58
A . M O S C O N A , O . A . T R O W E L L A N D E . N . W I L L M E R
tions, or with low concentrations of Versene, does not seem to impair
cells of embryonic origin irreversibly (Dornfeld and Owczarzak, 1958),
extensive extraction of cations from cells by Versene, or citrate, may
cause serious metabolic lesions in the dissociated cells (Laws and Stickland, 1956; Kalant and Young, 1957). There are probably significant
changes in permeability and electrolyte balance of cells thus treated
and the possible effects of these on subsequent cell behaviour must be
borne in mind, in view of the morphogenetic changes which can be induced in cells by variations in ionic balance (Willmer, 1956, 1960).
Zimmerman et al. (1960) reported loss of aldolase, lactic dehydrogenase,
and glycerophosphate dehydrogenase from adult rat liver and kidney
cells dissociated by perfusion with citrate and homogenization. There
was a significantly smaller loss from similarly treated thymocytes and
various tumor cells. The authors ascribe the differences in enzyme
"leakage" to "a fundamental difference in the membrane structure
of the cells depending on the degree of organisation of the tissue
source". Cytological examinations, viability or recovery tests were
not reported, nor are comparative studies along these lines available,
using various embryonic cells and different dissociation procedures.
Brief treatment with trypsin seems to be remarkably non-injurious
to living cells (Rinaldini, 1958). Nerve axons exposed to trypsin remain
excitable (Tobias, 1960). Careful studies on the viability (Hanks and
Wallace, 1958) and osmotic behaviour (Rosenbluth and Pappas, 1958)
of trypsin-treated cells support, in general, Northrop's (1947) notion that
intact cells are not rapidly nor adversely affected by tryptic enzymes.
Preliminary electron-microscopic studies by Lesseps (1961) on trypsin
dispersed embryonic cells indicate that the double membrane remains
intact. There are, however, other observations which, in demonstrating
that on a more subtle level tryptic treatment does affect some kinds
of cells, justify caution. Trypsin affects fertilization of sea urchin eggs
(Runnstrom, Hagstrom and Perlmann, 1959), presumably by interfering with the configuration of the cell surface and the effectiveness
of cortical processes. It may cause animalization of sea urchin embryos
(Moore, 1952) and otherwise interfere with specific biosynthesis in
early embryonic development (Runnstrom, 1961). It affects the location
of the cleavage furrow in grasshopper neuroblasts (Kawamura and
Carlson, 1962). It is also of interest that trypsin-dissociated cells from
an amphibian gastrula or neurula are unable to recohere (Townes,
1953; Feldman, 1955)—a finding at complete variance with the behaviour of similarly dissociated tissue cells from higher vertebrate
embryos. All this stresses the need for detailed information on the
differential responses of cells to trypsin and other proteases.
The characteristic "bubbling" of trypsin-dissociated cells may be
A . M O S C O N A , O . A . T R O W E L L A N D E . N . W I L L M E R
tions, or with low concentrations of Versene, does not seem to impair
cells of embryonic origin irreversibly (Dornfeld and Owczarzak, 1958),
extensive extraction of cations from cells by Versene, or citrate, may
cause serious metabolic lesions in the dissociated cells (Laws and Stickland, 1956; Kalant and Young, 1957). There are probably significant
changes in permeability and electrolyte balance of cells thus treated
and the possible effects of these on subsequent cell behaviour must be
borne in mind, in view of the morphogenetic changes which can be induced in cells by variations in ionic balance (Willmer, 1956, 1960).
Zimmerman et al. (1960) reported loss of aldolase, lactic dehydrogenase,
and glycerophosphate dehydrogenase from adult rat liver and kidney
cells dissociated by perfusion with citrate and homogenization. There
was a significantly smaller loss from similarly treated thymocytes and
various tumor cells. The authors ascribe the differences in enzyme
"leakage" to "a fundamental difference in the membrane structure
of the cells depending on the degree of organisation of the tissue
source". Cytological examinations, viability or recovery tests were
not reported, nor are comparative studies along these lines available,
using various embryonic cells and different dissociation procedures.
Brief treatment with trypsin seems to be remarkably non-injurious
to living cells (Rinaldini, 1958). Nerve axons exposed to trypsin remain
excitable (Tobias, 1960). Careful studies on the viability (Hanks and
Wallace, 1958) and osmotic behaviour (Rosenbluth and Pappas, 1958)
of trypsin-treated cells support, in general, Northrop's (1947) notion that
intact cells are not rapidly nor adversely affected by tryptic enzymes.
Preliminary electron-microscopic studies by Lesseps (1961) on trypsin
dispersed embryonic cells indicate that the double membrane remains
intact. There are, however, other observations which, in demonstrating
that on a more subtle level tryptic treatment does affect some kinds
of cells, justify caution. Trypsin affects fertilization of sea urchin eggs
(Runnstrom, Hagstrom and Perlmann, 1959), presumably by interfering with the configuration of the cell surface and the effectiveness
of cortical processes. It may cause animalization of sea urchin embryos
(Moore, 1952) and otherwise interfere with specific biosynthesis in
early embryonic development (Runnstrom, 1961). It affects the location
of the cleavage furrow in grasshopper neuroblasts (Kawamura and
Carlson, 1962). It is also of interest that trypsin-dissociated cells from
an amphibian gastrula or neurula are unable to recohere (Townes,
1953; Feldman, 1955)—a finding at complete variance with the behaviour of similarly dissociated tissue cells from higher vertebrate
embryos. All this stresses the need for detailed information on the
differential responses of cells to trypsin and other proteases.
The characteristic "bubbling" of trypsin-dissociated cells may be