2 . M E T H O D S
53
gressively less on divalent cations; it is also possible that the cations
become bound so as to be less readily removed by chelators.
Easty and Mutolo (1960) reported that the dispersive effect of Versene
on adult liver tissue and on cell-islands of the Walker ascites tumor was
not greater than that of normal saline solution, and they concluded that
the role of calcium in the adhesiveness of adult liver cells may have
been overemphasized. On the other hand, Anderson (1953), St.
George, Friedman and Byers (1954), and Laws and Stickland (1956,
1961) demonstrated that adult liver perfused with Versene or citrate
could be readily disrupted into cells by being pressed through sieves or
by grinding. Data on viability or plating efficiency of cells thus obtained have not been reported. However, certain metabolic lesions
found in cells expressed from Versene-perfused liver (Laws and Stickland, 1956; Kalant and Young, 1957; Zimmerman, Devlin and Pruss,
1960) indicate that the chelator, at the concentrations required for cell
detachment, may cause various lasting functional disturbances. Detachment of cells in Versene-perfused liver may be accompanied by (or
due to) structural damage other than at the cell surface (Coman, 1954).
Zwilling (1959) used Versene to remove the epidermis from embryonicduck limb-buds and found that many torn cells remained attached to
the basement membrane; effective removal of the epidermis from the
treated limb-bud required scraping in addition. Branster and Morton
(1957) found no difference in obtaining cells by mechanical crushing of
liver perfused either with various chelating or non-chelating solutions.
Easty and Mutolo (1960) concluded that the mechanical effects in such
dissociation procedures are probably more important than the presence
or absence of chelators. It is of interest that in Anderson's (1953) dissociations the proportion of whole cells to isolated nuclei varied
between 30% and 70°/o in different experiments, and that the proportion of broken cells did not depend on the concentration of chelator,
but on the duration of grinding.
With regard to the attachment of cells to glass, Dornfeld and O w c -
zarzak (1958) found that when chick fibroblasts in monolayer cultures
were washed with calcium- and magnesium-free saline or with Versene
solution the cells rounded off, showed "bubbling," and detached from
the glass. Rappaport (1956), Owens (1957), L. Weiss (1960) and others
found that attachment of various cells to glass was calcium dependent;
the differences in cell types used, in the kind and treatment of glassware,
and in the composition of the media, however, preclude detailed
comparison of the data in terms of calcium requirements alone.
Complete separation and dissociation of embryonic cells growing on
glass and particularly of epithelioid elements by gentle calcium depletion and shaking is feasible only when cell density is low. With increase
53
gressively less on divalent cations; it is also possible that the cations
become bound so as to be less readily removed by chelators.
Easty and Mutolo (1960) reported that the dispersive effect of Versene
on adult liver tissue and on cell-islands of the Walker ascites tumor was
not greater than that of normal saline solution, and they concluded that
the role of calcium in the adhesiveness of adult liver cells may have
been overemphasized. On the other hand, Anderson (1953), St.
George, Friedman and Byers (1954), and Laws and Stickland (1956,
1961) demonstrated that adult liver perfused with Versene or citrate
could be readily disrupted into cells by being pressed through sieves or
by grinding. Data on viability or plating efficiency of cells thus obtained have not been reported. However, certain metabolic lesions
found in cells expressed from Versene-perfused liver (Laws and Stickland, 1956; Kalant and Young, 1957; Zimmerman, Devlin and Pruss,
1960) indicate that the chelator, at the concentrations required for cell
detachment, may cause various lasting functional disturbances. Detachment of cells in Versene-perfused liver may be accompanied by (or
due to) structural damage other than at the cell surface (Coman, 1954).
Zwilling (1959) used Versene to remove the epidermis from embryonicduck limb-buds and found that many torn cells remained attached to
the basement membrane; effective removal of the epidermis from the
treated limb-bud required scraping in addition. Branster and Morton
(1957) found no difference in obtaining cells by mechanical crushing of
liver perfused either with various chelating or non-chelating solutions.
Easty and Mutolo (1960) concluded that the mechanical effects in such
dissociation procedures are probably more important than the presence
or absence of chelators. It is of interest that in Anderson's (1953) dissociations the proportion of whole cells to isolated nuclei varied
between 30% and 70°/o in different experiments, and that the proportion of broken cells did not depend on the concentration of chelator,
but on the duration of grinding.
With regard to the attachment of cells to glass, Dornfeld and O w c -
zarzak (1958) found that when chick fibroblasts in monolayer cultures
were washed with calcium- and magnesium-free saline or with Versene
solution the cells rounded off, showed "bubbling," and detached from
the glass. Rappaport (1956), Owens (1957), L. Weiss (1960) and others
found that attachment of various cells to glass was calcium dependent;
the differences in cell types used, in the kind and treatment of glassware,
and in the composition of the media, however, preclude detailed
comparison of the data in terms of calcium requirements alone.
Complete separation and dissociation of embryonic cells growing on
glass and particularly of epithelioid elements by gentle calcium depletion and shaking is feasible only when cell density is low. With increase