82
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
of the medium was harmful, but the cultures would tolerate a slow
streaming of medium across their lower surface. This was achieved
by a "rocking boat" type of culture apparatus which maintained a
gentle flow of fully-oxygenated medium beneath the cultures by a
reversing siphon action. This procedure did improve oxygenation of
the basal part of the culture and slightly larger cultures could be used,
but it had no other advantage and it was not adopted for routine use.
By what other means could oxygen supply be improved ? The most
obvious way would be to increase the oxygen pressure in the gas-phase
by culturing in a pressurized vessel. But, since Dickens (1946), in
experiments on slices of a variety of tissues, had apparently shown that
certain respiratory enzymes are irreversibly poisoned by high-pressure
oxygen, this did not appear to be a very promising procedure. Nevertheless, MacDougall (1963) has recently cultured pieces of liver and
kidney from adult rats, under 3 atmospheres pressure of oxygen, for up
to 6 days without any histological sign of oxygen toxicity. He also
found that the limiting culture size was increased to almost exactly the
extent predicted by the above-mentioned formula. It seems possible
therefore that culture under high-pressure oxygen may become
routinely adopted for the maintenance of certain adult organs, at any
rate for short periods, and further experimental results will be awaited
with interest. The solubility of oxygen in culture media is very low,
about 0-4 mM, and the volume dissolved in 5 ml of medium is consumed
by twenty cultures in less than 1 h. At first sight it would seem that if
the solubility of oxygen could be increased by adding haemoglobin to
the medium, for instance, the situation would be improved. But this is
uncertain. It has been shown that the diffusion rate of oxygen
through a static solution of haemoglobin depends on the oxygen concentration at the receiving end. If this concentration is very low,
diffusion is faster than through water, but with higher concentrations
there is no haemoglobin effect (Hemmingsen and Scholander, 1960;
Collins, 1961). In any case haemoglobin is too toxic to use for this
purpose; Trowell (1961a) added haemoglobin, prepared from rat red
cells and purified by dialysis to a chemically-defined medium, and
found that even 0*5 % was toxic to most organs. At least 2°/o would be
required for any worth-while increase in oxygen solubility. It is equally
questionable, on theoretical grounds, if addition to the medium of
"carriers" such as cytochrome, ascorbic acid, glutathione or methylene
blue would be of any benefit in a strictly stationary system, and there is
also reason to think that the concentrations required would be toxic.
There remains one "trick" by which larger cultures can be used and
this exploits the fact that, among mammalian species, the inherent rate
of oxygen consumption by isolated tissues is inversely proportional to
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
of the medium was harmful, but the cultures would tolerate a slow
streaming of medium across their lower surface. This was achieved
by a "rocking boat" type of culture apparatus which maintained a
gentle flow of fully-oxygenated medium beneath the cultures by a
reversing siphon action. This procedure did improve oxygenation of
the basal part of the culture and slightly larger cultures could be used,
but it had no other advantage and it was not adopted for routine use.
By what other means could oxygen supply be improved ? The most
obvious way would be to increase the oxygen pressure in the gas-phase
by culturing in a pressurized vessel. But, since Dickens (1946), in
experiments on slices of a variety of tissues, had apparently shown that
certain respiratory enzymes are irreversibly poisoned by high-pressure
oxygen, this did not appear to be a very promising procedure. Nevertheless, MacDougall (1963) has recently cultured pieces of liver and
kidney from adult rats, under 3 atmospheres pressure of oxygen, for up
to 6 days without any histological sign of oxygen toxicity. He also
found that the limiting culture size was increased to almost exactly the
extent predicted by the above-mentioned formula. It seems possible
therefore that culture under high-pressure oxygen may become
routinely adopted for the maintenance of certain adult organs, at any
rate for short periods, and further experimental results will be awaited
with interest. The solubility of oxygen in culture media is very low,
about 0-4 mM, and the volume dissolved in 5 ml of medium is consumed
by twenty cultures in less than 1 h. At first sight it would seem that if
the solubility of oxygen could be increased by adding haemoglobin to
the medium, for instance, the situation would be improved. But this is
uncertain. It has been shown that the diffusion rate of oxygen
through a static solution of haemoglobin depends on the oxygen concentration at the receiving end. If this concentration is very low,
diffusion is faster than through water, but with higher concentrations
there is no haemoglobin effect (Hemmingsen and Scholander, 1960;
Collins, 1961). In any case haemoglobin is too toxic to use for this
purpose; Trowell (1961a) added haemoglobin, prepared from rat red
cells and purified by dialysis to a chemically-defined medium, and
found that even 0*5 % was toxic to most organs. At least 2°/o would be
required for any worth-while increase in oxygen solubility. It is equally
questionable, on theoretical grounds, if addition to the medium of
"carriers" such as cytochrome, ascorbic acid, glutathione or methylene
blue would be of any benefit in a strictly stationary system, and there is
also reason to think that the concentrations required would be toxic.
There remains one "trick" by which larger cultures can be used and
this exploits the fact that, among mammalian species, the inherent rate
of oxygen consumption by isolated tissues is inversely proportional to