2 Basic Information and Overview
21
heat-treated insect hemolymph, the further addition of vitamins improved insect tissue culture.
Thereafter, culture media have been formulated by a combination of
inorganic salts, sugars, amino acids, vitamins, and hemolymph or sera.
Instead of using individual chemicals, natural substances that contain groups of nutrients can be used. For example, lactalbumin hydrolysate or other protein hydrolysates have been used instead of a mixture of
free amino acids, and TC-yeastolate (Difco) or other yeast extracts have
been used instead of a mixture of vitamins. It is possible to use diluted
seawater in lieu of a mixture of inorganic salts (Mitsuhashi, 1987).
Insect hemolymph can be obtained in large quantities from matured
larvae or pupae of the industrially raised commercial silkworm and
saturnid moth, but availability of these insects is limited. Thus, vertebrate sera were examined for their ability to replace insect hemolymph.
FBS has been used since the 1960s. Mitsuhashi and Maramorosch (1964)
tested several commercially available sera, and came to the conclusion
that FBS can be used instead of insect hemolymph. They even stressed
that the presence of FBS is a prerequisite to obtain cellular outgrowth in
insect cell cultures. Although less effective, newborn calf serum can also
support the outgrowth of cells, but other sera from calf, bovine, horse,
goat, and chicken were ineffective.
Some media can support cell growth without supplementation with
sera. Such media are termed serum-free media. Serum-free media are
not necessarily chemically defined media. Most of the hitherto developed serum-free media contain more or less natural substances, and most
media that consist of chemically defined substances, such as Grace's medium, cannot support cell growth unless supplemented with hemolymph
or sera.
Only a few media are strictly chemically defined. For example, the
chemically defined media of Wilkie et al. (1980), MTCM-1520
(Mitsuhashi, 1996), and MTCM -1583 (Mitsuhashi, 1998b) are completely
chemically defined media. The former two media contain lipid components, while the latter is free of proteins and lipids.
In order to formulate culture media, the concentration and balance
of each component is important. The chemical composition of insect
hemolymph gives an indication of the concentration of each component
that should be used. This is based on the idea that, in vivo, tissues and
cells are bathed in hemolymph that contains nutrients suitable for the
maintenance or multiplication of cells and tissues. However,it should be
noted that the hemolymph also contains metabolites that are not necessary for, or are even toxic to, cells. Although Wyatt (1956) succeeded in
formulating a favorable medium based on the chemical composition of
insect hemolymph, imitation of hemolymph composition does not necessarily result in an optimal medium for cells from insects whose
hemolymph has been analyzed.
Another indicator for the composition of the culture medium is the
consumption of nutrients by cells. If a component is consumed to a greater
extent, then it is considered better that the amount of that component is
21
heat-treated insect hemolymph, the further addition of vitamins improved insect tissue culture.
Thereafter, culture media have been formulated by a combination of
inorganic salts, sugars, amino acids, vitamins, and hemolymph or sera.
Instead of using individual chemicals, natural substances that contain groups of nutrients can be used. For example, lactalbumin hydrolysate or other protein hydrolysates have been used instead of a mixture of
free amino acids, and TC-yeastolate (Difco) or other yeast extracts have
been used instead of a mixture of vitamins. It is possible to use diluted
seawater in lieu of a mixture of inorganic salts (Mitsuhashi, 1987).
Insect hemolymph can be obtained in large quantities from matured
larvae or pupae of the industrially raised commercial silkworm and
saturnid moth, but availability of these insects is limited. Thus, vertebrate sera were examined for their ability to replace insect hemolymph.
FBS has been used since the 1960s. Mitsuhashi and Maramorosch (1964)
tested several commercially available sera, and came to the conclusion
that FBS can be used instead of insect hemolymph. They even stressed
that the presence of FBS is a prerequisite to obtain cellular outgrowth in
insect cell cultures. Although less effective, newborn calf serum can also
support the outgrowth of cells, but other sera from calf, bovine, horse,
goat, and chicken were ineffective.
Some media can support cell growth without supplementation with
sera. Such media are termed serum-free media. Serum-free media are
not necessarily chemically defined media. Most of the hitherto developed serum-free media contain more or less natural substances, and most
media that consist of chemically defined substances, such as Grace's medium, cannot support cell growth unless supplemented with hemolymph
or sera.
Only a few media are strictly chemically defined. For example, the
chemically defined media of Wilkie et al. (1980), MTCM-1520
(Mitsuhashi, 1996), and MTCM -1583 (Mitsuhashi, 1998b) are completely
chemically defined media. The former two media contain lipid components, while the latter is free of proteins and lipids.
In order to formulate culture media, the concentration and balance
of each component is important. The chemical composition of insect
hemolymph gives an indication of the concentration of each component
that should be used. This is based on the idea that, in vivo, tissues and
cells are bathed in hemolymph that contains nutrients suitable for the
maintenance or multiplication of cells and tissues. However,it should be
noted that the hemolymph also contains metabolites that are not necessary for, or are even toxic to, cells. Although Wyatt (1956) succeeded in
formulating a favorable medium based on the chemical composition of
insect hemolymph, imitation of hemolymph composition does not necessarily result in an optimal medium for cells from insects whose
hemolymph has been analyzed.
Another indicator for the composition of the culture medium is the
consumption of nutrients by cells. If a component is consumed to a greater
extent, then it is considered better that the amount of that component is