JPS6244173A - Method of culture and device therefor - Google Patents

Abstract

PURPOSE:To cultivate a cell or a plant tissue in high efficiency without applying shear force to a mixed system of a liquid medium and the cell or the plant tissue, by rotating steadily a culture container packed with the mixed system of the liquid medium and the cell or the plant cell round an axis inclined in a specific angle range. CONSTITUTION:The cylindrical closed culture container 1 is charged with the liquid medium 3 mixed with the cell or plant cell 2 in such a way that inner space of the container 1 is filed with it. Then, in this state, the axis X of the container is inclined at the angle alpha of 5-55 deg., especially 20-45 deg.C from the horizontal plane and rotated at a fixed speed. By making the container 1 turn on its axis in this way, after an initial period is passed, the medium 3 in the container 1 and the mixed system are rotated in one piece with the container by the viscosity of the medium 3 in a state free from mechanical flow. Consequently, the cell or plant cell 2 will hardly change a relative position to the medium 3 nor an existing position to the outside of the container 1. Gravity is successively applied to the cell or plant tissue 2 in the container 1 successively in different directions, the cell or plant tissue is dispersed into the medium 3 in a good dispersion degree and the culture is efficiently carried out.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a method and apparatus for culturing cells or plant tissue, and more specifically to cell or plant Mi fabrics suitable for producing physiologically active substances. The present invention relates to a culturing method and apparatus.

[Conventional technology]

In general, physiologically active substances are substances that impart a certain type of activity to cells or plants and tissue, causing specific physiological reactions in the cells or plant tissues, and although some can be artificially synthesized, they are usually must be produced by living cells or plant tissues to maintain their survival.

As a conventional method for producing a physiologically active substance, a method is known in which cells or plant Mi tissues that produce the desired physiologically active substance are cultured in order to survive and maintain them.

In general, when culturing cells or plant tissues, if the culture target is a suspension-propagating cell or plant tissue, that is, when the cells or plant tissue itself can grow in a suspended state in a liquid medium, the nutrient source is It is necessary to contact the culture medium by suspending it in a liquid medium, and when the culture target is an adhesion-dependent cell, that is, a cell that requires adhesion to a substrate in order to grow and proliferate in the liquid medium. For this purpose, it is necessary to attach the cells to the surface of a suitable substrate and then contact the cells with a liquid medium. As a substrate for adhesion-dependent cells, small-diameter carrier particles have recently been used because they can easily obtain a large adhesion area.

[Problem that the invention seeks to solve]

However, in order to culture the target cells with high efficiency, it is essential that the cells to be cultured are constantly in contact with fresh medium. It is necessary to disperse the carrier particles (hereinafter collectively referred to simply as "cells") in a liquid medium with a high degree of dispersion.

Furthermore, when adhesion-dependent cells are attached to carrier particles as a substrate, if the carrier particles can be dispersed with a high degree of dispersion in a liquid medium in which the cells are mixed, they can be used for adhesion of the cells. The effective area of the surface of the resulting carrier particles is increased, and therefore cells can be attached to the carrier particles with a high adhesion rate, which is extremely advantageous in increasing culture efficiency.

Conventionally, a common method for keeping cells to be cultured in constant contact with a fresh liquid medium is to use a rotary blade to stir a mixture of cells to be cultured in a liquid medium. Since this method applies shear force to a mixed system of culture medium and cells to disperse the cells, it is possible to avoid damage to the cells being cultured due to collisions with rotary blades or the action of large shear forces. However, there are problems in that the culture efficiency is low, and this method may not be applicable depending on the MN of the cells.

In addition, when constructing a large-capacity cell culture device for the purpose of mass culture in order to carry out the method described above, a large-sized stirrer is required for stirring, which inevitably increases the shearing force. Another problem is that the culture efficiency further decreases.

In order to keep the cells to be cultured constantly in contact with a new liquid medium, it is necessary to replace the liquid medium in the culture container with a new one as appropriate during the culture period.Therefore, it is advantageous to replace the liquid medium. Preferably, the cells are dispersed in a liquid medium.

The above situation is the same in culturing plant tissues.

Although various measures have been researched to solve these three problems, the current situation is that a method and device for culturing cells or plant tissue that essentially solves these problems has not yet been developed. It is.

The present invention was completed as a result of intensive research based on the above circumstances, and its purpose is to:
Dispersing cells or plant tissues in a liquid medium with a good degree of dispersion and in a state that is advantageous for exchanging the liquid medium without essentially imparting shearing forces to the mixed system of the liquid medium and the cells or plant tissues to be cultured. The object of the present invention is to provide a method and an apparatus that can culture cells or plant Mi tissue with high efficiency.

Means for Solving Problem C] The feature of the method of the present invention is that cells or plants 1
The mixed system is cultured by rotating the culture container filled with the mixed system with JIv6 so as to substantially fill it around an axis tilted at an angle of 5 to 55 degrees with respect to the horizontal plane. The particles to be dispersed, the suspended proliferative cells, or the plant tissue are dispersed in the liquid medium in the mixing system, preferably with a difference in the dispersion concentration between the upper and lower parts, by rotating the container constantly and 7 times. The point is that it has a process of forcing.

Furthermore, the feature of the device of the present invention is that 5
comprising a culture container having a rotating shaft supported so as to be tilted at an angle in the range of ~55°, and a drive mechanism that rotates the culture container so as to rotate around the rotating shaft,
The driving mechanism has a function of constantly rotating a mixed system of a liquid medium and cells or plant tissue, which is filled so as to substantially fill the culture container, together with the culture container.

According to such technical means, cells or plant tissues can be dispersed with a high degree of dispersion in a culture container without applying substantial shearing force, for example, by using cells or plant tissues that have a higher specific gravity than a liquid medium. Since the liquid medium in the culture container can be dispersed with a difference in dispersion concentration between the upper and lower parts, this difference in dispersion can be used to advantageously replace the old liquid medium with a new liquid medium. becomes possible. That is, the culture container is usually provided with a filter member that allows only the liquid medium to pass through, but not cells or plant tissues, and the liquid medium is exchanged through this filter member.
According to the above technical means, the medium can be replaced from the part of the liquid medium where the dispersion of cells or plant tissue is low, so the liquid medium can be exchanged efficiently without clogging the filter member. This makes it possible to exchange cells or plant tissues, and as a result, culturing cells or plant tissues with high efficiency can be achieved.

The present invention will be specifically explained below.

In the present invention, as shown in FIG. 1, for example, cells or plant tissues 2 are placed in a cylindrical sealed culture container 1
Fill the internal space of the culture container 1 with a liquid medium 3 mixed with the above so that it is substantially filled, and set the axis The culture container l is rotated around this axis X at a constant rotational speed while being tilted at an angle (α) more preferably in the range of 20 to 45°. At this time, if the angle (α) is smaller than 5°, there will be no difference in the dispersed concentration of cells or plant tissues in the culture container even if cells or plant tissues with higher specific gravity than the liquid medium are used. No matter where you replace the liquid medium, the filter member will become clogged, making it difficult to exchange the liquid medium efficiently.If the angle (α) is larger than 55″, the liquid medium and specific gravity Different cells or plant tissues are hardly dispersed in the liquid medium, making it difficult to perform efficient culture.

By rotating the culture container 1 in this way, after an initial period immediately after the start of rotation, the mixed system of the liquid medium 3 and the cells or plant fabric 2 in the culture container 1 is changed due to the viscosity of the liquid medium 3. The culture container 1 rotates steadily around the axis X without any mechanical flow, so that the cells or plant tissues 2 are transferred to the culture container 1.
The position relative to the outside of the culture container 1 is changed without changing the relative position with respect to the liquid medium 3 inside, and as a result, gravity acts on the cells or plant tissue 2 in the culture container 1 sequentially from different directions. This allows the cells or plant tissue 2 to be dispersed in the liquid medium 3 with a good degree of dispersion.

In culturing cells or plant tissues, replacement of the liquid medium is usually required. In the present invention, the liquid medium also needs to be replaced during long-term culture. Methods for replacing the liquid medium include, for example, stopping the rotation of the culture container and replacing part or all of the liquid medium, or installing a supply pipe and a discharge pipe for the liquid medium in the culture container and continuously replacing it during the culture period. Examples include a method of supplying a new liquid medium and discharging the old liquid medium. By supplying a new liquid medium in this manner, culture, which normally requires a period of several days or more, can be carried out successfully. If the new liquid medium is constantly supplied through the supply pipe during the culture period as described above, the amount of supply and discharge per unit time will be small, so the liquid medium in the culture container will have a laminar flow and turbulence. It is possible to create a state in which substantially no mechanical flow such as a stream is applied, and therefore the liquid medium can be exchanged without disturbing the desired dispersion state of cells or plant Mi tissue. In order to increase culture efficiency, it is preferable to constantly supply the liquid medium through a supply pipe.

Then, by rotating the culture container 1 while tilting the axis X of the culture container 1 at an angle within the above range with respect to the horizontal plane, the culture container 1
A difference occurs in the dispersion concentration of cells or plant tissues 2 in the upper and lower parts of the liquid medium 3 in the liquid medium 3. For example, if the specific gravity of the cells or plant tissues 2 is higher than the specific gravity of the liquid medium 3, The dispersion concentration in the upper part can be low and the dispersion concentration in the lower part can be high. Therefore, when replacing the liquid medium 3 in the culture container 1 with a new liquid medium as necessary, the old liquid medium should be drained from the area where the dispersion concentration is low and the new liquid medium should be supplied to the area where the dispersion concentration is high. By doing so,
Cells or plant tissue can be efficiently brought into contact with a new liquid medium, and when discharging an old liquid medium, clogging of the filter member by cells or plant tissue 2 can be suppressed. So, cultivate again stably! It becomes possible to continuously exchange the liquid medium during IJI, and as a result, it becomes possible to culture cells or plant tissues with high efficiency over a long period of time.

In the present invention, no special method is required to fill a culture container with a liquid medium and cells or plant tissues; for example, a mixed system of a liquid medium and cells or plant tissues that have been mixed in advance. All you have to do is fill it up. However, when culturing adhesion-dependent cells, rather than filling a culture container with carrier particles to which adhesion-dependent cells have been attached in advance, it is preferable to fill a culture container with a liquid medium, carrier particles, and adhesion-dependent cells as described above. Fill the mixed system with cells and rotate the culture container in the same manner as in the method of the present invention. As a result, the carrier particles and adhesion-dependent cells are dispersed in the liquid medium without applying shearing force, and the cells are transferred to each carrier. It is desirable to have an even and efficient adhesion to the surface of the particles. In this case, the present invention can be carried out subsequent to the adhesion step.

Furthermore, when culturing adhesion-dependent cells according to the present invention, if a method is applied in which a new liquid medium is constantly supplied into the culture container through a supply pipe and an old liquid medium is constantly discharged through a discharge pipe, the new liquid medium is A method is used in which new carrier particles and adhesion-dependent cells are supplied into a culture vessel, and the adhesion-dependent cells that have grown together with the old liquid medium that is discharged from the culture vessel extract part of the carrier particles that have adhered to the surface. By replacing the liquid medium,
Adhesion of adhesion-dependent cells to carrier particles, culturing, and extraction of the proliferated adhesion-dependent cells can be performed continuously.

In the application of the present invention, the cells to be cultured are not limited in any way, and for example, the cells shown in Table 1 can be exemplified.

Table 1 (Part 1) Table 1 (Part 2) Table 1 (Part 3) In the table, those marked with ☆ are adhesion-dependent cells, and the others are floating proliferative cells.

Suspension-proliferating cells other than those exemplified in Table 1 that can be cultured according to the present invention include, for example, lymphoblasts, Parkit-kumboma cells, acute lymphoblastic leukemia cells, and myeloma cells such as myeloma cells. Cells or hybridoma cells based thereon, and adhesion-dependent cells include human uterine cancer cells HeLa, Chinese hamster lung cells V-79°, human fetal lung cells MRC-5. Examples include chimpanzee liver fibroblasts, human foreskin cells, chicken fetal fibroblasts, primary monkey kidney cells, mouse metastatic fibroblasts, pituitary gland tumor cells, African green monkey kidney cells Vero, and adrenal gland tumor cells.

In the application of the present invention, the plant tissues to be cultured are not particularly limited, and include, for example, Seriba auren protoblast, Hanakirin protoblast, tobacco mesophyll cell protoplast, carrot protoplast, liverwort callus, soybean callus, and purple callus. , strawberry shoot tips, tomato shoot tips, wild varieties of potato (Sola
num gonioca-1yx), the shoot apex of Japanese pea, the shoot apex of carnation, the shoot apex of asparagus, protoplasts and callus in plants such as chlorella, and &1lVa forming part of the plant body.

The liquid medium for cell culture in the application of the present invention is not particularly limited, and any known medium can be used as is, such as RPM11640 medium, Eagle ME medium, etc.
Examples include M medium, Dulbecco's modified Eagle's medium, Earle medium 199, Ham's F12 medium, etc. These mediums contain, for example, fetal bovine serum, neonatal bovine serum, horse serum, It is preferable to add human serum. Serum-free medium without serum,
For example, HB102 (Hana Biologics), RI
TC55-9 medium etc. can also be used. The specific gravity of these liquid media is generally 1.00 to 1.05.

Note that it is usually necessary to dissolve oxygen and carbon dioxide gas in these liquid media, and cell culture usually requires
It is carried out at 30-40°C, preferably 36-37°C.

Further, the liquid medium for plant tissue culture in the application of the present invention is not particularly limited, and examples include Linsmaier-Skoog's liquid medium, Murashige-Skoog's liquid medium, and Murashige-3k.
oog) liquid medium, MG-5■ medium, etc.

The specific gravity of these liquid media is generally 1.00 to 1.05. Note that it is necessary to dissolve oxygen in these liquid media, and the temperature of the liquid medium during culturing of plant tissue is usually 10 to 40°C, preferably 20°C.
~30°C.

Further, there are no particular restrictions on the carrier particles used when culturing adhesion-dependent cells, as long as they are suitable for the adhesion and proliferation of adhesion-dependent cells, such as synthetic polymers such as polystyrene, or carrier particles. White matter and multi-IJ! 1
Examples include particles whose surfaces are made of natural polymers such as '4, but it is advantageous for the carrier particles to have magnetism, which allows the use of magnets to collect and move the carrier particles. It becomes possible to perform processing and other handling simply and quickly. Examples of magnetic carrier particles include those made by binding magnetic powder with a polymeric material;
There are products in which a core containing a magnetic substance is coated with a polymer material. Specific examples of the magnetic substance include iron, cobalt, nickel, alloys thereof, low carbon steel, silicon steel, r-iron oxide, ferrite, Magnetite and others can be mentioned. The specific gravity of the carrier particles varies depending on the viscosity and specific gravity of the liquid medium, but is generally higher than the specific gravity of the liquid medium, and is usually in the range of up to 1.5. Further, the particle size of the carrier particles is preferably about 40 to 500 μl, and the shape is preferably spherical, but it may also be granular, cylindrical, or irregularly shaped.

The seeding rate of cells or plant tissues per 1 m7 of liquid medium is generally between LXIO' and lXl0b, and the number of carrier particles used for culturing adhesion-dependent cells is usually lXl0 per m7 of liquid medium. The number is about 1×10.

In the present invention, it is desirable that the culture container is filled with a mixed system of a liquid medium and cells or plant tissues so that there are no spaces, so that the mixed system can be easily filled into the culture container. If there is a small space, the disturbance in the liquid medium due to the presence of the space will be limited to a small area of the upper part of the mixed system in the culture container. Therefore, the effects of the present invention are not effectively nullified. However, if the proportion of space in the culture container is large (if the proportion of space is large, it becomes difficult to rotate the mixing system together with the culture container without substantially disturbing the culture container, and the purpose of the present invention cannot be achieved.For this reason, in the present invention, A substantially full state is defined as a case where the filling ratio of the mixed system in the culture container is 70% by volume or more, preferably 90% by volume or more, particularly preferably 98% by volume or more.

In the present invention, the rotation speed of the culture container is
It is selected by taking into account the size and specific gravity of cells or plant tissues, the viscosity of the liquid medium, the shape and size of the culture container, etc. - Although it cannot be generally specified, it is usually 5 to 5
0 r, p, +a, preferably 10 to 30 r, ρ,
+1. Rotate the culture container at a rotation speed of

Further, the shape of the culture container is preferably cylindrical as in the above-mentioned example, but there is no problem if the culture container is prismatic or spherical, and the rotation method is not limited.

FIG. 2 shows an example of a culture apparatus according to the present invention, and in the example shown in this figure, a culture container is constituted by a bottom plate 30 and a bottomed cylindrical container body 31. That is, the container main body 31 is pressed against the bottom plate 30 via the O-ring 33 by a holding mechanism 32 provided on the bottom plate 30 according to a national standard, and the internal space thereof is used as a culture area.

The bottom plate 30 is fixed to a rotating sleeve 35, and this rotating sleeve 35 is connected to the stand 3 via a dry bearing 36.
The rotatable sleeve 35 is rotatably held by a mantle member 38 held by 7, and the axis Y of this rotating sleeve 35 is inclined at an angle (α) (11) in the range of 5 to 55 degrees with respect to the horizontal plane (H). has an opening in its center, and this opening is blocked by a filter member, for example, a mesh 40, which allows the liquid medium to pass through but not cells or plant tissue. An inner conduit 41 extending inside is fixed to the mesh 40. This inner conduit 41 has an opening 4 at one end located inside the container body 31.
3, the other end of which extends outward through the rotary sleeve 35, and is rotatably connected to a supply pipe 51 from the outside at a connection seal portion 50 so as to communicate with the supply pipe 51 from the outside.

An external hot tube 42 is fixedly provided on the inner periphery of the rotating sleeve 35 so as to have a double tube structure surrounding the inner conduit 41.
One end opening 44 of 2 is connected to communicate with the opening of the bottom plate 30, and the other end is rotatably connected to an external discharge pipe 52 at a connecting seal portion 50 so as to communicate therewith. 60 is a driven gear fixed to the rotating sleeve 35, and this driven gear 60 meshes with a driving gear 61 driven by a motor (not shown).

In the culture apparatus configured as described above, the rotation sleeve 35 is rotated by driving the drive gear 61, thereby rotating the culture container composed of the bottom plate 30 and the container body 31, and at the same time, the inner conduit 41 and Exothermal tube 4
2 also rotate together.

However, in this device, the culture is performed while rotating the culture container with the culture container filled with a mixed system of a liquid medium and cells or plant tissue, so the cell or plant tissue is can be dispersed in the liquid medium with a high degree of dispersion, and since the axis Y of the rotating sleeve 35, which is also the axis of rotation of the culture container, is inclined at an angle within a specific range with respect to the horizontal plane, the rotating sleeve 35 can be rotated. When the culture container is rotated, there will be a difference in the dispersed concentration of cells or plant Mi fabrics in the upper and lower parts of the liquid medium in the culture container due to the action of gravity or buoyancy. When the specific gravity is larger than the specific gravity, the dispersion concentration in the upper part of the liquid medium is low and the dispersion concentration in the lower part is high.
2, one end opening 43 of the inner conduit 41 is located below the liquid medium filled in the container body 31, and one end opening 44 of the outer conduit 42 contacts the upper part of the liquid medium in the mesh 40. Therefore, when replacing the liquid medium in the culture container with a new liquid medium, the old liquid medium that has lost nutrients can be drained from the part with low dispersion concentration, that is, the upper part of the liquid medium, and the dispersion Fresh liquid medium can be supplied to the concentrated part, ie, the lower part of the liquid medium, so that the cells or plant tissues can be brought into contact with the fresh liquid medium efficiently. In addition, when discharging the old liquid medium that has lost nutrients, it is possible to suppress the occurrence of clogging of the mesh 40 by cells or plant tissue7, and as a result, the liquid medium can be can be exchanged efficiently and continuously, and as a result, cells or plant tissues can be cultured with high efficiency over a long period of time.

FIG. 3 is an example of a system chart for culturing using the apparatus of the present invention, in which 70 is a liquid medium tank;
1 is a culture container, 72 is a liquid medium discharge tank, and a monitor mechanism 73 measures the pH of the liquid medium immediately after being discharged from the culture container 71, using a pH sensor 74, for example.
In addition, the amount of dissolved oxygen is detected by the Do sensor 75, and the amount of new liquid medium supplied to the culture container 71 is regulated by the control valve 76 according to the result. 77 and 78
are a pH sensor and a Do sensor provided in the liquid medium tank 70, respectively, and according to the pH of the liquid medium measured by the pH sensor 77, alkali is added at a controlled rate from the alkali tank 8o to adjust the pH'. is carried out, and air is supplied in accordance with the detection result of the Do sensor 78 via a control valve 82 inserted in an air supply pipe 81 extending into the liquid medium tank 70 and supplying air containing, for example, 5% carbon dioxide gas. Volume is controlled.

According to such a system, the culture conditions can always be maintained in a good constant state during the culture period, and the cells or plant Mi tissue can be dispersed with a high degree of dispersion in the liquid medium. This makes it possible to perform the desired culture with high efficiency on an industrial scale.

〔Example〕

Examples of culturing actually carried out based on the present invention will be described below.

Example 1 Cultured cells: rV-79J derived from Chinese hamster lung fibroblasts Liquid medium: 10V/V% dissolving oxygen and carbon dioxide
Eagle containing tallow serum (viscosity: 0.01 poise, specific gravity: 1.01)
Carrier particles: rcytodex IIIJ (Phar
(manufactured by Macia) (particle size: 180n, specific gravity: 1.
03) 9 by dry weight in a cylindrical culture vessel with a capacity of 300 d.
Add 00IIlr (approximately 3 x 10'') of the above carrier particles and fill the liquid medium, seed 6 x 10' of the above cultured cells, and seal it to prevent air from entering. At an angle of 45'' with respect to the horizontal plane, the culture vessel was rotated at a rotation speed of 15 r, p, m for 4 hours in an environment at a temperature of 37°C to allow cells to adhere to the carrier particles. .

As a result, the proportion of carrier particles to which cells did not adhere was approximately 5
%, and the number of cells on each carrier particle to which cells were attached was 2 to 4, with an average of 2.5, and the number of cells on all carrier particles was 4.8XlO'.

Subsequently, the culture container was rotated for 72 hours under the same conditions as above to culture the cells. During this time 2
The rotation of the culture container was stopped every 4 hours, and the entire liquid medium was replaced. As a result, cells on all carrier particles are 7.2X10'
It was multiplying individually.

Example 2 Using a culture apparatus having a culture container with an internal volume of 1° C. and having the configuration shown in FIG. 2, cultured cells similar to those in Example 1,
Cells were cultured using a liquid medium and carrier particles. In other words, the dry weight of liquid medium 11 is 3 g (approximately 1
．． Using OX 10' carrier particles, liquid medium 1-
Seed 1X10' cultured cells per cell, fill the culture container with a mixed system of cultured cells, liquid medium, and carrier particles while preventing air from entering, and adjust the angle of the rotation axis of the culture container with respect to the horizontal plane. 45", rotation speed 12 r, p, m for 144 hours in an environment with a temperature of 37 ° C.
, while rotating the culture container, a new liquid medium was supplied into the culture container at a rate of 80 d/hr, and the same amount of old liquid medium was discharged. As a result, the average number of cells on the carrier particles was 2.5×10′ cells per 117 liquid medium. Furthermore, during the exchange of the liquid medium, the liquid medium could be exchanged stably to the end without clogging the filter member provided in the culture container.

Comparative Example 1 Cells were cultured using a spinner bottle with an internal volume of 21 and the same cultured cells, liquid medium, and carrier particles as in Example 1. That is, 3 g of dry weight carrier particles were added to the liquid medium 11 in a spinner bottle, and then 1X10' cultured cells were seeded per 117 liquid medium, and the number of revolutions of the rotor was set to 30 in an environment at a temperature of 37°C.
Culture was carried out for 144 hours while rotating at r, p, m. During this time, air containing 5% carbon dioxide gas was continuously supplied to the top of the spinner bottle so that the liquid medium was constantly in contact with the air. As a result, the number of cells on the carrier particles was on average 5. There were 1×10' pieces.

Example 3 A cylindrical culture container with an internal volume of 100 tZ was filled with a Linsmeyer Skoog liquid medium (specific gravity 1.0), and 0.5 g of dry weight of purple violet (Lithospe-r) was added to this.
mum erythrorhizon 5ieb, etc.
, Zucc) was put in and sealed, and then the culture vessel was placed at a rotational speed of 15 r, p, at a temperature of 25°C for 14 days with the rotation axis of the culture container set at an angle of 30° with respect to the horizontal plane. The plant tissues were cultured while rotating the culture container.

During this period, the rotation of the culture container was stopped and the entire liquid medium was replaced every 24 hours.

When the thus obtained plant tissue was dried and its weight was measured, it was 1.3 g.

Example 4 Using a culture apparatus having a culture container with an internal volume of 120 cm as shown in FIG. 2, plants M similar to those in Example 3 were grown.
Plant Mi tissue was cultured using i tissue and a liquid medium.

That is, the dry weight is 0.6 g for 120 − of the liquid medium.
Lithospermum erythr
orhi-zon 5ieb, et, Zucc) and sealed, the culture container was rotated for 14 days at a temperature of 25°C with the rotational axis of the culture container set at an angle of 30'' with respect to the horizontal plane. Rotate the culture container with steps 15 r, p, m, and pour one new liquid medium into the culture container.
The plant tissue was cultured while supplying the medium at a rate of 2017/24 hours and discharging the old liquid medium at the same rate.

When the plant Mi fabric thus obtained was dried and its weight was measured, it was 1.7 g.

Comparative Example 2 Using a spinner bottle with an internal volume of 2001/2, the same liquid medium xoomz as in Example 3 and callus 0.5
The plant fabric was cultured in the same manner as in Example 3, using a rotor with a rotating blade of 15 r, p, m, and using a rotary blade of 15 r, p, m.

The dry weight of the resulting plant tissue was 1.0 g.

〔Effect of the invention〕

As explained in detail above, according to the present invention, since the culture container is rotated while the culture container is filled with a mixed system of a liquid medium and cells or plant tissues, the cells or plant tissues are rotated. can be dispersed with good dispersion without applying substantial shearing force, and because the culture vessel is rotated around an axis tilted at an angle of 5 to 55 degrees with respect to the horizontal plane. For example, by using cells or plant tissue that has a higher specific gravity than the liquid medium, it is possible to disperse the cells or plant tissue with a difference in the dispersion concentration between the upper and lower parts of the liquid medium in the culture container. By discharging the old liquid medium from the area with low concentration, the liquid medium can be discharged stably without clogging the filter member installed in the culture container, and the new liquid medium can be discharged from the area with high dispersion concentration. By supplying a liquid medium, a large number of cells or plant tissues can be efficiently brought into contact with a new liquid medium, and as a result, cells or plant tissue can be stably cultured over a long period of time. It is possible to provide a culture method and apparatus that can achieve high efficiency culture.

[Brief explanation of the drawing]

FIG. 1 is an explanatory perspective view of an example of the present invention, FIG. 2 is an explanatory sectional view of an example of a culture device according to the present invention, and FIG. 3 is a culture performed using the culture device according to the present invention. FIG. 2 is an explanatory diagram showing an example of a system chart for the case. ■...Culture container 2...Cells or plant tissue

Claims (1)

[Scope of Claims] 1) A culture vessel filled with a mixed system of a liquid medium and a mixed system of dispersed particles formed by adhering adhesion-dependent cells, suspended proliferating cells, or plant tissue on a horizontal surface. Against 5
A culture method characterized in that the mixing system is constantly rotated together with the culture container by rotating it so as to rotate around an axis tilted at an angle in the range of ~55 degrees. 2) A culture container having a rotation axis supported so as to be inclined at an angle in the range of 5 to 55 degrees with respect to a horizontal plane, and a drive mechanism that rotates the culture container so as to rotate around the rotation axis. The driving mechanism operates in a mixed system of a liquid medium filled to substantially fill the culture container, and dispersed particles formed by adhering adhesion-dependent cells, suspended proliferative cells, or plant tissues. A culture device characterized in that it has a function of constantly rotating together with the culture container.