16
General Methods
Inorganic
salts
is one in which the main constituents are chemically defined substances,
and this type of medium is used with supplementation of natural substances, such as sera or hemolymph. The third type of culture medium
consists of only chemically defined substances and can support cell
growth without any supplementation. The first and second types of media are usually supplemented with sera. However, some media can support cell growth without the addition of sera. These culture media are
called serum-free media. Serum-free media are not necessarily chemically defined media. The third type of culture medium sometimes contains proteins or lipids. However, formulation of media free of proteins,
lipids, or both is possible, even for the continuous culturing of cells.
2.1 Nutritional Requirements
Generally, cells require at least some inorganic ions, sugars, amino acids,
and vitamins for growth. In addition, most cells require proteins or lipids as growth promoters.
Inorganic salts are important for the maintenance of ionic balance and
osmotic pressure. In general,Na+, K+,Mg2+, Ca2+, CI-, and H 2 P0 4 - are considered essential for the survival and growth of cells. Chloride is the anion most frequently used. However, sulfide, carbonate and other anionic
forms can be used for at least part of the inorganic salts instead of chloride. It is known that cultured insect cells are relatively insensitive to
ionic balance. For example, many insect cell lines can grow in media over
a wide range of Na/K ratios. In extreme cases, cells of an insect cell line
can grow in a medium in which the Na/K ratio is larger than 1.0, as well
as in a medium in which the Na/K ratio is smaller than 1.0. (Mitsuhashi,
1989). From these facts, it can be said that the Na/K ratio is generally not
important for the growth of insect cells in culture, although it is reportedly important for the growth of some insect cells. In an embryonic cell
culture of the German cockroach Blattella germanica, active cell growth
was obtained only when the Na/K ratio of the medium was 10.0, which is
approximately the same as that of the hemolymph of this species (Ting
and Brooks, 1965). Krause et al. (1966) have reported that a Na/K ratio of
0.2 in molar concentration was a prerequisite for cellular outgrowth in
an ovarian cell culture of the silkworm Bombyx mori. However, the dependency of cell growth on the Na/K ratio may change according to the
composition of other components in the medium, because it has also
been reported that the growth of cells from B. mori ovaries in a different
medium was not affected by changes in the Na/K ratio (Wyatt, 1956).
Inorganic salts are important for the maintenance of the osmotic pressure of the culture medium. However, insect cells in vitro are known to
be tolerant of a wide range of osmotic pressures (Mitsuhashi, 1989). Insect hemolymph has an osmotic pressure of approximately 300 mOsmol/
kg, and most insect cell culture media have been designed so as to have
an osmotic pressure that does not differ much from this value. In extreme cases, cells can grow even in a medium that has been diluted more
General Methods
Inorganic
salts
is one in which the main constituents are chemically defined substances,
and this type of medium is used with supplementation of natural substances, such as sera or hemolymph. The third type of culture medium
consists of only chemically defined substances and can support cell
growth without any supplementation. The first and second types of media are usually supplemented with sera. However, some media can support cell growth without the addition of sera. These culture media are
called serum-free media. Serum-free media are not necessarily chemically defined media. The third type of culture medium sometimes contains proteins or lipids. However, formulation of media free of proteins,
lipids, or both is possible, even for the continuous culturing of cells.
2.1 Nutritional Requirements
Generally, cells require at least some inorganic ions, sugars, amino acids,
and vitamins for growth. In addition, most cells require proteins or lipids as growth promoters.
Inorganic salts are important for the maintenance of ionic balance and
osmotic pressure. In general,Na+, K+,Mg2+, Ca2+, CI-, and H 2 P0 4 - are considered essential for the survival and growth of cells. Chloride is the anion most frequently used. However, sulfide, carbonate and other anionic
forms can be used for at least part of the inorganic salts instead of chloride. It is known that cultured insect cells are relatively insensitive to
ionic balance. For example, many insect cell lines can grow in media over
a wide range of Na/K ratios. In extreme cases, cells of an insect cell line
can grow in a medium in which the Na/K ratio is larger than 1.0, as well
as in a medium in which the Na/K ratio is smaller than 1.0. (Mitsuhashi,
1989). From these facts, it can be said that the Na/K ratio is generally not
important for the growth of insect cells in culture, although it is reportedly important for the growth of some insect cells. In an embryonic cell
culture of the German cockroach Blattella germanica, active cell growth
was obtained only when the Na/K ratio of the medium was 10.0, which is
approximately the same as that of the hemolymph of this species (Ting
and Brooks, 1965). Krause et al. (1966) have reported that a Na/K ratio of
0.2 in molar concentration was a prerequisite for cellular outgrowth in
an ovarian cell culture of the silkworm Bombyx mori. However, the dependency of cell growth on the Na/K ratio may change according to the
composition of other components in the medium, because it has also
been reported that the growth of cells from B. mori ovaries in a different
medium was not affected by changes in the Na/K ratio (Wyatt, 1956).
Inorganic salts are important for the maintenance of the osmotic pressure of the culture medium. However, insect cells in vitro are known to
be tolerant of a wide range of osmotic pressures (Mitsuhashi, 1989). Insect hemolymph has an osmotic pressure of approximately 300 mOsmol/
kg, and most insect cell culture media have been designed so as to have
an osmotic pressure that does not differ much from this value. In extreme cases, cells can grow even in a medium that has been diluted more