JPH0534944B2 - - 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]

[Industrial Application Field] The present invention relates to a method and apparatus for culturing cells or plant tissues, and more particularly to a method and apparatus for culturing cells or plant tissues suitable for producing physiologically active substances, etc. It is. [Prior Art] In general, physiologically active substances are substances that impart a certain kind of activity to cells or plant tissues and cause them to produce specific physiological reactions, and some of them can be artificially synthesized. , which usually has to be produced by maintaining the survival of living cells or plant tissues. As a conventional method for producing a physiologically active substance, a method is known in which cells or plant tissues that produce the desired physiologically active substance are cultured to keep them alive. 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. Recently, small-diameter carrier particles have come to be used as substrates for adhesion-dependent cells because they can easily provide a large adhesion area. [Problems to be solved by the invention] However, in order to culture the target cells with high efficiency, it is important to constantly contact the cells to be cultured with a fresh medium. , or carrier particles to which adhesion-dependent cells have adhered (hereinafter also simply referred to as "cells") are required to be dispersed with a high degree of dispersion in a liquid medium. 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 surface area of the resulting carrier particles becomes larger, 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 has been to use a rotary blade to stir a system in which cells to be cultured are mixed in a liquid medium; however, this method Since this method applies shear force to a mixed system of liquid culture medium and cells to disperse the cells, it avoids 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 type of cells. Furthermore, 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 results in a large shearing force. Therefore, there is also the problem 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 in a state where they can be dispersed. The above situation is the same in culturing plant tissues. Although various measures have been researched to solve these problems, the current situation is that no cell or plant tissue culture method or device that essentially solves these problems has been developed. . The present invention was completed as a result of intensive research based on the above circumstances, and its purpose is to apply shearing force to the mixed system of a liquid medium and cells or plant tissues to be cultured. A method capable of culturing cells or plant tissues with high efficiency by dispersing cells or plant tissues in a liquid medium with a good degree of dispersion and in a state that is advantageous for exchange of the liquid medium without providing and equipment. [Means for Solving the Problems] The method of the present invention is characterized in that the culture vessel filled with the mixed system with cells or plant tissues is placed at an angle of 5 to 55 degrees with respect to the horizontal plane. The mixed system is constantly rotated together with the culture vessel by rotating around an axis inclined at an angle of is dispersed in the liquid medium, preferably with a difference in dispersion concentration in the upper and lower parts. The apparatus of the present invention is also characterized by a culture container having a rotating shaft supported so as to be inclined at an angle of 5 to 55 degrees with respect to a horizontal plane, and a culture container that rotates around the rotating shaft. and a drive mechanism that rotates a mixed system of cells or plant tissue and a liquid medium filled so as to substantially fill the culture container together with the culture container. It has the function of rotating the According to such technical means, cells or plant tissues can be dispersed with a high degree of dispersion in a culture container without applying substantially shearing force. For example, cells or plant tissues having a higher specific gravity than a liquid medium can be used. Therefore, it is possible to disperse the liquid medium in the culture container with a difference in dispersion concentration between the upper and lower parts, so this difference in dispersion concentration can be used to advantageously replace an old liquid medium with a new liquid medium. It becomes possible to exchange. That is, the culture container is usually provided with a filter member that allows only the liquid medium to pass through without passing through cells or plant tissues, and the liquid medium is exchanged through this filter member, but the above technical means According to , it is possible to exchange the medium from the part of the liquid medium where the dispersed concentration of cells or plant tissue is low, making it possible to exchange the liquid medium efficiently without clogging the filter member. As a result, culturing of cells or plant tissues can be achieved with high efficiency. The present invention will be specifically explained below. In the present invention, as shown in FIG. 1, for example, a cylindrical sealed culture container 1 is filled with a liquid medium 3 in which cells or plant tissues 2 are mixed, and the inner space of the culture container 1 is substantially filled. Fill the culture container so that the axis
This axis
The culture container 1 is rotated at a constant rotational speed around the . At this time, if the angle (α) is smaller than 5°, for example, even if cells or plant tissues with higher specific gravity than the liquid medium are used, there will be no difference in the dispersed concentration of cells or plant tissues in the culture container. 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 is larger than 55°, cells or plant tissues having a different specific gravity from the liquid medium will hardly be 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 tissues 2 in the culture container 1 becomes viscous. As a result, the culture container 1 rotates steadily around the axis X without any mechanical flow, so that the cells or plant tissues 2 are rotated relative to the liquid medium 3 in the culture container 1. As a result, the cell or plant tissue 2 in the culture container 1 is subjected to gravity from different directions sequentially, and as a result, the cell or plant tissue 2 in the culture container 1 is subjected to gravity from different directions. The tissue 2 becomes 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 satisfactorily. If 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 flow occurs, and therefore the liquid medium can be exchanged without disturbing the desired dispersion state of cells or plant tissues. In order to improve culture efficiency, it is preferable to constantly supply the liquid medium through a supply pipe. Then, with the axis X of the culture container 1 inclined at an angle within the above range with respect to the horizontal plane, the culture container
By rotating the cell or plant tissue 2, a difference will occur in the dispersed concentration of the cells or plant tissue 2 in the upper and lower parts of the liquid medium 3 in the culture container 1 due to the action of gravity or buoyancy. When the specific gravity is higher than that of the liquid medium 3, the dispersion concentration in the upper part of the liquid medium 3 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,
By discharging the old liquid medium from the area where the dispersion concentration is low and supplying new liquid medium to the area where the dispersion concentration is high, cells or plant tissues can be efficiently brought into contact with the new liquid medium. When discharging an old liquid medium, clogging of the filter member by cells or plant tissue 2 can be suppressed, so the liquid medium can be replaced stably and continuously during the culture period. This makes it 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 doing so, it is possible to continuously exchange the liquid medium, adhere the adhesion-dependent cells to the carrier particles, and extract the cultured and proliferated adhesion-dependent cells. 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】

〔Example〕

Examples of culturing actually carried out based on the present invention will be described below. Example 1 Cultured cells: "V-79" derived from Chinese hamster lung fibroblasts Liquid medium: "Eagle-" containing 10V/V% fetal bovine serum dissolving oxygen and carbon dioxide gas
MEM” (viscosity: 0.01 poise, specific gravity: 1.01) Carrier particles: “Cytodex” (manufactured by Pharmacia) (particle size: 180 μm, specific gravity: 1.03) 900 mg dry weight in a cylindrical culture vessel with a capacity of 300 ml
Add (approximately 3 x 10 ⁶ pieces) of the above carrier particles and fill the liquid medium, seed 6 x 10 ⁶ pieces of the above cultured cells, and seal it to prevent air from entering. The angle of the rotation axis relative to the horizontal plane
45° in an environment with a temperature of 37°C.
The culture vessel was rotated at a rotational speed of 15 rpm for a period of time to allow the 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 in each carrier particle to which cells did adhere was 2 to 4, with an average of 2.5, and the number of cells on all carrier particles was 2.5. The number was 4.8× ¹⁰⁶ . Continue to incubate the culture vessel under the same conditions as above.
Cells were cultured by rotating for hours. During this period, the rotation of the culture container was stopped and the entire liquid medium was replaced every 24 hours. As a result, the cells on all carrier particles had grown to 7.2×10 ⁷ cells. Example 2 Cells were cultured using the same cultured cells, liquid medium, and carrier particles as in Example 1 using a culture apparatus having a culture container with an internal volume of 1 as shown in FIG. . That is, using 3 g (approximately 1.0 x 10 ⁷ cells) of carrier particles in dry weight per 1 liquid medium, seeding 1 x 10 ⁴ cultured cells per 1 ml of liquid medium, and making sure that no air enters the culture container. Filled with a mixed system of cultured cells, liquid medium, and carrier particles, the culture vessel was rotated for 144 hours at a temperature of 37°C with the rotation axis of the culture vessel set at an angle of 45° with respect to the horizontal plane. While rotating the culture container at 12 rpm, a new liquid medium was supplied into the culture container at a rate of 80 ml/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 ⁶ per ml of 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 Using a spinner bin with an internal volume of 2, Example 1
Cells were cultured using the same cultured cells, liquid medium, and carrier particles as in . That is, 3 g of dry weight carrier particles were added to 1 ml of liquid medium in a spinner bottle, and then 1 x 10 ⁴ cultured cells were seeded per 1 ml of liquid medium, and the rotation speed of the rotor was adjusted at a temperature of 37°C. Culture was carried out for 144 hours while rotating at 30 rpm. 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 average number of cells on the carrier particles was 5.1×10 ⁵ cells per ml of liquid medium. Example 3 A cylindrical culture container with an internal volume of 100 ml was filled with Linsmeyer-Skoog's liquid medium (specific gravity 1.0), and 0.5 g of dry weight Lithospermum erythrorhizon Sieb.et, Zucc was added to this.
After putting in the wet callus and sealing it, with the rotation axis of the culture container set at an angle of 30° with respect to the horizontal plane,
Rotation speed over 14 days in an environment with a temperature of 25℃
Plant tissues were cultured while rotating the culture container at 15 rpm. 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 Plant tissue was cultured using the same plant tissue and liquid medium as in Example 3 using a culture apparatus having a culture container with an internal volume of 120 ml as shown in FIG. i.e. 0.6 dry weight for 120ml of liquid medium
Lithospermum erythrorhizon
After placing wet callus (Sieb.et, Zucc) and sealing, adjust the angle of the rotation axis of the culture container with respect to the horizontal plane.
14 in an environment with a temperature of 25℃ at a temperature of 30℃.
The culture vessel was rotated at a rotation speed of 15 rpm for days, and while supplying new liquid medium into the culture vessel at a rate of 120 ml/24 hours and discharging the old liquid medium at the same rate, the plant tissue was grown. Culture was carried out. When the thus obtained plant tissue was dried and its weight was measured, it was 1.7 g. Comparative Example 2 Using a spinner bottle with an internal volume of 200 ml, 100 ml of the same liquid medium as in Example 3 and callus were added.
Plant tissues were cultured in the same manner as in Example 3 using 0.5 g and using a rotating blade of 15 rpm. The dry weight of the resulting plant tissue was 1.0 g. [Effects of the Invention] As explained in detail above, according to the present invention, the culture container is rotated while the culture container is filled with a mixed system of a liquid medium and cells or plant tissues so as to be substantially filled. , cells or plant tissues can be dispersed with a good degree of dispersion without applying substantial shearing force, and 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 tissues that have a higher specific gravity than the liquid medium, the cells or plant tissues can be dispersed with a difference in the dispersion concentration between the upper and lower parts of the liquid medium in the culture container. Therefore, by discharging the old liquid medium from the part where the dispersion concentration is low, it is possible to stably discharge the liquid medium without clogging the filter member provided in the culture container, and By supplying new liquid medium to areas with high dispersed concentration, a large number of cells or plant tissues can be brought into contact with the new liquid medium efficiently, resulting in stable growth of cells or plant tissues over a long period of time. It is possible to provide a culture method and apparatus that can perform culture and achieve culture with high efficiency.

[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. 1... Culture container, 2... Cell or plant tissue, 3... Liquid medium, 30... Bottom plate, 31... Container body, 32... Holding mechanism, 33... O ring,
35... Rotating sleeve, 36... Dry bearing, 37... Stand, 38... Mantle member, 40...
...Meshyu, 41...Inner conduit, 42...Outer conduit,
43... One end opening of the inner conduit, 44... One end opening of the outer conduit, 50... Connecting seal portion, 51... Supply pipe, 52... Discharge pipe, 60... Driven gear, 61...
...Drive gear, 70...Liquid medium tank, 71...
Culture container, 72... Discharge tank, 73... Monitor mechanism, 74, 77... PH sensor, 75, 78
...DO sensor, 80 ...Alkaline tank, 8
1... Air pipe, 82... Control valve.

Claims (1)

[Scope of Claims] 1. A culture vessel filled with a mixed system of a liquid medium and a mixed system of particles to be dispersed with adhesion-dependent cells, suspended proliferative cells, or plant tissues is held against a horizontal surface. 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 inclined at an angle in the range of 5 to 55 degrees. 2. A culture container having a rotation shaft supported so as to be inclined at an angle of 5 to 55 degrees with respect to a horizontal plane, and a drive mechanism for rotating the culture container so as to rotate around the rotation shaft. The driving mechanism drives a mixed system of a liquid medium filled in the culture container so as 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 a culture container.