18
General Methods
amino acids can be utilized by insect cells. In many insect cell culture
media, serine is often used as the DL-isomer. However, the DL-isomer of
serine can be replaced with the L-isomer, reducing the amount required
by one-half. Non-essential amino acids can be omitted from the culture
media altogether. However, when non-essential amino acids are present
in culture media, some are consumed by the cells (Mitsuhashi, 1976).
Therefore, it may be that some non-essential amino acids contribute to
the nutrition of the cells.
Vitamins Determination of essential vitamins is difficult. Because vitamins act at
very low concentrations, the complete elimination of certain vitamins is
impossible in serum-containing media. Even if using serum-free media,
if the media contain natural substances, a similar situation will occur.
Therefore, chemically defined media are a prerequisite for the determination of essential vitamins. However, until now, very few chemically
defined media have been developed for invertebrate cell culture and, consequently, information about the necessity of vitamins is limited.
In cultures of the American cockroach Periplaneta americana cell line
EPa, Landureau (1969) has reported that folic acid and cyanocobalamin
are necessary for the synthesis of nucleic acids, and that pantothenate,
choline, and inositol are necessary for lipid synthesis.
It has also been shown that pantothenate, folate, riboflavin, and inositol
are required for the long-term survival of cell lines, and that carnitine,
choline, pyridoxine, thiamine, and niacinamide are essential for the proliferation of cells (Becker and Landureau, 1981). Recently, by culturing
the flesh fly Sarcophaga peregrina cell line NIH-SaPe-4 in a chemically
defined medium for over 50 passages, the essential vitamins for this cell
line were determined (Mitsuhashi, 1998a). These cells did not require
any lipid-soluble vitamins or vitamin C. Among the B-group vitamins,
only thiamine, riboflavin, pantothenate, and niacin were essential. Niacin was interchangeable with niacinamide.
Some vitamins have been reported to be growth stimulating. Grace
(1958) has reported that the addition of 10 vitamins to Wyatt's medium
was growth promoting at a concentration of 0.01 Ilg/ml, but was detrimental at concentrations above 10 Ilg/ml. Sanborn and Haskell (1960)
noticed stimulation of cellular outgrowth by choline, biotin, and inositol
in cell cultures of the cynthia moth Philosamia cynthia. Nagle (1969)
observed growth promotion of cells from the yellow fever mosquito Aedes
aegypti by increasing the concentration of choline or pyridoxal.
Lipids The necessity of lipids for the growth of insect cells has not yet been
investigated. Most insect cell culture media need supplementation with
insect hemolymph or vertebrate sera. Lipids may be supplied from the
blood components. Therefore, most insect cell culture media are free of
added lipids.
Sterols are constituents of biologic membranes. Therefore, sterols seem
to be essential. In fact, mammalian cells can synthesize sterol but, if cultured with a sterol synthesis inhibitor, cells cannot multiply continuously
General Methods
amino acids can be utilized by insect cells. In many insect cell culture
media, serine is often used as the DL-isomer. However, the DL-isomer of
serine can be replaced with the L-isomer, reducing the amount required
by one-half. Non-essential amino acids can be omitted from the culture
media altogether. However, when non-essential amino acids are present
in culture media, some are consumed by the cells (Mitsuhashi, 1976).
Therefore, it may be that some non-essential amino acids contribute to
the nutrition of the cells.
Vitamins Determination of essential vitamins is difficult. Because vitamins act at
very low concentrations, the complete elimination of certain vitamins is
impossible in serum-containing media. Even if using serum-free media,
if the media contain natural substances, a similar situation will occur.
Therefore, chemically defined media are a prerequisite for the determination of essential vitamins. However, until now, very few chemically
defined media have been developed for invertebrate cell culture and, consequently, information about the necessity of vitamins is limited.
In cultures of the American cockroach Periplaneta americana cell line
EPa, Landureau (1969) has reported that folic acid and cyanocobalamin
are necessary for the synthesis of nucleic acids, and that pantothenate,
choline, and inositol are necessary for lipid synthesis.
It has also been shown that pantothenate, folate, riboflavin, and inositol
are required for the long-term survival of cell lines, and that carnitine,
choline, pyridoxine, thiamine, and niacinamide are essential for the proliferation of cells (Becker and Landureau, 1981). Recently, by culturing
the flesh fly Sarcophaga peregrina cell line NIH-SaPe-4 in a chemically
defined medium for over 50 passages, the essential vitamins for this cell
line were determined (Mitsuhashi, 1998a). These cells did not require
any lipid-soluble vitamins or vitamin C. Among the B-group vitamins,
only thiamine, riboflavin, pantothenate, and niacin were essential. Niacin was interchangeable with niacinamide.
Some vitamins have been reported to be growth stimulating. Grace
(1958) has reported that the addition of 10 vitamins to Wyatt's medium
was growth promoting at a concentration of 0.01 Ilg/ml, but was detrimental at concentrations above 10 Ilg/ml. Sanborn and Haskell (1960)
noticed stimulation of cellular outgrowth by choline, biotin, and inositol
in cell cultures of the cynthia moth Philosamia cynthia. Nagle (1969)
observed growth promotion of cells from the yellow fever mosquito Aedes
aegypti by increasing the concentration of choline or pyridoxal.
Lipids The necessity of lipids for the growth of insect cells has not yet been
investigated. Most insect cell culture media need supplementation with
insect hemolymph or vertebrate sera. Lipids may be supplied from the
blood components. Therefore, most insect cell culture media are free of
added lipids.
Sterols are constituents of biologic membranes. Therefore, sterols seem
to be essential. In fact, mammalian cells can synthesize sterol but, if cultured with a sterol synthesis inhibitor, cells cannot multiply continuously