JP3217451B2 - Shaking culture method of microorganisms, plant cells or animal cells - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method of shaking culture using a flexible container having a filter which does not allow various bacteria to pass therethrough and has an excellent gas supply ability. More specifically, the present invention relates to a method for shaking and culturing aerobic microorganisms, plant cells, or animal cells , which has excellent gas exchange ability between the inside and outside of a container and between a culture solution and gas in a container and has improved culturing efficiency.

[0002]

2. Description of the Related Art Conventionally, when performing liquid culture, a method of increasing the gas-liquid contact area by shaking has been adopted in order to improve the uptake of gas into the culture solution. A glass container such as an Erlenmeyer flask was used. However, when these glass containers are shaken, there is a limit to increasing the gas-liquid contact area.In some cases, obstacles are provided in the container to generate turbulence, and the gas-liquid Although such methods as increasing the contact area have been adopted, there is a drawback that stress or damage is given to the cultured cells.

[0003] In addition, instead of using a glass container, Japanese Utility Model Laid-Open No. 3-12
In the case of using an irregular container made of a synthetic resin such as No. 6,850, gas exchange cannot be sufficiently performed depending on the type of filter material, and cultured cells may be necrotic, or the rate of generation of cultured metabolic gas. In some cases, the gas exchange capacity of the filter material could not keep up, and the container sometimes expanded or burst.

[0004]

OBJECTS OF THE INVENTION It is therefore an object of the present invention, aerobic microorganisms to improve the culture efficiency without burdening the superior and cultured cells to a gas supply capacity, plant fine
It is an object of the present invention to provide a method for shaking culture of vesicles or animal cells.

[0005]

SUMMARY OF THE INVENTION An object of the present invention is to provide two opposing side members having a heat-fusible synthetic resin layer on at least the inner surface thereof, and a mountain fold sandwiched between the side members near a lower portion thereof. more becomes Jo bottom member, and mountain fold shape bottom of the bag-like objects comprising in succession at two side members between Oyo BiAmane edge and is housed in the pouch material and within the bag-like object A sheet-like material having a rigidity for releasing a mountain fold and flattening the material; and a pore having a maximum diameter of 0.1 to 50 μm , which is provided near an upper portion of at least one of the side members and does not pass various bacteria.
At a rate of 30 to 90%, air permeability of 1 to 30 seconds / 100cc or less
After storing the culture solution by flattening the bottom material of a flexible container having an opening closed with a filter, closing the opening of the flexible container, and culturing while shaking. This is achieved by a method of shaking culture of aerobic microorganisms, plants or animals.

[0006]

Next, the present invention will be described in detail with reference to the drawings. That is, as shown in FIGS. 1 to 3, two side members 1 and 2 having a heat-fusible synthetic resin layer at least on the inner surface and facing each other, and a lower portion between the side members 1 and 2. A mountain-folded bottom member 3 sandwiched in the vicinity, and the two side members 1 and 2 and the side member 1 or 2 and the bottom member 3 have peripheral portions 4, 5, 6 Is a continuous bag-like material 7. A sheet-like material 8 having rigidity for releasing the mountain-fold of the bag-like material and flattening the bag-like material is inserted into the bag-like material 7. An opening 10 closed by a filter member 9 is provided near the upper part of at least one of the side members 1 and 2. The bag-like material 7 is formed by converting one rectangular sheet into a mountain 11 of the bottom material 3.
This is obtained by bending at the peripheral edge 6 of the bottom member 3 and thermally bonding the side members 1 and 2 to the bottom member 3 at the peripheral edge 5 and the side members at the peripheral edge 4. Also, the side members 1 and 2 made of two sheet-like objects
It can also be obtained by heat-sealing the peripheral folds 4, 5 and 6 with the mountain-folded bottom material 3 which is a separate body.

The sheet-like material forming the side members 1 and 2 and the bottom member 3 only needs to have at least the inner surface (the surface to be fused to each other) made of a heat-fusible synthetic resin and have water resistance. For example, a single sheet of polyethylene, polypropylene, a polyethylene-polypropylene mixture, an ethylene-vinyl acetate copolymer, or the like, or a sheet obtained by laminating or coating them with another material may be used. In order to allow light to pass therethrough and to observe the state of culture, it is desirable that all of them be transparent.

The sheet-like material 8 having rigidity only needs to be water-resistant, and has a width protruding from the mountain-folded bottom material 3, that is, the bottom-fold material 3 is flattened by releasing the mountain-fold when used. In this case, it is desirable to have a width substantially corresponding to the width. The sheet-like material 8 may be simply stored in the bag-like material 7, but is desirably inserted between any one side material and the bottom material of the bag-like material. It is desirable that the shape of the bottom surface of the bag-shaped material 8 when the mountain-folding is released and flattened is a square having a long side: short side of 1: 1 to 3: 2.

The mounting opening 10 of the filter member 9 is provided at a position high enough that the liquid does not reach even if the culture solution is shaken after storage. As the filter material 9, air and culture metabolic gas can flow freely, but it is necessary that the filter material 9 has a diameter that does not allow passage of various bacteria.
Specifically, the maximum pore diameter is 0.1 to 50 μm, preferably 0.4 to 20 μm, and the porosity is 30 to 90%, preferably 5
It has a performance of 0 to 90% and an air permeability of 1 to 30 seconds / 100 cc, preferably 1 to 10 seconds / 100 cc.
Further, the filter material 9 is a single layer or a multilayer, and it is preferable that at least one layer has hydrophobicity in order to prevent permeation of various bacteria. Here, the maximum pore diameter is ASTM E128-61, and the porosity is ASTM D2.
873, and the air permeability is a value measured according to JIS P-8117 (Gurley method).

The bag-like material 7 of the flexible container used in the present invention may have a shape in which the lower corner portions 13 of the side members 1 and 2 and the bottom member 3 are cut off as shown in FIG. Good.

The flexible container of the present invention thus formed is in a sheet shape having no space inside when not in use, as shown in FIG. 2, but as shown in FIG. 3, when in use, as shown in FIG. Since the sheet-like material 8 presses and flattens the mountain-folded portion of the bottom material 3 and, due to its rigidity, prevents the mountain-folded portion from returning to its original position, the bag-shaped material 7 has a sufficient space. And allows the side members 1, 2 to maintain their independence.

After the culture solution is stored in the bag-like material 7 whose bottom material 3 is flattened in this way, the opening 14 of the bag-like material 7 is opened.
Is sealed by heat fusion or by using a fitting-type sealing jig 12 as shown in FIG. In this way, microorganisms such as aerobic bacteria, plant fine
Vesicles and animal cells can be cultured.

According to the present invention, any microorganisms, plants and animals can be cultured as long as they are aerobic. For example, examples of the microorganisms include fermentation microorganisms such as Pseudomonas ovalis and Saccharomyces cerevisiae. Saccharomyces cerevisiae, Saccharomyces carlsbergensis (Saccharomyces car
lsbergensis), yeast, and oyster mushroom (Pleurotus ost)
reatus), Shiitake mushroom (Lentinus edodes), Enokitake mushroom (F
aerobic microorganisms such as basidiomycetes such as lammulina velutipes), poplar (Populus nigra), larch (Larix leptolepis),
Phalaenopsis (Phalaenopsis amabilis), Catharanthus roseus
(Catharantus roseus), Purple (Lithospermum erythr
orhizon) there is a plant cell such as. In animal cells , Namalba cells (lymphoblasts), human kidney cells and the like can be cultured for the purpose of producing useful substances such as interferon.

As the medium, any liquid medium usually used in this field can be used. For example, as a medium for microbial cells, for example, a potato dextrose medium, a tryptoso broth medium, There are ordinary bouillon medium, YCC liquid medium, heart infusion medium, etc., and as a medium for plants, MS medium (Murashige medium) is used.
& Skoog), KC medium (Knudson, C.), VW medium (Vacio &
Went), B5 medium (Gamborg etc. B5), White medium, etc., and as an animal cell medium, Eagle medium,
There are an RPMI medium and a medium modified by adding serum or the like to these mediums.

[0015]

Next, the present invention will be described in more detail with reference to examples.

Example 1 Maximum pore diameter 40 μm, porosity 90%, air permeability 1 sec / 1
100 ml containing 0.5 mg / l of 2.4-D and 0.1 mg / l of kinetin in a 500 ml capacity cell culture vessel according to the invention having a structure shown in FIGS. After supplying an MS medium consisting of an aqueous solution of Catharanthus roseus (C
atharantus roseus), inoculate the opening, and add 8
The culture was performed at a temperature of 0 rpm / 27 ° C. FIG. 5 shows the relationship between the passage of time and the amount of cell proliferation at this time.

Comparative Example 1 A similar method was carried out in Example 1, except that a 500 ml Erlenmeyer flask was used instead of the cell culture container according to the present invention, and the container was sealed with a cotton plug. FIG. 5 shows the relationship between the passage of time and the amount of cell proliferation at this time.

Example 2 Maximum pore diameter 40 μm, porosity 90%, air permeability 1 sec / 1
00cc the cell culture vessel according to the invention of 500ml capacity having the structure shown in Figures 1-3 having a polypropylene filter having a glucose 50 g, KH ₂ PO
₄ 1 g / l, after supplying the medium 100ml consisting aqueous solution containing _{MgSO 4 · 7H 2 O 1g /} l and ammonium acetate 1 g / l, were inoculated with Shudomanasu-Ovarisu (Pseudomonas ovalis), 210 rotate, 30
The culture was carried out at a temperature of ° C., and the relationship between the amount of cell growth and the amount of gluconic acid and the culture time was examined.

Comparative Example 2 The same method as in Example 2 was carried out except that a 500 ml Erlenmeyer flask was used in place of the cell incubator according to the present invention, and the cap was closed with a cotton plug. The results shown in FIG. 6 were obtained. Was done.

[0020]

According to the present invention, two opposing side members having a heat-fusible synthetic resin layer on at least the inner surface thereof, and a mountain-folded bottom member sandwiched between the side members near a lower portion thereof, and wherein the two side members between Oyo BiAmane edge bag-like objects comprising continuously in, to release the crease of mountain fold shape bottom member of the housing in the pouch material and within the bag-like object a sheet-like material having rigidity for flattening, without passing through the and bacteria is provided near the top of at least one of the side members, the maximum
Pore diameter 0.1-50 μm, porosity 30-90%, air permeability 1
A method for shaking culture of aerobic microorganisms, plant cells or animal cells , characterized by using a flexible container having an opening closed with a filter of up to 30 seconds / 100 cc, so that the container was folded. Not only space saving because it can be stored in a state, but also light weight is advantageous for transportation and handling, there is no fear of breakage.In addition, in the case of conventional Erlenmeyer flask culture, it is inclined upward because it has a slope The gas-liquid contact area was reduced, and even when the shaking culture was performed, the gas-liquid contact area did not increase so much, and the gas supply was insufficient.However, the flexible container had a rectangular shape having a rigid bottom surface. Since the space inside the container is held by the sheet-like material, the gas-liquid contact area hardly changes even when it is turned upward, and the culture solution does not scatter even when shaking culture is performed. When compared to the flask, in the case of the same shaking speed becomes about two times of the gas-liquid contact area of the Erlenmeyer flask, it is possible to perform sufficiently well gas supply.

Further, by the filter having the above performance,
Gas exchange between the inside and the outside of the container can be performed efficiently without filling the bag with the culture metabolic gas.

[Brief description of the drawings]

FIG. 1 is an exploded perspective view of a cell culture container according to the present invention.

FIG. 2 is a cross-sectional view of a cell culture container according to the present invention.

FIG. 3 is a perspective view showing a use state of the cell culture solution of FIG. 2;

FIG. 4 is a front view showing another embodiment of the present invention.

FIG. 5 is a graph showing the growth state of cells.

FIG. 6 is a graph showing the proliferation state of other cells.

[Explanation of symbols]

1, 2, ... side material, 3: bottom material, 4, 5, 6 ... peripheral part, 7 ... bag-like material, 8 ... sheet-like material, 9 ... filter member, 10 ... filter opening, 11 ... mountain part, 12 ... closing jig, 14 ... opening of bag-like material.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI C12M 3/00 C12N 5/00 F (72) Inventor Mitsunori Mori 56 Imaiuecho, Nakahara-ku, Kawasaki-shi, Kanagawa Prefecture Fujimori Kogyo Co., Ltd. (56) References JP-A-4-20281 (JP, A) JP-A-3-126850 (JP, U) (58) Fields investigated (Int. Cl. ⁷ , DB name) C12N 1/00-5 / 28 C12M 1/00-3/10

Claims (2)

(57) [Claims] 1. A pair of opposed side members having a heat-fusible synthetic resin layer on at least an inner surface thereof, and a mountain-folded bottom member sandwiched between the side members near a lower portion thereof. and wherein the two side members between Oyo BiAmane edge bag-like objects comprising continuously in, to release the crease of mountain fold shape bottom member of the housing in the pouch material and within the bag-like object a sheet-like material having rigidity for flattening, without passing through the and bacteria is provided near the top of at least one of the side members, the maximum
Pore diameter 0.1-50 μm, porosity 30-90%, air permeability 1
After flattening the bottom material of a flexible container having an opening closed with a filter of ３０30 sec / 100 cc and storing a culture solution, the opening of the flexible container is sealed and shaken. Aerobic microorganisms and plants characterized by being cultured
A method for shaking culture of cells or animal cells . 2. The shape of the bottom surface when the mountain-folded bottom material of the bag-like material is flattened by releasing the mountain-folding is long side: short side = 1: 1.
The shaking culture method according to claim 1, wherein a flexible container having a rectangular shape of up to 3: 2 is used.