42
A . M O S C O N A , O . A . T R O W E L L A N D E . N . W l L L M E R
growth-inhibitors have all been investigated in flask cultures with more
or less success.
In the long run, however, in all experiments of this sort it is desirable,
if not absolutely necessary, to know how much tissue is present and if
it is all behaving in the same way, and whether or not it is increasing
in amount and how rapidly. Flask cultures in plasma clots do not
readily allow this. It is very difficult to get any reliable estimate of the
amount of tissue present once the tissue has become embedded in the
clot, and there is always the difficulty that the centre of the explant is
behaving differently from the peripheral outgrowth. Growth in the
periphery may be balanced by necrosis in the centre. Measurements
of the D N A content of the tissue can to some extent now overcome the
difficulty of the measurement of weight, since the D N A content per
cell, in normal non-growing tissues, is reasonably constant and the total
D N A can therefore be used to estimate the number of cells (Davidson,
Leslie and Waymouth, 1949; Healy, Fisher and Parker, 1954); but
the other difficulties remain, and dead cells may still contain DNA,
thus confusing the issue.
Total D N A content can, of course, only be measured once in such
cultures, and this limits its usefulness as a measure of growth for example.
DNA content of individual cells in the zone of outgrowth can, however,
be estimated by u.v. microscopy in living cells (Walker and Yates, 1952),
and this technique has shown how the D N A increases from n to 2n
during the interkinetic period. Since cells in a growing culture may
contain anything between n and 2n values for their DNA, estimates of
DNA in cell populations have only limited value in estimating cell
numbers when such populations are growing. The increase in D N A
content itself is, of course, a good measure of growth (see Chapters 7,
10 and 12).
It may be relevant here to point out that in all metabolic experiments
with cells in vitro, results which are apparently very different are obtained according to the method by which the amount of tissue present
is estimated, or rather according to the unit in which it is expressed.
Thus, activity expressed in terms of wet weight of tissue, dry weight of
tissue, number of cells, D N A content, total protein, protein nitrogen and
the like can all give quite different results. There are, in addition to the
errors involved in estimating these, very obvious differences in the
quantities being measured. For example, a piece of growing tissue
can vary the number of its cells, the size of its cells, the water content
of its cells and the amount of inter-cellular material, and the method
of growth-measurement selected must take account of all these factors
and, in addition, must allow for the fact that cells in the outgrowth
exposed to the medium may migrate, grow or function quite differently
A . M O S C O N A , O . A . T R O W E L L A N D E . N . W l L L M E R
growth-inhibitors have all been investigated in flask cultures with more
or less success.
In the long run, however, in all experiments of this sort it is desirable,
if not absolutely necessary, to know how much tissue is present and if
it is all behaving in the same way, and whether or not it is increasing
in amount and how rapidly. Flask cultures in plasma clots do not
readily allow this. It is very difficult to get any reliable estimate of the
amount of tissue present once the tissue has become embedded in the
clot, and there is always the difficulty that the centre of the explant is
behaving differently from the peripheral outgrowth. Growth in the
periphery may be balanced by necrosis in the centre. Measurements
of the D N A content of the tissue can to some extent now overcome the
difficulty of the measurement of weight, since the D N A content per
cell, in normal non-growing tissues, is reasonably constant and the total
D N A can therefore be used to estimate the number of cells (Davidson,
Leslie and Waymouth, 1949; Healy, Fisher and Parker, 1954); but
the other difficulties remain, and dead cells may still contain DNA,
thus confusing the issue.
Total D N A content can, of course, only be measured once in such
cultures, and this limits its usefulness as a measure of growth for example.
DNA content of individual cells in the zone of outgrowth can, however,
be estimated by u.v. microscopy in living cells (Walker and Yates, 1952),
and this technique has shown how the D N A increases from n to 2n
during the interkinetic period. Since cells in a growing culture may
contain anything between n and 2n values for their DNA, estimates of
DNA in cell populations have only limited value in estimating cell
numbers when such populations are growing. The increase in D N A
content itself is, of course, a good measure of growth (see Chapters 7,
10 and 12).
It may be relevant here to point out that in all metabolic experiments
with cells in vitro, results which are apparently very different are obtained according to the method by which the amount of tissue present
is estimated, or rather according to the unit in which it is expressed.
Thus, activity expressed in terms of wet weight of tissue, dry weight of
tissue, number of cells, D N A content, total protein, protein nitrogen and
the like can all give quite different results. There are, in addition to the
errors involved in estimating these, very obvious differences in the
quantities being measured. For example, a piece of growing tissue
can vary the number of its cells, the size of its cells, the water content
of its cells and the amount of inter-cellular material, and the method
of growth-measurement selected must take account of all these factors
and, in addition, must allow for the fact that cells in the outgrowth
exposed to the medium may migrate, grow or function quite differently