54
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
in cell density and in the number of intercellular connexions, the cells
usually come off as sheets; these may be broken up mechanically into
fragments and some individual cells, but total dispersion becomes
increasingly difficult. The reaction in this respect varies somewhat with
the type of cells and with their history in culture. Various views on the
role of calcium in the attachment of cells in culture to various substrata have been discussed by Rinaldini (1958) and L. Weiss (1960).
Whether they are relevant to the situation in tissues is unknown.
Enzymic dissociation. The most effective current methods of dissociating
tissues into viable single cells are based on treatment with tryptic
enzymes. Most embryonic tissues or organ rudiments yield readily
to dispersion by trypsin; in general, tissues from later embryos and
adults are more difficult to dissociate, and usually require considerable
flushing or gently mechanical treatment for complete cell dispersion.
However, not all adult tissues can be reduced by tryptic digestion to
single cells which, in view of the diversity and changes with development
of cell-binding mechanisms, is not unexpected. The introduction of
enzymic dissociation of tissues (Moscona, 1951, 1952) was based on
prior findings by Willmer (1945) of a dispersive effect of trypsin on
embryonic-chick heart cells, which could then be used in culture.
Rous and Jones (1916) first described the release of cells from the outgrowth of plasma-clot cultures by digesting the clot with crude trypsin.
Trypsin has also been shown to separate epidermis (Medawar, 1941) and
corneal epithelium from their underlying connective tissue elements
(Herrmann and Hickman, 1948; Buschke, 1949); it has been used in
conjunction with mechanical disruption to obtain cells from whole
chick-embryos (Dulbecco, 1952) and from adult organs (Dulbecco and
Vogt, 1954; Younger, 1954; Melnick, Rappaport, Banker and Bhatt,
1955) for virus propagation, and from embryonic heart and muscle
(Rinaldini, 1954, 1959) for quantitative cell cultures.
Trypsin acts on peptide linkages adjacent to arginine and lysine;
therefore proteins or peptides with these linkages are presumably involved in cell attachment. The cell-dispersive effect of trypsin could be
due either to its typical proteolytic activity or to other physico-chemical
properties of the enzyme molecule. It was shown that diisopropylfluorophosphate, which inhibits the proteolytic action of trypsin without appreciably altering its physico-chemical characteristics, prevents
tissue dissociation (Easty and Mutolo, 1960); ovomucoid trypsininhibitor and soy-bean trypsin-inhibitor also inhibit dissociation
(Moscona, 1963a). It is therefore likely that the proteolytic activity
of the enzyme is causally involved in cell dissociation, rather than
alterations of charge at the cytomembrane or other physicochemical effects. There is no precise information on the identity of any
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
in cell density and in the number of intercellular connexions, the cells
usually come off as sheets; these may be broken up mechanically into
fragments and some individual cells, but total dispersion becomes
increasingly difficult. The reaction in this respect varies somewhat with
the type of cells and with their history in culture. Various views on the
role of calcium in the attachment of cells in culture to various substrata have been discussed by Rinaldini (1958) and L. Weiss (1960).
Whether they are relevant to the situation in tissues is unknown.
Enzymic dissociation. The most effective current methods of dissociating
tissues into viable single cells are based on treatment with tryptic
enzymes. Most embryonic tissues or organ rudiments yield readily
to dispersion by trypsin; in general, tissues from later embryos and
adults are more difficult to dissociate, and usually require considerable
flushing or gently mechanical treatment for complete cell dispersion.
However, not all adult tissues can be reduced by tryptic digestion to
single cells which, in view of the diversity and changes with development
of cell-binding mechanisms, is not unexpected. The introduction of
enzymic dissociation of tissues (Moscona, 1951, 1952) was based on
prior findings by Willmer (1945) of a dispersive effect of trypsin on
embryonic-chick heart cells, which could then be used in culture.
Rous and Jones (1916) first described the release of cells from the outgrowth of plasma-clot cultures by digesting the clot with crude trypsin.
Trypsin has also been shown to separate epidermis (Medawar, 1941) and
corneal epithelium from their underlying connective tissue elements
(Herrmann and Hickman, 1948; Buschke, 1949); it has been used in
conjunction with mechanical disruption to obtain cells from whole
chick-embryos (Dulbecco, 1952) and from adult organs (Dulbecco and
Vogt, 1954; Younger, 1954; Melnick, Rappaport, Banker and Bhatt,
1955) for virus propagation, and from embryonic heart and muscle
(Rinaldini, 1954, 1959) for quantitative cell cultures.
Trypsin acts on peptide linkages adjacent to arginine and lysine;
therefore proteins or peptides with these linkages are presumably involved in cell attachment. The cell-dispersive effect of trypsin could be
due either to its typical proteolytic activity or to other physico-chemical
properties of the enzyme molecule. It was shown that diisopropylfluorophosphate, which inhibits the proteolytic action of trypsin without appreciably altering its physico-chemical characteristics, prevents
tissue dissociation (Easty and Mutolo, 1960); ovomucoid trypsininhibitor and soy-bean trypsin-inhibitor also inhibit dissociation
(Moscona, 1963a). It is therefore likely that the proteolytic activity
of the enzyme is causally involved in cell dissociation, rather than
alterations of charge at the cytomembrane or other physicochemical effects. There is no precise information on the identity of any