2 . M E T H O D S
37
and studied by all the usual histological, cytological, and histochemical
techniques. The fixed cells, even in the outgrowth, can also be studied
by means of the electron-microscope so that the same cell can be studied
in life and after fixation at all magnifications possible for the living and
the dead. The earlier methods for electron-microscope studies involved
growing the cells on a "Formvar" film on the coverslip (Porter,
Claude and Fullam, 1945; Porter, 1953) but the more recent techniques allow individual cells to be embedded in situ and then detached
from the glass (Howatson and Almeida, 1958; Bloom, 1960).
Against these positive advantages of the hanging-drop culture must
be placed several serious disadvantages. In the first place the volume
of medium relative to that of the tissue is very small; in other words,
the medium : cell ratio is low; food substances are quickly reduced
below subsistence levels, and metabolic products quickly accumulate
to such an extent that they may become toxic. Thus, during the
three days or so which can be set as a limit to the useful life of a hangingdrop culture, the medium may be changing rapidly in composition.
Some of this may be beneficial modification, but the changes are
largely uncontrollable and liable to be harmful in the long run.
Moreover, events in the centre of the explant, where oxygen supplies
may be limited, often culminate in necrosis and autolysis with the
liberation of many unknown substances with varying degrees of biological activity on the more peripheral and still living cells. Even in
the outgrowth, the distribution of cells does not become uniform so that
the variation in the environments of cells in different parts of the outgrowth may be very considerable. All this, of course, means that certain
classes of cells from the original tissue may be favoured while other
classes are inhibited or even killed, and it is very difficult to make any
assessment of the actual environment of individual cells.
Another feature of cell behaviour which, though not by any means
limited to hanging-drop cultures, is nevertheless more easily studied
in such cultures than elsewhere is the change of form which the cells
undergo as they migrate out from the original tissue. This change of
form makes the identification of the cells extremely difficult (see
Chapter 4) and, since the environments of the cells are not constant, the
causes of the change of form are not easy to evaluate. Some are purely
mechanical, as for example the flattened shapes of the cells which are
characteristic of growth on a flat surface, or the fusiform shapes which
are found in the substance of a plasma clot. Others may result from
changed cellular interactions, caused by the outgrowth of the more
motile cells and the gradual transition of the tissue from its original
organization to something more nearly approaching a colony of
undifferentiated cells.
37
and studied by all the usual histological, cytological, and histochemical
techniques. The fixed cells, even in the outgrowth, can also be studied
by means of the electron-microscope so that the same cell can be studied
in life and after fixation at all magnifications possible for the living and
the dead. The earlier methods for electron-microscope studies involved
growing the cells on a "Formvar" film on the coverslip (Porter,
Claude and Fullam, 1945; Porter, 1953) but the more recent techniques allow individual cells to be embedded in situ and then detached
from the glass (Howatson and Almeida, 1958; Bloom, 1960).
Against these positive advantages of the hanging-drop culture must
be placed several serious disadvantages. In the first place the volume
of medium relative to that of the tissue is very small; in other words,
the medium : cell ratio is low; food substances are quickly reduced
below subsistence levels, and metabolic products quickly accumulate
to such an extent that they may become toxic. Thus, during the
three days or so which can be set as a limit to the useful life of a hangingdrop culture, the medium may be changing rapidly in composition.
Some of this may be beneficial modification, but the changes are
largely uncontrollable and liable to be harmful in the long run.
Moreover, events in the centre of the explant, where oxygen supplies
may be limited, often culminate in necrosis and autolysis with the
liberation of many unknown substances with varying degrees of biological activity on the more peripheral and still living cells. Even in
the outgrowth, the distribution of cells does not become uniform so that
the variation in the environments of cells in different parts of the outgrowth may be very considerable. All this, of course, means that certain
classes of cells from the original tissue may be favoured while other
classes are inhibited or even killed, and it is very difficult to make any
assessment of the actual environment of individual cells.
Another feature of cell behaviour which, though not by any means
limited to hanging-drop cultures, is nevertheless more easily studied
in such cultures than elsewhere is the change of form which the cells
undergo as they migrate out from the original tissue. This change of
form makes the identification of the cells extremely difficult (see
Chapter 4) and, since the environments of the cells are not constant, the
causes of the change of form are not easy to evaluate. Some are purely
mechanical, as for example the flattened shapes of the cells which are
characteristic of growth on a flat surface, or the fusiform shapes which
are found in the substance of a plasma clot. Others may result from
changed cellular interactions, caused by the outgrowth of the more
motile cells and the gradual transition of the tissue from its original
organization to something more nearly approaching a colony of
undifferentiated cells.