20
General Methods
sect hemolymph, the active substance(s) in FBS has not been isolated.
Although some insect cell lines can be cultured in serum-free media,
many cell lines cannot multiply without FBS.
Tissue Chicken egg extract, chicken egg ultrafiltrate, and chicken egg yolk have
extracts been used previously in insect cell cultures. However, they are seldom
used at present. In addition, insect extracts made from eggs, fat bodies,
ovaries, and other tissues, or from whole animals, have been used sporadically. These extracts are growth stimulating to some insect cells, but
not to all cells.
Growth Growth factors of insect cells have not been isolated, except for the growth
factors factor from flesh fly cell cultures (Homma et aI., 1996). This growth factor has been said to act in an autocrine manner. Because flesh fly cells
can synthesize this growth factor and secrete it into the culture medium,
the cells can be cultured in a chemically defined medium (Mitsuhashi,
1996) without the addition of growth factor if the cells are seeded at a
sufficiently high density. None of the vertebrate growth factors has been
demonstrated to be effective on the invertebrate cells so far examined.
2.2 Design of Culture Media
During the very early stages of insect tissue culture, insect blood or
hemolymph itself was used as the culture medium. Insect blood has
strong phenol oxidase activity, and, consequently, the blood becomes dark
soon after exposure to air. The dark pigment formed is melanin, which is
detrimental to cultured cells. In order to prevent the melanization of
blood, phenylthiourea, a phenol oxidase inhibitor, was used. However,
phenylthiourea itself, or an accompanying impurity, was found to be toxic
to cultured cells, and now phenol oxidase is inactivated mostly by heating.
Simple physiological salt solutions were also used as the culture medium with or without glucose during the early stages of insect tissue
culture. Naturally, the results of these cultures were a short survival of
the cells.
Following this early period in the culture of insect tissues, the media
used for vertebrate cell cultures, especially mixtures of natural substances,
have been used for insect tissue cultures.
A medium proper for insect cell culture was developed by Wyatt (1956)
on the basis of the chemical composition of the hemolymph of several
insects. This medium consists of inorganic salts, sugars and organic acids including amino acids and is used with the addition of heat -treated
hemolymph.
Grace (1962) successfully improved the medium developed by Wyatt,
mainly by introducing 10 water-soluble vitamins. In Wyatt's medium,
vitamins were supplied by the added hemolymph. It is probable that the
vitamins contained in heat-treated hemolymph were not sufficient for
cell growth. Although Grace's medium also needs to be fortified with
General Methods
sect hemolymph, the active substance(s) in FBS has not been isolated.
Although some insect cell lines can be cultured in serum-free media,
many cell lines cannot multiply without FBS.
Tissue Chicken egg extract, chicken egg ultrafiltrate, and chicken egg yolk have
extracts been used previously in insect cell cultures. However, they are seldom
used at present. In addition, insect extracts made from eggs, fat bodies,
ovaries, and other tissues, or from whole animals, have been used sporadically. These extracts are growth stimulating to some insect cells, but
not to all cells.
Growth Growth factors of insect cells have not been isolated, except for the growth
factors factor from flesh fly cell cultures (Homma et aI., 1996). This growth factor has been said to act in an autocrine manner. Because flesh fly cells
can synthesize this growth factor and secrete it into the culture medium,
the cells can be cultured in a chemically defined medium (Mitsuhashi,
1996) without the addition of growth factor if the cells are seeded at a
sufficiently high density. None of the vertebrate growth factors has been
demonstrated to be effective on the invertebrate cells so far examined.
2.2 Design of Culture Media
During the very early stages of insect tissue culture, insect blood or
hemolymph itself was used as the culture medium. Insect blood has
strong phenol oxidase activity, and, consequently, the blood becomes dark
soon after exposure to air. The dark pigment formed is melanin, which is
detrimental to cultured cells. In order to prevent the melanization of
blood, phenylthiourea, a phenol oxidase inhibitor, was used. However,
phenylthiourea itself, or an accompanying impurity, was found to be toxic
to cultured cells, and now phenol oxidase is inactivated mostly by heating.
Simple physiological salt solutions were also used as the culture medium with or without glucose during the early stages of insect tissue
culture. Naturally, the results of these cultures were a short survival of
the cells.
Following this early period in the culture of insect tissues, the media
used for vertebrate cell cultures, especially mixtures of natural substances,
have been used for insect tissue cultures.
A medium proper for insect cell culture was developed by Wyatt (1956)
on the basis of the chemical composition of the hemolymph of several
insects. This medium consists of inorganic salts, sugars and organic acids including amino acids and is used with the addition of heat -treated
hemolymph.
Grace (1962) successfully improved the medium developed by Wyatt,
mainly by introducing 10 water-soluble vitamins. In Wyatt's medium,
vitamins were supplied by the added hemolymph. It is probable that the
vitamins contained in heat-treated hemolymph were not sufficient for
cell growth. Although Grace's medium also needs to be fortified with