56
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
somal contacts resist tryptic digestion longer than attachments along the
rest of the cellular periphery. Desmosomes are characteristic of many
epithelial cells (Porter, 1956; Fawcett, 1961) and this may account for
the fact that, in trypsin, epithelia tend, at first, to separate from neighbouring mesenchyme as whole sheets, and only by longer treatment
do they fall apart into single cells. Such differential susceptibilities can
be turned to advantage for clean splitting and separation of epithelial
from mesenchymal constituents in embryonic organs, for the purpose
of separate cultivation, for various recombination studies, or for subsequent dissociation into cells. By controlled tryptic digestion, limb
epidermis can be cleanly separated from the mesoblast (Moscona and
Moscona, 1952; Zwilling, 1959; Sengel, 1958; McLoughlin, 1961;
Wessells, 1962); salivary, nephric and pancreatic epithelia (Grobstein,
1953, 1962), and somites (Holtzer and Detwiler, 1953; Grobstein and
Parker, 1954; Grobstein and Holtzer, 1955; Lash, Holtzer and Holtzer,
1957) from surrounding mesenchyme; optic lobe epithelium from the
ectoderm (Stroeva, 1960); uropygial epidermis from underlying mesenchyme (Gomot, 1961); germinal epithelium from gonadal medullary
tissue (Haffen, 1961); oviduct (Moscona, 1961b), thymus (Auerbach,
1961) and lung (Grover, 1962) epithelia from their mesenchymes. In
most of these studies, viability and the retention of histotypic activity by
the isolated cells was demonstrated by cultivation and by various
tissue-recombination and interaction tests.
Since trypsin does not attack collagen fibres, tissues rich in collagen
do not readily yield to tryptic dissociation. This may be one of the
reasons for the difficulty in obtaining free cells from later embryonic
and adult tissues. According to Lasfargues (1957) careful treatment
with purified collagenase makes it possible to separate ducts, acini, and
cells of the mammary gland from the adipose tissue surrounding them,
presumably by breaking up the collagenous network. Hinz and Syverton (1959) used collagenase to obtain single cells from lung tissue for
monolayer cultures. Sobel (1958) used collagenase to aid in the dissociation of trypsin-treated pituitary rudiments of the chick embryo;
Grover (1962) used this combination on embryonic-chick lung. Laws
and Stickland (1961) found that collagenase loosened up cells in
slices of adult liver tissue, but the cells were very fragile; their viability
and metabolic activity were not tested. According to Rinaldini (1958)
pure collagenase by itself does not readily dissociate cells from embryonic tissues; he expresses the need for caution in using collagenasecontaining preparations in view of the highly toxic contaminants, or
the inherent toxicity of this enzyme to some cells.
Other enzymes have been tested for cell-dispersing activity but with
largely negative results. Hyaluronidase seems to have no practically
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
somal contacts resist tryptic digestion longer than attachments along the
rest of the cellular periphery. Desmosomes are characteristic of many
epithelial cells (Porter, 1956; Fawcett, 1961) and this may account for
the fact that, in trypsin, epithelia tend, at first, to separate from neighbouring mesenchyme as whole sheets, and only by longer treatment
do they fall apart into single cells. Such differential susceptibilities can
be turned to advantage for clean splitting and separation of epithelial
from mesenchymal constituents in embryonic organs, for the purpose
of separate cultivation, for various recombination studies, or for subsequent dissociation into cells. By controlled tryptic digestion, limb
epidermis can be cleanly separated from the mesoblast (Moscona and
Moscona, 1952; Zwilling, 1959; Sengel, 1958; McLoughlin, 1961;
Wessells, 1962); salivary, nephric and pancreatic epithelia (Grobstein,
1953, 1962), and somites (Holtzer and Detwiler, 1953; Grobstein and
Parker, 1954; Grobstein and Holtzer, 1955; Lash, Holtzer and Holtzer,
1957) from surrounding mesenchyme; optic lobe epithelium from the
ectoderm (Stroeva, 1960); uropygial epidermis from underlying mesenchyme (Gomot, 1961); germinal epithelium from gonadal medullary
tissue (Haffen, 1961); oviduct (Moscona, 1961b), thymus (Auerbach,
1961) and lung (Grover, 1962) epithelia from their mesenchymes. In
most of these studies, viability and the retention of histotypic activity by
the isolated cells was demonstrated by cultivation and by various
tissue-recombination and interaction tests.
Since trypsin does not attack collagen fibres, tissues rich in collagen
do not readily yield to tryptic dissociation. This may be one of the
reasons for the difficulty in obtaining free cells from later embryonic
and adult tissues. According to Lasfargues (1957) careful treatment
with purified collagenase makes it possible to separate ducts, acini, and
cells of the mammary gland from the adipose tissue surrounding them,
presumably by breaking up the collagenous network. Hinz and Syverton (1959) used collagenase to obtain single cells from lung tissue for
monolayer cultures. Sobel (1958) used collagenase to aid in the dissociation of trypsin-treated pituitary rudiments of the chick embryo;
Grover (1962) used this combination on embryonic-chick lung. Laws
and Stickland (1961) found that collagenase loosened up cells in
slices of adult liver tissue, but the cells were very fragile; their viability
and metabolic activity were not tested. According to Rinaldini (1958)
pure collagenase by itself does not readily dissociate cells from embryonic tissues; he expresses the need for caution in using collagenasecontaining preparations in view of the highly toxic contaminants, or
the inherent toxicity of this enzyme to some cells.
Other enzymes have been tested for cell-dispersing activity but with
largely negative results. Hyaluronidase seems to have no practically