2 . M E T H O D S
59
indicative of intensified pinocytosis or of release of materials. The
possibility of uptake by such cells of large molecular constituents or
cellular particulates released from other cells cannot be dismissed;
the influence of such inclusions on the subsequent performance of
embryonic cells, though at present a highly speculative matter, must
therefore be kept in mind, particularly in view of the findings by Niu
(1959) and Benitez, Murray and Chargaff (1959) of "inductive" or
"infective" effects of nucleoprotein on cells in vitro (also Dumont,
Ayvazian and McCluskey, 1962).
That cell surfaces may be significantly affected by exposure to
trypsin is also suggested first by the increased ability of trypsin-treated
erythrocytes to become serologically agglutinated (Coffin and Pickles,
1953; Coombs, 1954), presumably due to exposure of active sites, and
secondly by the liberation from the surface of trypsinized erythrocytes of
a mucoid containing sialic acid (Cook, Heard and Seaman, 1960;
Uhlenbruck, 1961). Mucoids of undefined nature appear also around
embryonic cells (Moscona and Moscona, 1952; Rinaldini, 1958) or
adult cells (Laws and Stickland, 1961) dissociated with trypsin. Also,
L. Weiss (1958) found that Sarcoma 37 ascites cells incubated in trypsin-liberated material referred to as "extraneous coating". It is not
clear whether these materials represent changes in existing cell-surface
constituents, whether they may be classed as truly (i.e. physiologically)
newly elaborated or released products, or are liberated by cell damage.
Increase in cytoplasmic basophilia was observed in trypsin dissociated
cells (Moscona and Moscona, 1952) and alkali-dissociated cells (Holtfreter, 1943).
The appearance of highly viscous materials around trypsin-treated
embryonic cells is particularly noticeable when the cells are dispersed
in serum-free media (Moscona, 1962). It is possible that residual trypsin, retained by the dissociated cells, is responsible, since serum,
which has a trypsin-inhibiting activity, considerably reduces this
effect. It is also possible that the treated cells are "leaky" and that
protein is essential for repair of the cell surface. Noticeably less mucoid
is produced when crude pancreatin is used in dissociation; this could
be due to the trypsin inhibitor present in such preparations (Kunitz
and Northrop, 1936), to their content of non-enzymic proteins or to
DNA-ase (Moscona, 1962). According to Rinaldini (1958) a pancreatic
mucase, isolated by Banga and Balo (1956), rapidly hydrolyzed the
mucoids released by trypsin-treated cells; he suggested that the
absence of mucoidal materials around cells treated with crude pancreatin was due to the presence of this mucase in pancreatin, and
suggested that the viscous product of trypsinized cells contained a
mucoprotein. It has recently been found that this product is also
59
indicative of intensified pinocytosis or of release of materials. The
possibility of uptake by such cells of large molecular constituents or
cellular particulates released from other cells cannot be dismissed;
the influence of such inclusions on the subsequent performance of
embryonic cells, though at present a highly speculative matter, must
therefore be kept in mind, particularly in view of the findings by Niu
(1959) and Benitez, Murray and Chargaff (1959) of "inductive" or
"infective" effects of nucleoprotein on cells in vitro (also Dumont,
Ayvazian and McCluskey, 1962).
That cell surfaces may be significantly affected by exposure to
trypsin is also suggested first by the increased ability of trypsin-treated
erythrocytes to become serologically agglutinated (Coffin and Pickles,
1953; Coombs, 1954), presumably due to exposure of active sites, and
secondly by the liberation from the surface of trypsinized erythrocytes of
a mucoid containing sialic acid (Cook, Heard and Seaman, 1960;
Uhlenbruck, 1961). Mucoids of undefined nature appear also around
embryonic cells (Moscona and Moscona, 1952; Rinaldini, 1958) or
adult cells (Laws and Stickland, 1961) dissociated with trypsin. Also,
L. Weiss (1958) found that Sarcoma 37 ascites cells incubated in trypsin-liberated material referred to as "extraneous coating". It is not
clear whether these materials represent changes in existing cell-surface
constituents, whether they may be classed as truly (i.e. physiologically)
newly elaborated or released products, or are liberated by cell damage.
Increase in cytoplasmic basophilia was observed in trypsin dissociated
cells (Moscona and Moscona, 1952) and alkali-dissociated cells (Holtfreter, 1943).
The appearance of highly viscous materials around trypsin-treated
embryonic cells is particularly noticeable when the cells are dispersed
in serum-free media (Moscona, 1962). It is possible that residual trypsin, retained by the dissociated cells, is responsible, since serum,
which has a trypsin-inhibiting activity, considerably reduces this
effect. It is also possible that the treated cells are "leaky" and that
protein is essential for repair of the cell surface. Noticeably less mucoid
is produced when crude pancreatin is used in dissociation; this could
be due to the trypsin inhibitor present in such preparations (Kunitz
and Northrop, 1936), to their content of non-enzymic proteins or to
DNA-ase (Moscona, 1962). According to Rinaldini (1958) a pancreatic
mucase, isolated by Banga and Balo (1956), rapidly hydrolyzed the
mucoids released by trypsin-treated cells; he suggested that the
absence of mucoidal materials around cells treated with crude pancreatin was due to the presence of this mucase in pancreatin, and
suggested that the viscous product of trypsinized cells contained a
mucoprotein. It has recently been found that this product is also