2 . M E T H O D S
23
quite widely in this respect, but it is only recently that indications have
appeared that, within the limits of any one animal, the cells of different
tissues may require, or be constantly subjected to, solutions whose
osmotic pressure is different from that of the blood as a whole. For
example, the cells in the pyramids of the mammalian kidney are
normally subjected to salt concentrations hypertonic to those in most
other parts of the body (Wirz, Hargitay and Kuhn, 1951). In recent
experiments on lymphocytes in organ culture (Trowell, 1962) (see
Vol. 2, Chapter 2) it has been observed that mammalian lymphocytes
survive better in media containing only 0-4 °/o NaCl in place of the
more usual 0-8 °/o NaCl. The significance of these findings is still somewhat obscure, but they certainly point towards a greater diversity than
was hitherto suspected.
While mammalian and avian tissues in culture may be able to withstand "abnormal" osmotic pressures for a time, i.e. 3 or 4 days, it has
long been known (Ebeling, 1914; Lewis and Lewis, 1924; Willmer,
1927) that tissues and cells which have to battle against unfavourable
osmotic pressures sooner or later go down hill and eventually succumb.
In this connexion it is important to differentiate between the effects of
ionic concentrations and of osmotic pressure as such. The two may be
very different. In the body, the fluid bathing the individual cells is to
some extent a dialysate from the blood, but it nevertheless contains proteins and other molecules capable of exerting a "colloid osmotic pressure". How far this is important in determining the activity of cells in culture is uncertain; but, as will be mentioned elsewhere, methyl cellulose
and polyvinylpyrrolidone when added to the fluid for suspension cultures increase the viability of the cells (Merchant, Hellman, Schneider
and Muirhead, 1962), though whether their action is osmotic, adsorptive
or dependent on viscosity is not clear.
D. H Y D R O G E N - I O N C O N C E N T R A T I O N
The regulation of the hydrogen-ion concentration in the immediate
vicinity of the living cells is very important, and many technical
problems are set both in determining the optimum p H required
in the medium for any particular tissue and in maintaining the p H at
that level. In the body, the p H of the various fluids is maintained not
only by direct buffering systems in the fluids themselves, but also
indirectly by such physiological regulation as the controlled removal
of C O 2 from the lungs or the elimination of acid or alkali by the appropriate activity of the kidneys. Only the first of these methods
(direct buffering) is usually available in Tissue Culture though control
of the composition of the gas phase is becoming increasingly possible
23
quite widely in this respect, but it is only recently that indications have
appeared that, within the limits of any one animal, the cells of different
tissues may require, or be constantly subjected to, solutions whose
osmotic pressure is different from that of the blood as a whole. For
example, the cells in the pyramids of the mammalian kidney are
normally subjected to salt concentrations hypertonic to those in most
other parts of the body (Wirz, Hargitay and Kuhn, 1951). In recent
experiments on lymphocytes in organ culture (Trowell, 1962) (see
Vol. 2, Chapter 2) it has been observed that mammalian lymphocytes
survive better in media containing only 0-4 °/o NaCl in place of the
more usual 0-8 °/o NaCl. The significance of these findings is still somewhat obscure, but they certainly point towards a greater diversity than
was hitherto suspected.
While mammalian and avian tissues in culture may be able to withstand "abnormal" osmotic pressures for a time, i.e. 3 or 4 days, it has
long been known (Ebeling, 1914; Lewis and Lewis, 1924; Willmer,
1927) that tissues and cells which have to battle against unfavourable
osmotic pressures sooner or later go down hill and eventually succumb.
In this connexion it is important to differentiate between the effects of
ionic concentrations and of osmotic pressure as such. The two may be
very different. In the body, the fluid bathing the individual cells is to
some extent a dialysate from the blood, but it nevertheless contains proteins and other molecules capable of exerting a "colloid osmotic pressure". How far this is important in determining the activity of cells in culture is uncertain; but, as will be mentioned elsewhere, methyl cellulose
and polyvinylpyrrolidone when added to the fluid for suspension cultures increase the viability of the cells (Merchant, Hellman, Schneider
and Muirhead, 1962), though whether their action is osmotic, adsorptive
or dependent on viscosity is not clear.
D. H Y D R O G E N - I O N C O N C E N T R A T I O N
The regulation of the hydrogen-ion concentration in the immediate
vicinity of the living cells is very important, and many technical
problems are set both in determining the optimum p H required
in the medium for any particular tissue and in maintaining the p H at
that level. In the body, the p H of the various fluids is maintained not
only by direct buffering systems in the fluids themselves, but also
indirectly by such physiological regulation as the controlled removal
of C O 2 from the lungs or the elimination of acid or alkali by the appropriate activity of the kidneys. Only the first of these methods
(direct buffering) is usually available in Tissue Culture though control
of the composition of the gas phase is becoming increasingly possible