52
A . M O S C O N A , O . A . T R O W E L L A N D E . N . W l L L M E R
release of calcium ions from a bound state in the cell periphery; this
is of obvious interest, in view of the role of calcium in cell cohesion.
It is thus difficult to distinguish between the purely mechanical and the
accompanying chemical effects of such procedures. Disruption of
tissues by focused ultrasound (Lutz and Lutz-Ostertag, 1959; Bell,
1960) offers promise with regard to precision of application and quantitative regulation. However, the consequences of cavitation and degassing upon the ultrastructure and functional properties of cells need
further investigation, particularly in view of the reported developmental abnormalities following exposure to high frequency waves
(Selman and Counce, 1953). The combined mechanical and chemical
dissociation-procedure of Auerbach and Grobstein (1958) is based on
shearing trypsin-treated tissue in a rapidly vibrating tube. It is useful
for the dissociation of small embryonic rudiments and permits considerable standardization and control of the process.
2. Chemical Dissociation
Removal of divalent cations. That calcium is required for the maintenance of normal intercellular contacts has been known, at least, since
Ringer's work in 1880, and since Herbst's (1900) demonstration that
sea urchin larvae disintegrated into separate cells in calcium-free sea
water. Gray (1926) showed that magnesium ions have a related function
and that the two ions may not be fully interchangeable. Zweifach's
(1940) demonstration of the "loosening" of the intercellular cement in
capillary endothelium by depletion of calcium and Coman's (1954)
findings that metastasizing neoplasms have a lower calcium content than
normal tissues drew further attention to the role of calcium in cell
binding. Depletion of divalent cations suggested itself, therefore, as a
possible means of dissociating tissues.
Gentle depletion of divalent cations from avian or mammalian
tissues, i.e. soaking in calcium- and magnesium-free balanced saline
solution, does not, in general, bring about complete dissociation of the
cells; their attachment is however "loosened" and such treatment
enhances dissociation by subsequent treatment with tryptic enzymes
(Moscona, 1952). Versene (EDTA) and alkaline p H may increase the
effect of calcium- and magnesium-free solutions and they have been
used to dissociate chick blastoderms (Zwilling, 1954) and rabbit blastomeres (Brochart, 1954). However, tissues from older embryos cannot
be effectively dissociated with Versene or alkaline solutions without
extensive damage. Versene-treated tissues from later embryos can be
disrupted only by strong agitation and then yield, at best, a mixture
of shredded large and small tissue-fragments and cells. It seems that,
with advancing development, the stability of cell contact depends pro-
A . M O S C O N A , O . A . T R O W E L L A N D E . N . W l L L M E R
release of calcium ions from a bound state in the cell periphery; this
is of obvious interest, in view of the role of calcium in cell cohesion.
It is thus difficult to distinguish between the purely mechanical and the
accompanying chemical effects of such procedures. Disruption of
tissues by focused ultrasound (Lutz and Lutz-Ostertag, 1959; Bell,
1960) offers promise with regard to precision of application and quantitative regulation. However, the consequences of cavitation and degassing upon the ultrastructure and functional properties of cells need
further investigation, particularly in view of the reported developmental abnormalities following exposure to high frequency waves
(Selman and Counce, 1953). The combined mechanical and chemical
dissociation-procedure of Auerbach and Grobstein (1958) is based on
shearing trypsin-treated tissue in a rapidly vibrating tube. It is useful
for the dissociation of small embryonic rudiments and permits considerable standardization and control of the process.
2. Chemical Dissociation
Removal of divalent cations. That calcium is required for the maintenance of normal intercellular contacts has been known, at least, since
Ringer's work in 1880, and since Herbst's (1900) demonstration that
sea urchin larvae disintegrated into separate cells in calcium-free sea
water. Gray (1926) showed that magnesium ions have a related function
and that the two ions may not be fully interchangeable. Zweifach's
(1940) demonstration of the "loosening" of the intercellular cement in
capillary endothelium by depletion of calcium and Coman's (1954)
findings that metastasizing neoplasms have a lower calcium content than
normal tissues drew further attention to the role of calcium in cell
binding. Depletion of divalent cations suggested itself, therefore, as a
possible means of dissociating tissues.
Gentle depletion of divalent cations from avian or mammalian
tissues, i.e. soaking in calcium- and magnesium-free balanced saline
solution, does not, in general, bring about complete dissociation of the
cells; their attachment is however "loosened" and such treatment
enhances dissociation by subsequent treatment with tryptic enzymes
(Moscona, 1952). Versene (EDTA) and alkaline p H may increase the
effect of calcium- and magnesium-free solutions and they have been
used to dissociate chick blastoderms (Zwilling, 1954) and rabbit blastomeres (Brochart, 1954). However, tissues from older embryos cannot
be effectively dissociated with Versene or alkaline solutions without
extensive damage. Versene-treated tissues from later embryos can be
disrupted only by strong agitation and then yield, at best, a mixture
of shredded large and small tissue-fragments and cells. It seems that,
with advancing development, the stability of cell contact depends pro-