JPH067791B2 - Method for culturing insect cells in autoclave sterilized serum-free medium - Google Patents

Description

The present invention relates to a serum-free insect cell culture medium sterilized by a moist heat sterilization method, preferably an autoclave heating method, for culturing insect cells, in particular, Spodoptera frugiperda-derived cells. A technique for culturing insect cells using the same.

[Conventional technology]

The production of insect viruses using insect cells and the production of useful substances produced in animal cells such as the production of human interferon using immunologically unrelated insect cells have been studied.

Generally, in culturing insect cells, serum obtained from calves or fetal calves is used as a medium component.
However, this serum is not only expensive, but also due to the non-uniformity of quality, instability, and difficulty in obtaining in large quantities, studies have been conducted on a medium that can culture insect cells without containing serum. As a result, several serum-free media have been developed.
For example, a medium for Drosophila cells [The Ecaria: E., Carstuck, K. Malamoloche ed. "Invertebrate Tissue Culture Application In Medicine Biology And
Agriculture "(Invertabrate Tissue Culture, Ap
replications in Medicine, Biology and Agriculture.) 1
Pages 31-150, Academic Press, New York 19
Issued in 1976; Kei. Di. Davis, A. Shaun:
"Science" 196, 438-440, 1977],
Mosquito medium [S. Kitamura et al. "Journal of Medical Entomology" (J.Med.Entomol.) Volume 10
488-489, 1973], as a medium for insect cells of Lepidoptera and Diptera, Mitsuhashi and Mara Morosh
morosch) medium [J. Mitsuhashi, Kei. Malamo Roche “Contribution of Voice Thompson
`` Institute '' (Contrib.Boyce Thompson Inst.)
22 Vol. 435-460, 1964], serum-free medium and the like are used.

[Problems to be solved by the invention]

As a method for sterilizing these serum-free media, filter sterilization is performed. The method usually uses a sterilized membrane filter. After the medium is prepared, if it is passed through a filter for sterilization directly, clogging will be serious and it will not be efficient. Therefore, as a pretreatment, precipitates are removed from the medium immediately after preparation with a high-speed centrifuge and filtered with filters having different pore sizes step by step, but operations such as filter replacement and container preparation during each operation are complicated. Is. Further, it is necessary to carry out the aseptic dispensing operation for transferring the medium after the filter sterilization to each incubator, and it is necessary for a person skilled in the aseptic operation technique to perform the operation. Particularly, in order to dispense a large amount of medium without contamination with various bacteria, a high degree of aseptic technique is required. Large-scale culture requires a dedicated sterile filtration device, which increases equipment investment and maintenance costs.

Under such circumstances, it is desired to develop an inexpensive medium that does not require a high degree of aseptic operation and can be prepared by anyone with a simple operation, not limited to a person who has acquired a particular technical skill. An object of the present invention is to provide a serum-free method for culturing insect cells, which uses a serum-free medium for culturing insect cells, which meets these needs.

[Means for solving problems]

Based on such circumstances, the present inventors have conducted research to solve such problems, and have conducted autoclave sterilization of the medium from which serum has been removed from Mitsuhashi and Maramorosch (Mitsuhashi and Maramorosch) medium. It was found that it is possible to cultivate insect cells in this medium in a sterile condition, and the present invention was accomplished. That is, instead of the conventionally used medium for culturing insect cells, a medium containing yeast powder and albumin hydrolyzate (hydrolyzate) and containing no serum component is used for culturing insect cells by heat treatment. It was found that insect cells can be sufficiently grown without losing necessary components.

Therefore, according to the present invention, a medium containing sodium chloride, potassium chloride, sodium dihydrogen phosphate, sodium hydrogen carbonate, magnesium chloride, calcium chloride, saccharides, yeast powder and albumin hydrolyzate, which is substantially serum-free. A method for culturing insect cells in a medium that has been sterilized by a moist heat sterilization method is provided.

The medium used in the method of the present invention is Mitsuhashi maramoros
(Mitsuhashi and Maramorosch) Yeast powder and albumin hydrolyzate were added to the remaining components excluding serum from the medium, namely sodium chloride, potassium chloride, sodium dihydrogen phosphate, sodium bicarbonate, magnesium chloride, calcium chloride, and sugars. Using a medium having the composition of, like the conventional medium, the pH value can be adjusted to a range of 5.6 to 7.0 using potassium hydroxide, hydrochloric acid, etc., and then sterilized by moist heat to prepare a sterile serum-free medium. .

Among the above components, magnesium chloride anhydrous and magnesium chloride hexahydrate can be used as magnesium chloride, but magnesium chloride hexahydrate is preferable. As calcium chloride, anhydrous calcium chloride, calcium chloride dihydrate and calcium chloride hexahydrate can be used, but calcium chloride dihydrate is preferred.

As the saccharide, D-glucose or fructose can be used, but D-glucose is preferred. As yeast powder, baker's yeast powder and brewer's yeast powder can be used, but baker's yeast powder is preferable. As the albumin hydrolyzate, serum albumin hydrolyzate, lactalbumin hydrolyzate and ovalbumin hydrolyzate can be used, but lactalbumin hydrolyzate is preferred.

As the wet heat sterilization method, an autoclave sterilization method and an intermittent heating method can be used, but the autoclave sterilization method is preferable.

Furthermore, serum can be added to the above medium components before or after the sterilization operation.

Since each component of the serum-free medium for insect cell culture used in the method of the present invention can be used as it is as a commercial product, it can be stably obtained at a low cost, and further, it is highly sterilized by filtration. Since the object can be achieved by a steam sterilization method in a simple autoclave without depending on the operation, anyone who is not skilled in aseptic operation can prepare it.

To prepare the serum-free medium used in the method of the present invention,
The following inorganic salt components known as conventional components of conventional serum-free medium for insect cells, namely, sodium chloride (6.0 to 8.0 g /, suitable addition amount per medium: hereinafter the same), potassium chloride ( 0.1-0.3 g /), disodium phosphate (0.1-0.4 g /), sodium hydrogen carbonate (0.
1-0.4 g /), magnesium chloride hexahydrate (0.05-0.
2 g /) and calcium chloride dihydrate (0.1-0.3 g /)
), And D-glucose (2.0 to 8.0 g /), albumin hydrolyzate (3.0 to 15.0 g /) and yeast powder (1.0 to 8.0 g /) are added to the medium to prepare a medium. After that, adjust the pH with potassium hydroxide, hydrochloric acid, etc.
It may be adjusted within the range of 5.6 to 7.0. As these medium components, commercially available products may be used as they are.

In addition, other components, namely magnesium chloride anhydrous, calcium chloride anhydrous, calcium chloride dihydrate,
Etc. can be used as well.

As the wet heat sterilization method, the method of autoclave sterilization consists of a usual method, that is, heating at 110 to 130 ° C. for 15 to 30 minutes in a steam atmosphere of 1.0 to 1.2 atm. In this way, the sterile serum-free medium used in the present invention can be prepared.

Hereinafter, the present invention will be described in detail with reference to Examples, but these are merely examples and do not limit the present invention.

Example 1 A serum-free medium containing each component having the composition shown in Table 1 below was prepared,
5 m of sterilized sterile medium was heated in an autoclave (120 ° C., 20 minutes) and dispensed into a Corning plastic flask (25 cm ² ). On the other hand, four types of Spodoptera frugiperda-derived cells obtained by culturing in the autoclave-sterilized medium of Table 1 for 4 days, SES-MaBr-1; SES-MaBr-
2; SES-MaBr-3; SES-MaBr-4 and two types of blood cell-derived cells NIAS-
MaBr-92; NIAS-MaBr-93 and one type of ovary-derived cells, NIAS-Ma
Br-32 and the initial cell concentration were 2.0 × 10 ⁵ cells / m 2, respectively.
Was inoculated so that

The flasks were cultured on standing in a cell incubator which is maintained at 25 ° C., the number of cells after 9 days, 21.6 × 10 ⁵ cells each /M;18.1×10 ⁵ pieces / m;
21.7 × 10 ⁵ cells /m;16.4×10 ⁵ pieces /m;11.9×
10 ⁵ /M;16.5×10 ^5; 18.3 × 10 ⁵ cells / m
Up to.

Example 2 A serum-free medium having the composition shown in Table 1 was prepared and sterilized by the sterilization method used in Example 1, and then fetal bovine serum (Difco Di
(manufactured by fco) was aseptically added to a serum concentration of 3% to prepare a serum-containing medium. 5m of this serum-containing medium
To a Corning plastic flask (25 cm ² )
Dispensed. On the other hand, 4 in advance in the above serum-containing medium
Spodoptera frugiperda-derived cells NIAS-MaBr-93 obtained by culture for one day
Were inoculated so that the initial cell concentration would be 2.0 × 10 ⁵ cells / m 2.

When this flask was left standing in a cell incubator kept at 25 ° C and cultured, the number of cells after 9 days was 18.2 × 1
0 increased to up to ^five / m.

Example 3 A serum-free medium having the composition shown in Table 1 was prepared, sterilized by the sterilization method used in Example 1, and dispensed in 100 m aliquots into a glass spinner stirred incubator with an internal volume of 250 m. Spodoptera frugiperda blood cell-derived cells NIAS-MaBr-93 obtained by culturing in an autoclave-sterilized medium for 4 days in advance
Were inoculated so that the initial cell concentration was 2.0 × 10 ⁵ cells / m 2.

In a cell incubator kept at 25 ° C., stirring was carried out at a stirring rate of 60 times per minute, and the cells were cultured for 10 days. The number of cells after culturing increased to 9.07 × 10 ⁵ cells / m, and 9.07 × 10 ⁷ cells were obtained by serum-free culture from one spinner incubator.

Example 4 Serum-free cultured blood having the composition shown in Table 1 was prepared, sterilized by the sterilization method used in Example 1, and dispensed into a spinner stirred incubator made of glass having an inner volume of 250 m by 100 m. Spodoptera frugiperda-derived cells SES-MaBr-1 obtained by culturing for 4 days in the medium of Table 1 that had been autoclaved in advance.
Were inoculated so that the initial cell concentration was 2.0 × 10 ⁵ cells / m 2.

In a cell incubator kept at 25 ° C., stirring was carried out at a stirring rate of 60 times per minute, and the cells were cultured for 10 days. The number of cells after culturing was increased to 10.8 × 10 ⁵ cells / m, and 1.08 × 10 ⁸ cells were obtained by serum-free culture from one spinner incubator.

Example 5 A serum-free medium having the composition shown in Table 1 was prepared, sterilized by the sterilization method used in Example 1, and poured into a glass spinner stirred incubator with an internal volume of 1.3 for 500 m. The Spodoptera frugiperda-derived cells NIAS-MaBr-93 obtained by culturing in an autoclave-sterilized medium for 4 days in advance were inoculated so that the initial cell concentration was 1.0 × 10 ⁵ cells / m 2.
In a cell incubator kept at 25 ° C., the cells were stirred at a stirring rate of 40 times per minute and cultured for 12 days. The number of cells after culture was increased to 8.87 × 10 ⁵ cells / m, and 4.44 × 10 ⁸ cells were obtained by serum-free culture from one spinner incubator.

Example 6 A serum-free medium having the composition shown in Table 1 was prepared, sterilized by the sterilization method used in Example 1, and poured into a glass mini jar incubator having an internal volume of 5 every 3 minutes. The Spodoptera frugiperda-derived cells NIAS-MaBr-93 obtained by culturing in an autoclave-sterilized medium for 4 days in advance had an initial cell concentration of 1.0 × 1.
0 ^five were inoculated so that the / m. The mixture was kept at 25 ° C., stirred at a stirring rate of 150 times per minute, and cultured for 16 days. The number of cells after culturing was increased to 3.83 × 10 ⁵ cells / m, and 1.15 × 10 ⁹ cells were obtained by serum-free culture from one Miniger incubator.

〔The invention's effect〕

 According to the present invention, the following operational effects are brought about.

First of all, as a method of aseptic processing of a medium, a wet heat sterilization method, for example, an autoclave sterilization has become possible without relying on the conventional filter sterilization. Therefore, a high degree of aseptic operation of the filter sterilization method is not required, and a dedicated filtration device is not required even in mass culture. As a result, in the present invention, insect cells can be cultivated in a sterile serum-free medium that can be prepared at a low cost by a simple operation, not limited to a person who has mastered a particular technique.

Secondly, the components of the medium used in the method of the present invention are all commercially available, easily available and inexpensive raw materials are used, and the medium preparation is extremely easy as compared with the conventional medium using expensive serum. .

Thirdly, according to the serum-free medium used in the present invention, the growth of insect cells can be promoted to about 3.8 to 11 times the initial inoculation concentration, although there are some differences depending on the type of insect cells. . This is as shown in Examples 1 to 6.

Claims (1)

[Claims] 1. A medium containing sodium chloride, potassium chloride, sodium dihydrogen phosphate, sodium hydrogen carbonate, magnesium chloride, calcium chloride, saccharides, yeast powder and albumin hydrolyzate, which is substantially serum-free and is sterilized by moist heat. A method of culturing insect cells in a medium made sterile by the method.