2 . M E T H O D S
31
in physiological salt solution (Simms and Stillman, 1937) or if treated
with a "growth-promoting" extract such as a saline extract of minced
embryo tissues which may be either homologous or heterologous, though
preferably the former for reasons which will be discussed later (p. 35).
Similar extracts of certain adult tissues, e.g. brain (Trowell and Willmer,
1939), heart (Doljanski and Hoffman, 1939), cartilage (Davidson and
Waymouth, 1943), and a few others, are also effective in encouraging
the outgrowth of cells or the synthesis of nucleoprotein which is often
linked with increased cell migration. The extent of the migration and
growth is thus dependent on factors which are both intrinsic and extrinsic to the tissue.
The cells do not emerge readily into the fluid medium itself; if
they do so, they tend to sink to the bottom of the drop, round up and
become relatively inactive, though they may eventually spread out
along the fluid-air interface. Most of the "growth" of a culture in a
fluid medium takes place along the interface between the medium and
the coverslip, and a number of important points must be considered both
in relation to the extent of the outwandering and with regard to the
nature and condition of the cells which emerge. In "standing-drop
cultures" the contact between the glass and the tissue is increased,
sometimes with beneficial results.
It was remarked earlier that the glass coverslip has to be specially
cleaned. Traces of fatty or non-wettable substances, with the rather
surprising exception of cholesterol (Sato, Fisher and Puck, 1957),
generally inhibit cells from making the proper contact with glass which
is necessary for their adhesion. For most tissues the substratum must be
wettable, and if it is, then the cells can adhere, at least at certain parts
of their surface, and can migrate along the substratum at speeds and
with movements which are characteristic of different classes of cells.
Different classes of cells require different properties in the substrate for
their activity (Rappaport, 1960). Some of the cells which have been
studied (e.g. fibrocytes) do not adhere to the substratum simultaneously
all over their surface but only at certain points of contact (see p. 179)
which are themselves perpetually changing (Ambrose, 1961). Mechanocytes emerging from chick connective tissues on a glass surface can
migrate at about 20^/h, or rather faster if the concentration of embryo
extract is raised. Macrophages and monocytes move faster but with less
uniformity of direction. Lymphocytes, on the other hand, cannot make
proper contact with clean glass, and if they emerge from the tissues at
all in the sort of cultures under discussion they usually sink to the bottom
of the drop (see, however, p. 153). Many of the difficulties which arise
in the use of glass coverslips can be overcome by dipping the coverslips
in a dilute solution of celloidin and allowing them to dry in a vertical
31
in physiological salt solution (Simms and Stillman, 1937) or if treated
with a "growth-promoting" extract such as a saline extract of minced
embryo tissues which may be either homologous or heterologous, though
preferably the former for reasons which will be discussed later (p. 35).
Similar extracts of certain adult tissues, e.g. brain (Trowell and Willmer,
1939), heart (Doljanski and Hoffman, 1939), cartilage (Davidson and
Waymouth, 1943), and a few others, are also effective in encouraging
the outgrowth of cells or the synthesis of nucleoprotein which is often
linked with increased cell migration. The extent of the migration and
growth is thus dependent on factors which are both intrinsic and extrinsic to the tissue.
The cells do not emerge readily into the fluid medium itself; if
they do so, they tend to sink to the bottom of the drop, round up and
become relatively inactive, though they may eventually spread out
along the fluid-air interface. Most of the "growth" of a culture in a
fluid medium takes place along the interface between the medium and
the coverslip, and a number of important points must be considered both
in relation to the extent of the outwandering and with regard to the
nature and condition of the cells which emerge. In "standing-drop
cultures" the contact between the glass and the tissue is increased,
sometimes with beneficial results.
It was remarked earlier that the glass coverslip has to be specially
cleaned. Traces of fatty or non-wettable substances, with the rather
surprising exception of cholesterol (Sato, Fisher and Puck, 1957),
generally inhibit cells from making the proper contact with glass which
is necessary for their adhesion. For most tissues the substratum must be
wettable, and if it is, then the cells can adhere, at least at certain parts
of their surface, and can migrate along the substratum at speeds and
with movements which are characteristic of different classes of cells.
Different classes of cells require different properties in the substrate for
their activity (Rappaport, 1960). Some of the cells which have been
studied (e.g. fibrocytes) do not adhere to the substratum simultaneously
all over their surface but only at certain points of contact (see p. 179)
which are themselves perpetually changing (Ambrose, 1961). Mechanocytes emerging from chick connective tissues on a glass surface can
migrate at about 20^/h, or rather faster if the concentration of embryo
extract is raised. Macrophages and monocytes move faster but with less
uniformity of direction. Lymphocytes, on the other hand, cannot make
proper contact with clean glass, and if they emerge from the tissues at
all in the sort of cultures under discussion they usually sink to the bottom
of the drop (see, however, p. 153). Many of the difficulties which arise
in the use of glass coverslips can be overcome by dipping the coverslips
in a dilute solution of celloidin and allowing them to dry in a vertical