2 . M E T H O D S
81
term experiments on small amounts of tissue. Many problems in
morphogenesis, physiology, biochemistry, pharmacology and radiobiology have been or could be fruitfully investigated in this way. But
the wider application of the organ-culture method in these and other
fields is at present limited by two circumstances, the size of the cultures,
and the time for which they will survive and function in vitro. If larger
cultures could be maintained for a longer time the scope of the method
could be considerably extended. In this section we shall examine these
limitations in some detail and consider what attempts have been or
could be made to overcome them.
1. Culture Size
If the culture exceeds a certain size the central cells die, either from
lack of oxygen or glucose, or from poisoning by accumulated C O a ,
lactic acid or other waste product. The diffusion rate of one or other
of these substances must be the size-limiting factor. In the case of
embryonic organs it is not known which one is size-limiting. W e shall
consider here only mature organs, for in these it has been shown both
in theory and by experiment that the limiting factor is oxygen (Trowell,
1952, 1959, 1961a). If the rate of oxygen consumption (A), the external
oxygen concentration (C), and the diffusion coefficient of oxygen
through the tissue (D) are known, the limiting culture size can be
calculated. For a spherical culture, the limiting radius (r) for zero oxygen concentration at the centre is given by r = ^/6DCjA. The diffusion
coefficient of oxygen through living mammalian tissue is not known, so
the value through water has to be used. On this basis the calculated
value of r turns out to be somewhat less than that found by actual experiment. Trowell (1961a) found that for most organs the observed
value of r was roughly 5 0 % greater than the predicted value. This must
mean that the rate of diffusion of oxygen through living tissue is faster
than through water. Longmuir and Bourke (1960) have also deduced,
from experiments on tissue slices, that the diffusion coefficient of oxygen
through respiring tissue is considerably greater than that through nonrespiring tissue or through water. T o explain their findings they postulated an active intracellular transport of oxygen possibly by protoplasmic streaming. Whatever the transport mechanism, it is evidently
maintained by respiratory energy. This is an interesting hypothesis
which may have an important bearing on organ culture-technique.
If the oxygen supply could be improved, correspondingly larger
cultures could be maintained. In a culture on a static medium the centre
of the base receives less oxygen than other parts of the surface because
it is furthest removed from the gas-phase. This could be corrected
by stirring the medium. Trowell (1961a) found that any rapid movement
81
term experiments on small amounts of tissue. Many problems in
morphogenesis, physiology, biochemistry, pharmacology and radiobiology have been or could be fruitfully investigated in this way. But
the wider application of the organ-culture method in these and other
fields is at present limited by two circumstances, the size of the cultures,
and the time for which they will survive and function in vitro. If larger
cultures could be maintained for a longer time the scope of the method
could be considerably extended. In this section we shall examine these
limitations in some detail and consider what attempts have been or
could be made to overcome them.
1. Culture Size
If the culture exceeds a certain size the central cells die, either from
lack of oxygen or glucose, or from poisoning by accumulated C O a ,
lactic acid or other waste product. The diffusion rate of one or other
of these substances must be the size-limiting factor. In the case of
embryonic organs it is not known which one is size-limiting. W e shall
consider here only mature organs, for in these it has been shown both
in theory and by experiment that the limiting factor is oxygen (Trowell,
1952, 1959, 1961a). If the rate of oxygen consumption (A), the external
oxygen concentration (C), and the diffusion coefficient of oxygen
through the tissue (D) are known, the limiting culture size can be
calculated. For a spherical culture, the limiting radius (r) for zero oxygen concentration at the centre is given by r = ^/6DCjA. The diffusion
coefficient of oxygen through living mammalian tissue is not known, so
the value through water has to be used. On this basis the calculated
value of r turns out to be somewhat less than that found by actual experiment. Trowell (1961a) found that for most organs the observed
value of r was roughly 5 0 % greater than the predicted value. This must
mean that the rate of diffusion of oxygen through living tissue is faster
than through water. Longmuir and Bourke (1960) have also deduced,
from experiments on tissue slices, that the diffusion coefficient of oxygen
through respiring tissue is considerably greater than that through nonrespiring tissue or through water. T o explain their findings they postulated an active intracellular transport of oxygen possibly by protoplasmic streaming. Whatever the transport mechanism, it is evidently
maintained by respiratory energy. This is an interesting hypothesis
which may have an important bearing on organ culture-technique.
If the oxygen supply could be improved, correspondingly larger
cultures could be maintained. In a culture on a static medium the centre
of the base receives less oxygen than other parts of the surface because
it is furthest removed from the gas-phase. This could be corrected
by stirring the medium. Trowell (1961a) found that any rapid movement