24
A . M O S G O N A , O . A . T R O W E L L A N D E . N . W l L L M E R
and its effects beneficial. The direct buffering of the culture fluid
is generally much less efficient than the buffering of the blood, for
the latter depends largely on the high concentration of haemoglobin
available in the erythrocytes. Again, the question of local p H values
may be of great importance, and it is likely that some cells in the
body are adapted to different hydrogen-ion concentrations from others;
to give an example which is perhaps extreme, the venous bloods leaving
the pancreas and the stomach mucosa during periods of active secretion
present the endothelia of their respective venous channels with media
which must at least have very different alkali reserves if not actually
different hydrogen-ion concentrations. Epithelial cells frequently
have fluids of different pHs bathing their external and internal surfaces.
The pancreatic duct, the respiratory passages and the urinary passages
are examples of these. In the stagnant conditions in many tissue
cultures the local pH in the vicinity of the cells may often differ quite
widely from that of the surrounding fluids. The occurrence and extent
of such local differences may be very important in relation to the
choice of fluids suitable for perfusion methods as opposed to those
suitable for stagnant cultures. Moreover, the cells at the centre of a
large explant are often observed to become necrotic. The immediate
cause of this necrosis is not known. The diffusion of oxygen towards
the centre of the tissue is probably the limiting factor and could act
adversely on the cells both directly and also indirectly by throwing them
on to their glycolytic mechanisms with the inevitable production of
more acid metabolites, e.g. lactic acid, which then may have difficulty
in diffusing away quickly enough to prevent a fall in pH.
In attempting to maintain the hydrogen-ion concentration of
artificial media the nature of the buffer used is very important. Bicarbonate-C0 2 , phosphate, phosphate-bicarbonate, veronal and tris
(hydroxymethyl) amino-methane systems have all been tried from time
to time. In general, it is safe to say that greater trust should be placed
in the more nearly physiological systems relying on bicarbonatephosphate mixtures, as used for example in Tyrode's solution, than in
most others. Such a system is, of course, rather delicately poised in
equilibrium with the C 0 2 in the gas phase above the culture which, if
left uncontrolled, may lead to wild fluctuations in the pH of the
medium. At the same time, it provides the opportunity for very delicate
control of pH since the gas composition of the mixture (e.g. usually
around 5% C 0 2 in 0 2 or air) and the rate of its renewal can be
accurately regulated, thus in some way simulating the physiological
action of the lungs. Phenol red in the medium is relatively harmless
and provides good visual evidence of approximate p H values. Gas-tight
culture vessels are, of course, obligatory when any degree of accuracy
A . M O S G O N A , O . A . T R O W E L L A N D E . N . W l L L M E R
and its effects beneficial. The direct buffering of the culture fluid
is generally much less efficient than the buffering of the blood, for
the latter depends largely on the high concentration of haemoglobin
available in the erythrocytes. Again, the question of local p H values
may be of great importance, and it is likely that some cells in the
body are adapted to different hydrogen-ion concentrations from others;
to give an example which is perhaps extreme, the venous bloods leaving
the pancreas and the stomach mucosa during periods of active secretion
present the endothelia of their respective venous channels with media
which must at least have very different alkali reserves if not actually
different hydrogen-ion concentrations. Epithelial cells frequently
have fluids of different pHs bathing their external and internal surfaces.
The pancreatic duct, the respiratory passages and the urinary passages
are examples of these. In the stagnant conditions in many tissue
cultures the local pH in the vicinity of the cells may often differ quite
widely from that of the surrounding fluids. The occurrence and extent
of such local differences may be very important in relation to the
choice of fluids suitable for perfusion methods as opposed to those
suitable for stagnant cultures. Moreover, the cells at the centre of a
large explant are often observed to become necrotic. The immediate
cause of this necrosis is not known. The diffusion of oxygen towards
the centre of the tissue is probably the limiting factor and could act
adversely on the cells both directly and also indirectly by throwing them
on to their glycolytic mechanisms with the inevitable production of
more acid metabolites, e.g. lactic acid, which then may have difficulty
in diffusing away quickly enough to prevent a fall in pH.
In attempting to maintain the hydrogen-ion concentration of
artificial media the nature of the buffer used is very important. Bicarbonate-C0 2 , phosphate, phosphate-bicarbonate, veronal and tris
(hydroxymethyl) amino-methane systems have all been tried from time
to time. In general, it is safe to say that greater trust should be placed
in the more nearly physiological systems relying on bicarbonatephosphate mixtures, as used for example in Tyrode's solution, than in
most others. Such a system is, of course, rather delicately poised in
equilibrium with the C 0 2 in the gas phase above the culture which, if
left uncontrolled, may lead to wild fluctuations in the pH of the
medium. At the same time, it provides the opportunity for very delicate
control of pH since the gas composition of the mixture (e.g. usually
around 5% C 0 2 in 0 2 or air) and the rate of its renewal can be
accurately regulated, thus in some way simulating the physiological
action of the lungs. Phenol red in the medium is relatively harmless
and provides good visual evidence of approximate p H values. Gas-tight
culture vessels are, of course, obligatory when any degree of accuracy