2 . M E T H O D S
57
useful dispersive effect on embryonic-chick organ-rudiments (Moscona,
1952; Rinaldini, 1958) or on adult tissues (Easty and Mutolo, 1960;
Laws and Stickland, 1961). The effectiveness of elastase and elastomucase for cell dispersion was briefly described by Rinaldini in 1958
and more fully evaluated, with many useful practical details concerning
the preparation and treatment of cell-suspensions in general, in a
further paper in 1959. The possible usefulness of papain protease as a
cell dispersive agent merits reinvestigation in view of the findings
that the enzyme injected into rabbits "softens" the matrix in ear
cartilages (Thomas, 1956); it has a similar effect when applied to
bone rudiments cultured in vitro (Fell, 1961). Easty and Mutolo
(1960) found that exposure to activated papain resulted in partial
separation of adult liver cells and of Walker tumor cells, but the
enzyme was less effective than trypsin. It is obvious that further
progress with enzymic dissociations of tissues, particularly for obtaining
cells for developmental studies, hinges upon more information on the
nature of intercellular bonds and on the availability of a wider range
of enzymes with lytic activity on cell-binding materials. Such materials
are generally thought to contain, in addition to proteins, also polysaccharides, yet surprisingly little is known about the possible usefulness
of "mucolytic" enzymes for tissue dissociation (see pp. 376, 520). Some of
the sugar-splitting bacterial enzymes used in studies on virus receptor
substances and blood group antigens (Burnet and Stone, 1947; Watkins,
1959; Morgan, 1960) could be suitable for the separation of tissue cells
and might be of value in further work on the nature of intercellular
materials.
3. Side Effects of Dispersive Treatments
A variety of developmental studies using dissociated embryonicchick or -mouse cells have failed to reveal any persistent, readily
noticeable effects that could be attributed to the direct or specific action
of the dispersive agent. However, it is possible that the criteria of
detection may not have been sufficiently refined. With increasing use
of cell suspensions, a more searching attitude seems advisable both
towards the action of dispersive agents and procedures, and to the
effects of the removal of the cells from their original environment.
There is practically no detailed information on the differential sensitivities of various kinds of embryonic cells to enzymes and chelators
used in dissociation, or on the ability of these agents to penetrate into
cells and to react with cell-surface or intracellular constituents. Equally
little is known about cellular products appearing during and after
dissociation.
Although brief treatment with calcium- and magnesium-free solu-
57
useful dispersive effect on embryonic-chick organ-rudiments (Moscona,
1952; Rinaldini, 1958) or on adult tissues (Easty and Mutolo, 1960;
Laws and Stickland, 1961). The effectiveness of elastase and elastomucase for cell dispersion was briefly described by Rinaldini in 1958
and more fully evaluated, with many useful practical details concerning
the preparation and treatment of cell-suspensions in general, in a
further paper in 1959. The possible usefulness of papain protease as a
cell dispersive agent merits reinvestigation in view of the findings
that the enzyme injected into rabbits "softens" the matrix in ear
cartilages (Thomas, 1956); it has a similar effect when applied to
bone rudiments cultured in vitro (Fell, 1961). Easty and Mutolo
(1960) found that exposure to activated papain resulted in partial
separation of adult liver cells and of Walker tumor cells, but the
enzyme was less effective than trypsin. It is obvious that further
progress with enzymic dissociations of tissues, particularly for obtaining
cells for developmental studies, hinges upon more information on the
nature of intercellular bonds and on the availability of a wider range
of enzymes with lytic activity on cell-binding materials. Such materials
are generally thought to contain, in addition to proteins, also polysaccharides, yet surprisingly little is known about the possible usefulness
of "mucolytic" enzymes for tissue dissociation (see pp. 376, 520). Some of
the sugar-splitting bacterial enzymes used in studies on virus receptor
substances and blood group antigens (Burnet and Stone, 1947; Watkins,
1959; Morgan, 1960) could be suitable for the separation of tissue cells
and might be of value in further work on the nature of intercellular
materials.
3. Side Effects of Dispersive Treatments
A variety of developmental studies using dissociated embryonicchick or -mouse cells have failed to reveal any persistent, readily
noticeable effects that could be attributed to the direct or specific action
of the dispersive agent. However, it is possible that the criteria of
detection may not have been sufficiently refined. With increasing use
of cell suspensions, a more searching attitude seems advisable both
towards the action of dispersive agents and procedures, and to the
effects of the removal of the cells from their original environment.
There is practically no detailed information on the differential sensitivities of various kinds of embryonic cells to enzymes and chelators
used in dissociation, or on the ability of these agents to penetrate into
cells and to react with cell-surface or intracellular constituents. Equally
little is known about cellular products appearing during and after
dissociation.
Although brief treatment with calcium- and magnesium-free solu-