2 . M E T H O D S
33
liberated from it may have considerable effects on the nature of any
adjacent cell surfaces. These ionic effects could be purely superficial
or they could alter the distribution of charges within the cell membranes
and thus alter the distribution and packing of the molecules into the
cell surface, thus perhaps changing its permeability and even its
enzymic systems. In any case, for purely mechanical reasons, the
interchange of metabolites through the surface of the cell next to the
glass must be very restricted and abnormal as compared with the interchange which normally goes on across the surface of the same cell in
the body. The normal polarity of the cell may thus be considerably
altered.
It must be emphasized therefore that the cells which emerge from a
tissue, cultured in a hanging drop of fluid medium, are limited to those
which can tolerate juxtaposition to a surface like that of glass, and thus
they may be derived only from certain classes of cells present within the
original explant. Moreover, their subsequent behaviour must depend
upon the nature of their interaction with the glass or other surface, and
they are inevitably to some extent abnormal. Finally, their original
interplay with neighbouring cells, perhaps of different types from themselves is interfered with, if not actually prevented; and this again may
provoke uncharacteristic behaviour. Such considerations emphasize the
severe limitations of the method and must always be kept in mind when
results obtained by this type of culture are being evaluated.
On the other hand, a cell which has thus flattened itself against the
glass is under excellent optical conditions for direct observation under
the highest powers of the light-microscope. By suitably mounting such
cultures on flat slides or in slide chambers (pp. 38-9), to eliminate the
disturbing effects of the concavity in the slide, studies can readily be
made with the polarizing and interference microscopes and also with
dark-field illumination and with the phase-contrast microscope. By
means of time-lapse cinematography it is possible to study the behaviour, movement and division of cells living under these conditions
in more detail than can be readily achieved by any other method.
As a cytological method, therefore, the hanging-drop culture in a
fluid medium is excellent for certain purposes, but it has very definite
limitations for others. As a further and interesting example of a limitation, it may be mentioned that when cells are seen in division in
the zone of outgrowth in hanging-drop cultures in fluid media the
rnetaphase plate of chromosomes is always orientated at right angles
to the plane of the glass surface. Because of this peculiarity it was not
until the technique was evolved of treating cells with a hypotonic
solution before fixation to disperse their chromosomes (Hughes, 1952;
Hsu and Pomerat, 1953a) that cultures could be used for even approxo
33
liberated from it may have considerable effects on the nature of any
adjacent cell surfaces. These ionic effects could be purely superficial
or they could alter the distribution of charges within the cell membranes
and thus alter the distribution and packing of the molecules into the
cell surface, thus perhaps changing its permeability and even its
enzymic systems. In any case, for purely mechanical reasons, the
interchange of metabolites through the surface of the cell next to the
glass must be very restricted and abnormal as compared with the interchange which normally goes on across the surface of the same cell in
the body. The normal polarity of the cell may thus be considerably
altered.
It must be emphasized therefore that the cells which emerge from a
tissue, cultured in a hanging drop of fluid medium, are limited to those
which can tolerate juxtaposition to a surface like that of glass, and thus
they may be derived only from certain classes of cells present within the
original explant. Moreover, their subsequent behaviour must depend
upon the nature of their interaction with the glass or other surface, and
they are inevitably to some extent abnormal. Finally, their original
interplay with neighbouring cells, perhaps of different types from themselves is interfered with, if not actually prevented; and this again may
provoke uncharacteristic behaviour. Such considerations emphasize the
severe limitations of the method and must always be kept in mind when
results obtained by this type of culture are being evaluated.
On the other hand, a cell which has thus flattened itself against the
glass is under excellent optical conditions for direct observation under
the highest powers of the light-microscope. By suitably mounting such
cultures on flat slides or in slide chambers (pp. 38-9), to eliminate the
disturbing effects of the concavity in the slide, studies can readily be
made with the polarizing and interference microscopes and also with
dark-field illumination and with the phase-contrast microscope. By
means of time-lapse cinematography it is possible to study the behaviour, movement and division of cells living under these conditions
in more detail than can be readily achieved by any other method.
As a cytological method, therefore, the hanging-drop culture in a
fluid medium is excellent for certain purposes, but it has very definite
limitations for others. As a further and interesting example of a limitation, it may be mentioned that when cells are seen in division in
the zone of outgrowth in hanging-drop cultures in fluid media the
rnetaphase plate of chromosomes is always orientated at right angles
to the plane of the glass surface. Because of this peculiarity it was not
until the technique was evolved of treating cells with a hypotonic
solution before fixation to disperse their chromosomes (Hughes, 1952;
Hsu and Pomerat, 1953a) that cultures could be used for even approxo