JPS63148982A - Cultivation of insect cell using autoclave-sterilized serum-free culture medium - Google Patents

Abstract

PURPOSE:To readily and efficiently cultivate insect cells, particularly cells derived from cabbage armyworm at a low cost, by using a culture medium prepared by converting a culture medium substantially containing no blood serum into a germ-free state by a wet-heat sterilizing method. CONSTITUTION:A culture medium containing residual components after removing blood serum from a Mitsuhashi.Maramorosch culture medium, i.e. culture medium containing 6.0-8.0g/l sodium chloride, 0.1-0.3g/l potassium chloride, 0.1-0.4g/l disodium phosphate, 0.1-0.4g/l sodium hydrogencarbonate, 0.05-0.2g/l magnesium chloride hexahydrate, 0.1-0.3g/l calcium chloride dihydrate and 2.0-8.0g/l D-glucose, 3.0-15.0g/l albumin hydrolyzate and 1.0-8.0g/l yeast flour is prepared and adjusted to a pH range of 5.6-7.0. The adjusted culture medium is then sterilized at 110-130 deg.C in a steam atmosphere under 1.0-1.2atm for 15-30min in an autoclave. Insect cells, e.g. cells derived from cabbage armyworm, etc., can be propagated in the above-mentioned culture medium.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention provides a serum-free insect cell culture medium sterilized by a moist heat sterilization method, preferably an autoclave heating method, for culturing insect cells, particularly cells derived from armyworm moth. The present invention relates to a technique for culturing insect cells using the present invention.

(Prior Art) Research has been conducted on the production of insect viruses using insect cells, as well as the production of useful substances produced in animal cells, such as the production of human interferon, using immunologically unrelated insect cells. ing.

Generally, when culturing insect cells, serum obtained from calves or bovine fetuses is used as a medium component.

However, this serum is not only expensive, but also has uneven quality, instability, and is difficult to obtain in large quantities. Therefore, research is being conducted on a medium that can culture insect cells without containing serum. As a result, several serum-free media have been developed.

For example, media for Drosophila cells (G.E.Kalianii, Karstack, K., Maramoros, eds., "Inverted Tissue Culture Applications in Medicine Biology and Applications J. -te
Ti5st+e Culture, Applica
tions in Medicine.

Biology and Agriculture,)
pp. 131-150, Academic Press, New York, 1976. K., D., Davis, N., Shaan: Science J.
196, pp. 438-440, 1977], medium for force cells [Niss, Kitamura et al., “Journal Medical-I
Entomology j (J, Hed, Entom-01,)
10, pp. 488-489, 1973], Mitsuhashi and Maramoros (M.
Itsuhashi and Maramorosch
) Medium [J.E., Mitsuhashi, K., Maramolos, Contribute to Boyce Thompson Institute J (Contrib, Boyce Thompson
n In5t, ) Vol. 22, pp. 435-460, 196
Since [2004], a culture medium with a serum-free composition has been used.

[Problem that the invention seeks to solve]

Filtration sterilization is used as a method for sterilizing these serum-free media. The method typically uses sterile membrane filters.

If you pass the culture medium directly through a sterilization filter after preparing it, it will become extremely clogged and inefficient. Therefore, as a pretreatment, the precipitate is removed from the culture medium immediately after preparation using a high-speed centrifuge, and the precipitate is filtered step by step through filters with different pore sizes, but operations such as filter replacement and container preparation during each operation are complicated. It is. Furthermore, the dispensing operation of transferring the filter-sterilized culture medium to each incubator must be performed under aseptic conditions, and the operation must be carried out by a person skilled in aseptic operation techniques. In particular, dispensing a large amount of culture medium without contaminating bacteria requires a high degree of aseptic technique. For mass culture, iJ'-3 requires a dedicated sterile filtration device, which increases equipment investment and maintenance costs.

Under these circumstances, there is a demand for the development of an inexpensive culture medium that does not require a high degree of sterile operation and can be prepared by anyone, not just those with specific technical skills, with simple operations. An object of the present invention is to provide a serum-free culture method for insect cells using a serum-free medium for culturing insect cells that meets such demands.

Means for Solving Problem C) In view of the above circumstances, the present inventors conducted research to solve the problem, and the present inventors conducted research to solve the problem, and the present inventors conducted research to solve the problem.
The present invention was based on the recognition that it is possible to culture insect cells in a medium obtained by removing serum from a medium (from which the serum has been removed) by sterilizing it in an autoclave. In other words, instead of the conventionally used insect cell culture medium, a medium containing yeast powder and albumin hydrolyzate (hydrolyzate) and containing no serum components can be used for culturing insect cells by heat treatment. It was discovered that insect cells can be sufficiently grown without losing necessary components.

According to the invention, therefore, a substantially serum-free medium containing sodium chloride, potassium chloride, sodium dihydrogen phosphate, sodium hydrogen chloride, magnesium chloride, calcium chloride, sugars, yeast powder and albumin hydrolyzate, Provided is a method for culturing insect cells in a medium made sterile by moist heat sterilization.

The medium used in the method of the present invention is Mitsuhashi and Maramoro
sch) A composition made by adding yeast powder and albumin hydrolyzate to the remaining components after removing serum from the medium, that is, sodium chloride, potassium chloride, sodium dihydrogen phosphate, sodium hydrogen carbonate, magnesium chloride, calcium chloride, and sugars. Using the medium, i
)H value to 5,6-7 using potassium hydroxide, hydrochloric acid, etc.
．． Once adjusted to a range of 0, a sterile serum-free medium can be prepared by sterilizing with moist heat.

Among the above components, as magnesium chloride, magnesium chloride anhydride and magnesium chloride hexahydrate can be used, but magnesium chloride hexahydrate is preferable. Further, as calcium chloride, calcium chloride anhydrate, calcium chloride dihydrate, and calcium chloride hydrate can be used, but calcium chloride dihydrate is preferable.

Further, as the sugar, D-glucose and fructose can be used, but D-glucose is preferable. Further, as the 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 egg albumin hydrolyzate can be used, but lactalbumin hydrolyzate is preferable.

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

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

Each component of the serum-free medium for insect cell culture used in the method of the present invention can be used as commercially available products, so it can be obtained stably at low cost. Since the purpose can be achieved by steam sterilization using a simple autoclave without any sterilization, anyone who is not an expert in aseptic techniques can prepare the product.

To prepare the serum-free medium used in the method of the invention,
The following inorganic salt components are known as common components of serum-free media for insect cells, namely, sodium chloride (
6.0-8.0'ii/Q, suitable addition maximum per 1Q of medium: the same applies below), potassium chloride (0.1-0.3g/Q
>, disodium phosphate (0.1-0.4 p/litre), sodium hydrogen carbonate (0.1-0.47/2), magnesium chloride hexahydrate (0.05-0.2 g/Q) and calcium chloride dihydrate (0,1~0.3y#2), and D-glucose (2,0~8.09/Q), and albumin hydrolyzate (3,0~15.09/Q) to these. Prepare a medium with a composition by adding Q> and yeast powder (1.0 to 8.09/Q>. Then, adjust the pH to a pti range of 5.6 to 7.0 using potassium hydroxide, hydrochloric acid, etc. Just adjust it.

Commercially available products may be used as they are for these culture medium components.

In addition, other ingredients, namely magnesium chloride anhydride, calcium chloride anhydride, calcium chloride dihydrate,
etc. can be used similarly.

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

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

Example 1 A serum-free medium containing each component with the composition shown in Table 1 below was prepared,
5 d of sterile medium heated in an autoclave (120' Cl2O min) and sterilized was dispensed into Corning plastic flasks (25 cm).

On the other hand, four types of Spodoptera fat pad-derived cells obtained by culturing for 4 days in the medium shown in Table 1, which had been sterilized in an autoclave in advance,
5ES-HaBr-1; 5ES-HaBr-2; 5
ES-) faBr-3; 5ES-HaBr-4 and two types of blood cell-derived cells NIAS-) 1aBr-92; NIA
S-HaBr-93 and NIAS-, a type of ovary-derived cell
and MaBr-32 at an initial cell concentration of 2.0x.
105 pieces/m! ! It was inoculated so that

When these flasks were placed and cultured in a cell incubator kept at 25°C, the number of cells after 9 days was 21.8 x 10" cells/rrd!: 18.1.
X10 pieces/lrd! ; 21.7x 105 pieces/
7: 16.4X10 pieces/ml+11.9X
'l o5 pieces/d: 1B, 5x10 pieces: 18
．． It increased to 3X 105 cells/ml.

Table 1 Sodium chloride 7°Oc+/Q Sodium dihydrogen phosphate hydrate 0.2 9IQ Sodium hydrogen carbonate 0.12q/Q Potassium chloride 0.2 gIQ chloride Magnesium chloride hydrate 0.1 a/Q chloride Calcium chloride hydrate 0.2 g/QD-glucose 4. Og, 1 Lactalbumin Hydrolyzate 6.5g/Q Yeastrate
5. Oa! Q (manufactured by Difco) pH 6.5 ± 0.2 By the way, the lactalbumin hydrolyzate in the above table is manufactured by Nutritional Biochemicals Corporation (
Nutritional Biochemicals
Corp.).

After preparing a serum-free medium with the composition shown in Table 1 and sterilizing it using the sterilization method used in Example 1, add beef tallow serum (Difco
(manufactured by Difco) was added aseptically to give a serum concentration of 3% to prepare a serum-containing medium.

5 ml of this serum-containing medium was dispensed into a Corning plastic flask (25cIA). On the other hand, armyworm hemocyte-derived cells NIAS-HaBr-93, which had been previously cultured in the serum-containing medium for 4 days, were incubated at an initial cell concentration of 2. It was inoculated at 105 OX/d.

When this flask was placed in a cell culture vessel kept at 25°C and cultured, the number of cells after 9 days was 18.2.
It increased to X 10” pieces/d.

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 10 (M) portions into glass spinner-stirred culture vessels with a capacity of 1250 ml.

Spodoptera hemocyte-derived cells N IAS-)1 obtained by culturing for 4 days in the medium shown in Table 1, which had been sterilized in an autoclave in advance.
aBr-93 at an initial cell concentration of 2. OX 105 pieces/
It was inoculated so that it became d.

In a cell culture #L vessel kept at 25°C, stir the
The mixture was stirred at 60 times per minute and cultured for 10 days. The number of cells after culture increased to 9.07 x 105 cells/d, and 1
9.07×10 7 cells were obtained by serum-free culture from a book spinner incubator.

Chimney dust colony-1 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 ml portions into a glass spinner-stirred culture vessel with an internal capacity of 250 liters. Spodoptera fat pad-derived cells 5ES-) 1aB obtained by culturing for 4 days in the medium shown in Table 1 which had been sterilized in an autoclave in advance
r-1 with an initial cell concentration of 2. The seedlings were inoculated at 10" Ox/d.

The cells were stirred at a rate of 60 times per minute in a cell culture vessel kept at 25° C. and cultured for 10 days. The number of cells after culturing increased to 10.8 x 10'' cells/d, and 1.08 x 108 cells were obtained from one spinner incubator by serum-free culture.

Daishikoshi-2 A serum-free medium with the composition shown in Table 1 was prepared, sterilized by the sterilization method used in Example 1, and poured into a glass spinner-stirred culture vessel with an internal capacity of 1.3Q in 500d portions. . Spodoptera-derived cells NIAS-MaBr- obtained by culturing for 4 days in the medium shown in Table 1, which had been sterilized in an autoclave in advance.
93, with an initial cell concentration of 1.0 x 10" cells/mlG:
I was vaccinated accordingly. The cells were stirred at 40 times per minute and cultured for 12 days in a cell culture vessel kept at 25°C. The number of cells after culture is 8.87 x 1Q5
4.4 cells/d from one spinner incubator.
4×'lQ8 cells were obtained by serum-free culture.

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 in 3Q portions into glass mini-jar culture vessels with a content of ff15Q.

Armworm-derived cells NIAS-HaBr- obtained by culturing for 4 days in the medium shown in Table 1, which was sterilized by caulking and autoclaved.
93 was inoculated so that the initial cell concentration was 1.0 x 10'' cells/d.The cells were kept at 25°C, stirred at a rate of 150 times per minute, and cultured for 16 days.Number of cells after culture increased to 3.83 X 10 cells/d, and 1.15 X 09 cells were obtained from one mini-jar incubator by serum-free culture.

〔Effect of the invention〕

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

First, as a method for aseptically treating a culture medium, it has become possible to use moist heat sterilization, such as autoclave sterilization, instead of the conventional filtration sterilization. Therefore, the highly aseptic operation of the filtration sterilization method is not required, and the Ohmukai culture also does not require a dedicated filtration device.

As a result, according to the present invention, insect cells can be cultured in a sterile, serum-free medium that is prepared easily and inexpensively by anyone, not only those with specific technical skills.

Second, compared to conventional media that use expensive serum, the components of the medium used in the method of the present invention are all commercially available, easily available, and inexpensive raw materials that can be used, making the medium preparation much easier. It is.

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

Claims (1)

[Claims] A substantially serum-free medium containing sodium chloride, potassium chloride, sodium dihydrogen phosphate, sodium bicarbonate, magnesium chloride, calcium chloride, sugars, yeast powder, and albumin hydrolyzate was sterilized by moist heat sterilization. A method of culturing insect cells in a culture medium.