDIATED AGGREGATION
Among the extrinsic factors affecting aggregation of dissociated
embryonic cells in rotating flasks, speed of rotation is of interest in that
it permits control of the size-distribution (and sometimes the cellular
composition) of the resulting aggregates, and the ratio of aggregated to
free cells. Needless to stress, the effects of rotation-speed vary with the
kind, concentration and treatment of the cells, and also with the size
and shape of flask, the quantity and composition of culture medium,
and the temperature. In general, under otherwise uniform conditions,
at higher speeds of rotation the shearing effects are greater and the
collision-frequency of the cells is lower; the gross effect is a decrease in
the size of aggregates, and an increase both in their number and in the
number of free cells. For example: a suspension of cells from the liver
of a 7-day chick embryo, rotated at 70 rev/min for 24 h, aggregates
typically into a single mass, including practically all the cells; at 85
rev/min, other conditions being the same, numerous spherical aggregates form; at still higher speeds the number of aggregates and free
cells increases concurrently with a further decrease in the size of the
aggregates (Moscona, 1961a). The rate and pattern of change for a
given tissue varies according to differences in the age of the donor
embryo; other pertinent variables have been mentioned above. In our
test-system compact aggregates do not form above 120 rev/min; cells
that are able to become mutually attached at this speed probably
represent the most mutually adhesive constituents of the tested cell
population. Thus, by exposing aliquots of the same cell suspension to
different speeds of rotation and harvesting the aggregates at suitable
intervals a cell suspension may be "fractionated" according to "differential adhesiveness" of cells. T o o high speeds, which cause frothing of
the medium and excessive cell damage, should be avoided.
Temperature has a controlling effect on aggregation of dissociated
cells in rotating cultures. At lower-than-optimal levels the formation of
compact cell clusters declines; at 25°C dispersed neural retina cells
(7-day chick embryos; 70 rev/min) yield minute clusters and free cells;
liver cells form amorphous, very loosely cohering masses. At about
15°C aggregation is persistently inhibited in spite of the cells being
continuously brought into contact by rotation, irrespective of their
individual mobilities (Fig. 3). Raising the temperature of such cultures
to optimal levels results in aggregation (Moscona, 1961a, c, 1962c;
A. A. MOSCONA
to cell aggregation phenomena is not clear, and their implications might
be misleading.
D. EXTRINSIC FACTORS IN
ROTATION-MEDIATED AGGREGATION
Among the extrinsic factors affecting aggregation of dissociated
embryonic cells in rotating flasks, speed of rotation is of interest in that
it permits control of the size-distribution (and sometimes the cellular
composition) of the resulting aggregates, and the ratio of aggregated to
free cells. Needless to stress, the effects of rotation-speed vary with the
kind, concentration and treatment of the cells, and also with the size
and shape of flask, the quantity and composition of culture medium,
and the temperature. In general, under otherwise uniform conditions,
at higher speeds of rotation the shearing effects are greater and the
collision-frequency of the cells is lower; the gross effect is a decrease in
the size of aggregates, and an increase both in their number and in the
number of free cells. For example: a suspension of cells from the liver
of a 7-day chick embryo, rotated at 70 rev/min for 24 h, aggregates
typically into a single mass, including practically all the cells; at 85
rev/min, other conditions being the same, numerous spherical aggregates form; at still higher speeds the number of aggregates and free
cells increases concurrently with a further decrease in the size of the
aggregates (Moscona, 1961a). The rate and pattern of change for a
given tissue varies according to differences in the age of the donor
embryo; other pertinent variables have been mentioned above. In our
test-system compact aggregates do not form above 120 rev/min; cells
that are able to become mutually attached at this speed probably
represent the most mutually adhesive constituents of the tested cell
population. Thus, by exposing aliquots of the same cell suspension to
different speeds of rotation and harvesting the aggregates at suitable
intervals a cell suspension may be "fractionated" according to "differential adhesiveness" of cells. T o o high speeds, which cause frothing of
the medium and excessive cell damage, should be avoided.
Temperature has a controlling effect on aggregation of dissociated
cells in rotating cultures. At lower-than-optimal levels the formation of
compact cell clusters declines; at 25°C dispersed neural retina cells
(7-day chick embryos; 70 rev/min) yield minute clusters and free cells;
liver cells form amorphous, very loosely cohering masses. At about
15°C aggregation is persistently inhibited in spite of the cells being
continuously brought into contact by rotation, irrespective of their
individual mobilities (Fig. 3). Raising the temperature of such cultures
to optimal levels results in aggregation (Moscona, 1961a, c, 1962c;