2 . M E T H O D S
63
which is consistent with achieving the necessary separation. Anything
which is done to a cell surface is liable to change the function of the cell
until the cell can restore its former condition. Consequently, it must
be considered that cells prepared in suspension are abnormal cells
unless or until they re-establish their normal surroundings and relationships; monolayers of cells are quite different from re-aggregates of
cells and may have very different activities. Treatment of the cells with
separating agents may leave them incapable of reforming the required
surface for their reaggregation, and recent work has emphasized that
simple physiological salt solutions are not necessarily the best basic
media in which to carry out experiments on separation and re-aggregation. The exact ionic concentration and the presence of non-electrolytes
including colloids of high molecular weight, like albumen and even
methyl cellulose (Earle, Bryant, Schilling and Evans, 1956), are both
becoming recognized as important factors in the survival and behaviour
of isolated cells and tissues. For example, the retinal rods of the frog are
stable in a sucrose solution, while they disintegrate rapidly in Ringer's
solution and the like (Arden, 1954). Sucrose also favours the survival
of dissociated retinal cells of the chick embryo (Moscona, 1960). The
addition of serine, inositol, glycine, cholesterol, certain glycoproteins of
serum and basic polypeptides have already been mentioned as important in this sort of way for preserving the stability and physiological
activity of cells in isolation, and in assisting them to adhere to or spread
on glass surfaces (see p. 32).
1. Clones
It was the hope of many of the early tissue culturists to be able to
grow animal cells in much the same way as bacteria are grown. This
aim has at last been achieved to a limited extent and it is now definitely
established that, from certain types of tissue, clones may be started
from single cells. With some cells, like the HeLa cell, which is a carcinomatous human cell originally derived from the cervix uteri, it is
comparatively easy to obtain clonal colonies by plating out dilute
suspensions of the cells in suitable flasks or dishes. With many normal
cells the problem is more difficult for reasons which have already been
discussed, unless the cells have been previously adapted to life in vitro.
An ingenious method has been used by Puck and his colleagues (1955,
1956) for this purpose. A copious suspension of cells is allowed to settle
from a nutrient solution and to form a monolayer on the bottom of a
dish. When these cells are firmly established as a uniform layer, they
are irradiated with a sufficient dose of X-rays to stop all cell division,
though growth in size of the cells continues. When a dilute suspension
of normal cells is then plated out on the top of such an inhibited colony
63
which is consistent with achieving the necessary separation. Anything
which is done to a cell surface is liable to change the function of the cell
until the cell can restore its former condition. Consequently, it must
be considered that cells prepared in suspension are abnormal cells
unless or until they re-establish their normal surroundings and relationships; monolayers of cells are quite different from re-aggregates of
cells and may have very different activities. Treatment of the cells with
separating agents may leave them incapable of reforming the required
surface for their reaggregation, and recent work has emphasized that
simple physiological salt solutions are not necessarily the best basic
media in which to carry out experiments on separation and re-aggregation. The exact ionic concentration and the presence of non-electrolytes
including colloids of high molecular weight, like albumen and even
methyl cellulose (Earle, Bryant, Schilling and Evans, 1956), are both
becoming recognized as important factors in the survival and behaviour
of isolated cells and tissues. For example, the retinal rods of the frog are
stable in a sucrose solution, while they disintegrate rapidly in Ringer's
solution and the like (Arden, 1954). Sucrose also favours the survival
of dissociated retinal cells of the chick embryo (Moscona, 1960). The
addition of serine, inositol, glycine, cholesterol, certain glycoproteins of
serum and basic polypeptides have already been mentioned as important in this sort of way for preserving the stability and physiological
activity of cells in isolation, and in assisting them to adhere to or spread
on glass surfaces (see p. 32).
1. Clones
It was the hope of many of the early tissue culturists to be able to
grow animal cells in much the same way as bacteria are grown. This
aim has at last been achieved to a limited extent and it is now definitely
established that, from certain types of tissue, clones may be started
from single cells. With some cells, like the HeLa cell, which is a carcinomatous human cell originally derived from the cervix uteri, it is
comparatively easy to obtain clonal colonies by plating out dilute
suspensions of the cells in suitable flasks or dishes. With many normal
cells the problem is more difficult for reasons which have already been
discussed, unless the cells have been previously adapted to life in vitro.
An ingenious method has been used by Puck and his colleagues (1955,
1956) for this purpose. A copious suspension of cells is allowed to settle
from a nutrient solution and to form a monolayer on the bottom of a
dish. When these cells are firmly established as a uniform layer, they
are irradiated with a sufficient dose of X-rays to stop all cell division,
though growth in size of the cells continues. When a dilute suspension
of normal cells is then plated out on the top of such an inhibited colony