474
CLIFFORD GROBSTEIN
in a single-cell system. Second, it is to be recalled that, at what may
prove to have been a comparable period in the development of technology for obtaining survival and growth of single cells, negative results
led to the assumption (Fischer, 1946) that single cells are not physiological units and can never initiate a culture. This assumption shortly
was belied (Sanford, Earle and Likely, 1948) by improvements of media
and technique which today permit almost routine cloning of cells from
a number of sources (Paul, 1959). Third, several lines of evidence seem
promising with respect to the achievement of differentiation in isolated
cultured cells at some time in the not too distant future. A brief look at
this evidence is in order.
Until recently, direct efforts to obtain differentiation of single or
restricted numbers of cells in vitro usually have been made with
amphibian cells. Wilde (1961) has reported extensive studies of the
behavior of single and small clusters of cells, obtained by disaggregation
of early amphibian embryos, in micro-drops of a simple culture medium.
In no instance did single cells in a drop show overt differentiation.
However, as the number of cells in a drop was increased the frequency
of differentiation among them also increased. In some instances, one of
two cells in a drop would differentiate. At low cell numbers per drop
the frequency of differentiation was markedly increased when the cells
were in the presence of the opalescent material exuded by them in the
disaggregation process. The results suggest that differentiation is
inhibited by a low ratio of cells to volume of medium, but that alteration
of the medium by materials exuded by the cells makes possible differentiation at lower ratios. It is obvious that this assumption provides a
mechanism for the so-called minimum mass effect, and is in conformity
with conditioning of media and other procedures reported to favor
survival and growth of single cells (Sanford et al., 1948; Powell, 1957;
Sato, Fisher and Puck, 1957; see also p. 174).
With respect to avian and mammalian cells, there do not yet appear
to be reports of isolated undifferentiated cells which undergo initiation,
progression and completion of differentiation in vitro. There are, however, reports of maintenance of existing differentiation in isolated cells,
and progression of differentiation in dispersed cells. For example,
Cavanaugh (1955a) dissociated embryonic-chick spinal cord in advanced stages of differentiation, and found that single nerve cells in
complete isolation developed typical nerve fibers over a period of
several days in plasma clots. A series of reports, beginning with Burrows
(1912) and more recently those of Rinaldini (1954) and Cavanaugh
(1955b), deal with the differentiative behavior of isolated or dispersed
muscle cells. Wollenberger and Halle (1960) described single cells,
isolated in semi-synthetic medium from dissociated ventricle of 13-day
CLIFFORD GROBSTEIN
in a single-cell system. Second, it is to be recalled that, at what may
prove to have been a comparable period in the development of technology for obtaining survival and growth of single cells, negative results
led to the assumption (Fischer, 1946) that single cells are not physiological units and can never initiate a culture. This assumption shortly
was belied (Sanford, Earle and Likely, 1948) by improvements of media
and technique which today permit almost routine cloning of cells from
a number of sources (Paul, 1959). Third, several lines of evidence seem
promising with respect to the achievement of differentiation in isolated
cultured cells at some time in the not too distant future. A brief look at
this evidence is in order.
Until recently, direct efforts to obtain differentiation of single or
restricted numbers of cells in vitro usually have been made with
amphibian cells. Wilde (1961) has reported extensive studies of the
behavior of single and small clusters of cells, obtained by disaggregation
of early amphibian embryos, in micro-drops of a simple culture medium.
In no instance did single cells in a drop show overt differentiation.
However, as the number of cells in a drop was increased the frequency
of differentiation among them also increased. In some instances, one of
two cells in a drop would differentiate. At low cell numbers per drop
the frequency of differentiation was markedly increased when the cells
were in the presence of the opalescent material exuded by them in the
disaggregation process. The results suggest that differentiation is
inhibited by a low ratio of cells to volume of medium, but that alteration
of the medium by materials exuded by the cells makes possible differentiation at lower ratios. It is obvious that this assumption provides a
mechanism for the so-called minimum mass effect, and is in conformity
with conditioning of media and other procedures reported to favor
survival and growth of single cells (Sanford et al., 1948; Powell, 1957;
Sato, Fisher and Puck, 1957; see also p. 174).
With respect to avian and mammalian cells, there do not yet appear
to be reports of isolated undifferentiated cells which undergo initiation,
progression and completion of differentiation in vitro. There are, however, reports of maintenance of existing differentiation in isolated cells,
and progression of differentiation in dispersed cells. For example,
Cavanaugh (1955a) dissociated embryonic-chick spinal cord in advanced stages of differentiation, and found that single nerve cells in
complete isolation developed typical nerve fibers over a period of
several days in plasma clots. A series of reports, beginning with Burrows
(1912) and more recently those of Rinaldini (1954) and Cavanaugh
(1955b), deal with the differentiative behavior of isolated or dispersed
muscle cells. Wollenberger and Halle (1960) described single cells,
isolated in semi-synthetic medium from dissociated ventricle of 13-day