2 . M E T H O D S
5 1
Rinaldini, 1958; Edds, 1958; Willmer, 1960), desmosomal attachments
(Porter, 1954, 1956; Fawcett, 1958,1961), and other cell-linking agents,
as well as variations in the susceptibility of different cells to experimental
treatments preclude a generally applicable and uniformly effective
single method of tissue-dissociation. Moreover, there is suggestive
evidence that the mechanisms involved in the mutual attachment of
cells and in stabilizing cell cohesion may vary not only with cell type
and function, but even around the perimeter of the same cell (Willmer,
1960). Electron-microscopy of epidermis (Weiss and Ferris, 1956;
Singer and Salpeter, 1961) has revealed marked differences in the
structure of lateral and basal cell surfaces indicative of different modes
of attachment. Corneal epithelium treated with fluoroacetate becomes
detached as a cell sheet, the separation taking place between the basal
epithelial layer and the stroma; on the other hand, histamine causes
disjunction between the basal and the superficial layers of the epithelium
(Herrmann, 1948; Herrmann and Hickman, 1948). In neither case
are the lateral attachments of the basal cells affected, which points to
a heterogeneity of contact mechanisms around the surface of these cells.
Probably the same holds true for other tissues, for instance limb epidermis of the chick embryo (Moscona, 1952; Zwilling, 1959). Some
of the current theoretical considerations on the nature of cellular
attachments and their disruption are based on work with adhesion of
cultured cells to glass; L. Weiss (1960) cautions against indiscriminate
comparison between adhesion of cells to various artificial substrates
and cohesion in tissues since the mechanisms may differ significantly.
1. Mechanical Dissociation
Disintegration of tissues by homogenizing, chopping, pressing through
sieves, etc., usually yields mixtures of cells and cell clumps. In critical
work as for example, on cell-aggregation, it is essential to start with
fully dispersed cells. Furthermore, such procedures are unavoidably
accompanied by extensive loss of cells, which restricts their practical
usefulness to large initial amounts of tissue. It is therefore frequently
inappropriate or difficult to apply conventional mechanical disintegration to embryonic material. The major advantage of such dissociation procedures is that the cells are not exposed to chemical treatments.
However, mechanical disruption may actually involve quite complex
effects; the possibility that tissue-damage and breakdown products
may affect cell properties and cell behaviour by lytic action, by activating intracellular enzymes, by binding calcium (Thomason and
Schofield, 1959) or simply by altering the cellular environments cannot
be disregarded. It has also been reported (Mazia and Clark, 1936)
that a variety of different stimuli, including mechanical shock, cause a
5 1
Rinaldini, 1958; Edds, 1958; Willmer, 1960), desmosomal attachments
(Porter, 1954, 1956; Fawcett, 1958,1961), and other cell-linking agents,
as well as variations in the susceptibility of different cells to experimental
treatments preclude a generally applicable and uniformly effective
single method of tissue-dissociation. Moreover, there is suggestive
evidence that the mechanisms involved in the mutual attachment of
cells and in stabilizing cell cohesion may vary not only with cell type
and function, but even around the perimeter of the same cell (Willmer,
1960). Electron-microscopy of epidermis (Weiss and Ferris, 1956;
Singer and Salpeter, 1961) has revealed marked differences in the
structure of lateral and basal cell surfaces indicative of different modes
of attachment. Corneal epithelium treated with fluoroacetate becomes
detached as a cell sheet, the separation taking place between the basal
epithelial layer and the stroma; on the other hand, histamine causes
disjunction between the basal and the superficial layers of the epithelium
(Herrmann, 1948; Herrmann and Hickman, 1948). In neither case
are the lateral attachments of the basal cells affected, which points to
a heterogeneity of contact mechanisms around the surface of these cells.
Probably the same holds true for other tissues, for instance limb epidermis of the chick embryo (Moscona, 1952; Zwilling, 1959). Some
of the current theoretical considerations on the nature of cellular
attachments and their disruption are based on work with adhesion of
cultured cells to glass; L. Weiss (1960) cautions against indiscriminate
comparison between adhesion of cells to various artificial substrates
and cohesion in tissues since the mechanisms may differ significantly.
1. Mechanical Dissociation
Disintegration of tissues by homogenizing, chopping, pressing through
sieves, etc., usually yields mixtures of cells and cell clumps. In critical
work as for example, on cell-aggregation, it is essential to start with
fully dispersed cells. Furthermore, such procedures are unavoidably
accompanied by extensive loss of cells, which restricts their practical
usefulness to large initial amounts of tissue. It is therefore frequently
inappropriate or difficult to apply conventional mechanical disintegration to embryonic material. The major advantage of such dissociation procedures is that the cells are not exposed to chemical treatments.
However, mechanical disruption may actually involve quite complex
effects; the possibility that tissue-damage and breakdown products
may affect cell properties and cell behaviour by lytic action, by activating intracellular enzymes, by binding calcium (Thomason and
Schofield, 1959) or simply by altering the cellular environments cannot
be disregarded. It has also been reported (Mazia and Clark, 1936)
that a variety of different stimuli, including mechanical shock, cause a