2 . M E T H O D S
5 5
breakdown products that may be released at the time of trypsindissociation of tissues. Studies on localization of trypsin by the use
of labelled enzyme or serological techniques are not available.
It is always possible that the enzyme may become secondarily attached to the cell surface or be taken up by the cells (Raut Hebb and
Wang Chu, 1960). Using a gelatin-film digestion-test and the D L
BAP A test for trypsin determination (Erlanger, Kokowsky and Cohen,
1961) Moscona has found that tissues incubated for 15 min in 0-5-1 °/o
solutions of crystalline trypsin, then rinsed thoroughly in saline, showed
very low, but measurable tryptic activity. This residual trypsin could
be rapidly inactivated by serum, by trypsin inhibitors, or by washing
the dispersed cells by centrifugation. The possible uptake or retention
of trypsin by trypsinized cells is a factor that must be fully considered
in all experiments with cells in serum-free media.
The standard procedure for cell dissociation by tryptic digestion
used by Moscona (1961a) in work with embryonic tissues involves
pre-incubation at 38°C of tissue fragments in a calcium- and magnesiumfree solution (CMF) for 10-15 min under 5 % C O a - a i r mixture, followed by 15-20 min (or longer, as needed) incubation at 38°C in the
enzyme (0.25-1-O^'o crystalline trypsin dissolved in C M F , under C O a -
air mixture). The treated fragments are rinsed three times in excess
C M F (pH 7-2), care being taken not to disrupt them at this stage.
Culture medium is then put on, and the fragments are dispersed in it
by brisk flushing through a fine pipette. Stock suspensions are sampled
for counting and for viability tests by staining (Hanks and Wallace,
1958) or plating on a plasma clot. This sequence eliminates dispersion
of the cells in the solution of the enzyme and rinsing by centrifugation,
thus minimizing cell damage and loss. The procedure can be modified
for various purposes but the effects of any modification on subsequent
results must be suitably controlled. Originally, crude trypsin (British
Drug Houses) was used (Moscona, 1952); however, for better standardization of procedures, crystalline trypsin is now employed whenever
possible. Pancreatin, may be more effective for some tissues, presumably
because it contains other agents which assist tissue disintegration. The
choice of enzyme and its optimal concentration must depend on the
type of tissue and the experimental requirements.
It is self-evident that different enzyme preparations vary not only
in their dispersive action on cells, but may also affect the subsequent
behaviour of the cells differently; simplicity of experimental design
and rigorous controls are therefore essential, since meaningful comparisons can only be made on the basis of strictly standardized and
reasonably clear conditions.
Electron-microscopic studies by Overton (1962) show that desmo-
5 5
breakdown products that may be released at the time of trypsindissociation of tissues. Studies on localization of trypsin by the use
of labelled enzyme or serological techniques are not available.
It is always possible that the enzyme may become secondarily attached to the cell surface or be taken up by the cells (Raut Hebb and
Wang Chu, 1960). Using a gelatin-film digestion-test and the D L
BAP A test for trypsin determination (Erlanger, Kokowsky and Cohen,
1961) Moscona has found that tissues incubated for 15 min in 0-5-1 °/o
solutions of crystalline trypsin, then rinsed thoroughly in saline, showed
very low, but measurable tryptic activity. This residual trypsin could
be rapidly inactivated by serum, by trypsin inhibitors, or by washing
the dispersed cells by centrifugation. The possible uptake or retention
of trypsin by trypsinized cells is a factor that must be fully considered
in all experiments with cells in serum-free media.
The standard procedure for cell dissociation by tryptic digestion
used by Moscona (1961a) in work with embryonic tissues involves
pre-incubation at 38°C of tissue fragments in a calcium- and magnesiumfree solution (CMF) for 10-15 min under 5 % C O a - a i r mixture, followed by 15-20 min (or longer, as needed) incubation at 38°C in the
enzyme (0.25-1-O^'o crystalline trypsin dissolved in C M F , under C O a -
air mixture). The treated fragments are rinsed three times in excess
C M F (pH 7-2), care being taken not to disrupt them at this stage.
Culture medium is then put on, and the fragments are dispersed in it
by brisk flushing through a fine pipette. Stock suspensions are sampled
for counting and for viability tests by staining (Hanks and Wallace,
1958) or plating on a plasma clot. This sequence eliminates dispersion
of the cells in the solution of the enzyme and rinsing by centrifugation,
thus minimizing cell damage and loss. The procedure can be modified
for various purposes but the effects of any modification on subsequent
results must be suitably controlled. Originally, crude trypsin (British
Drug Houses) was used (Moscona, 1952); however, for better standardization of procedures, crystalline trypsin is now employed whenever
possible. Pancreatin, may be more effective for some tissues, presumably
because it contains other agents which assist tissue disintegration. The
choice of enzyme and its optimal concentration must depend on the
type of tissue and the experimental requirements.
It is self-evident that different enzyme preparations vary not only
in their dispersive action on cells, but may also affect the subsequent
behaviour of the cells differently; simplicity of experimental design
and rigorous controls are therefore essential, since meaningful comparisons can only be made on the basis of strictly standardized and
reasonably clear conditions.
Electron-microscopic studies by Overton (1962) show that desmo-