JPS6167476A - Apparatus for vegetable cell culture - Google Patents

Abstract

PURPOSE:To provide the titled apparatus having excellent agitation effect and effective to prevent the damage of culture cells, by suspending an agitation blade above the medium surface in a vessel having a curved surface from the bottom to the upper part, and placing an aeration pipe at the bottom of the vessel eccentrically to the shaft of the agitation blade. CONSTITUTION:The vessel 11 is formed with curved surface free from flat part from the bottom to the top. A plurality of openings 13 attached with sleeves 14 protruded downward are formed at the positions deviated from the shaft of the agitation blade 13. An aeration pipe 15 having bubble-forming part 16 at the top is connected with each sleeve 14, and a rotary magnet is placed between the sleeves 14. The top end of the supporting pipe 20 placed rotatably to the outer circumference of the take-up pipe 8 is supported by the supporting mechanism formed around the take-up pipe 8 placed above the surface of the medium 18. The agitation blade 21 is suspended opposite to the magnet 17 at the lower end of the supporting pipe 20.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to a plant cell culturing device that can effectively carry out liquid suspension culture of cells of plants, animals, microorganisms, etc., especially plant cells.

[Prior art and its problems] In general, there are two types of cell culture methods: agar culture using agar and liquid culture using suspended culture medium. These liquid culture methods can be further divided into rotary culture methods. , shaking culture method,
It is classified into spin culture method and agitation culture method.

The rotational culture method is a method in which a nipple flask with multiple protrusions is attached to a large disk and rotated at a slow speed of 1 to 2 times per minute, submerging the cells inside the medium or exposing them to air for cultivation. be.

In the shaking culture method, a conical flask called an Erlenmeyer flask containing a cell suspension is placed on a rotary shaker. This is a method of culturing by placing the tube in a container and rocking it up and down in a circular motion.

The spin culture method uses a relatively large culture bottle, tilts the frame that holds the bottle at a 45 degree angle, and generates 80 to 120 cells per minute.
This is a method of culturing while rotating at the same speed.

The stirring culture method is a method of culturing cells while maintaining a dispersed state and performing gas exchange by passing air bubbles through the medium or stirring with a magnetic stirrer called a magnetic stirrer.

Discontinuous culture, called patch culture, in which cells are grown and multiplied in a fixed culture medium, can be carried out by any of the four methods described above.

However, when cells are grown and multiplied in a closed system, the amount of material cells increases after a certain period of time and reaches a state of maximum yield. At this point, either the nutrients in the soil are consumed or the growth stops due to the accumulation of toxic substances. If it is desired to continue culturing in this state, it is necessary to remove the culture medium in which a small number of cells have become sensitive to temperature and transfer this culture medium to a new medium.

In contrast to the discontinuous closed system described above, the so-called continuous culture system is an alternative system in which the culture solution is pumped into the culture bottle at a constant rate, and the culture solution containing the same amount of cells is taken out from the culture bottle. It is.

By the way, only the agitation culture method in which the container itself is stationary can be used as this system.

That is, the agitation culture method can be used both discontinuously and continuously.

When used continuously, constant culture with constant cell density, growth rate, chemical composition, metabolic activity, etc. can be achieved by measuring PH and oxygen concentration and adding an automatic control device for these.

In recent years, as cell culture has become more popular, the agitation culture method has become more important for the reasons mentioned above, and its problems have also come to the fore.

Fig. 2 shows a conventional plant cell culture device using such an agitation culture method. On a support stand 2 having a built-in one-rotation magnet 1, there is a container such as a culture bottle with a flat bottom 3a. 3 is placed.

A support portion 4 is provided at the center of the bottom portion 3a of the container 3, and a stirring blade 5 equipped with a magnet is supported on the support portion 4 so as to face the rotating magnet 1.
A ventilation pipe 6 is connected to the lower circumferential surface of the container 3, and microbubbles are brought out into the container 3 through a large number of micropores.

Further, from the upper part of the container 3, an injection tube 7 for a new culture solution and a take-out tube 8 for an old culture solution containing cultured cells face the inside of the container 3. Gases generated during culture are also discharged to the outside through a gas outlet pipe 9 interposed with a cotton plug filter IO.

Nao, Ko (1) Hokani PH measuring device, PHwAffi device, PHyA liquid saving injection joint inlet concentration measuring device, oxygen concentration 2y
Although there are other devices such as a n-type device, illustration thereof is omitted since they have no direct relation to the present invention.

According to the above-mentioned configuration, the magnet 1 is rotated once to rotate the stirrer 5, and fine bubbles are generated in the ventilation pipe 6, and a new culture solution is introduced into the container 3 from the injection pipe 7 by a pump (not shown). Plant cells can be continuously cultured by injecting it into the tube 8 and taking out the old culture solution from the same state from the tube 8.

However, in such a conventional plant cell culture device, the support part 4 of the stirring blade 5 is immersed in the culture solution, so heat and shear generated from the sliding part of the support part 4 and the stirring blade 5 are absorbed. Surrounding cells are adversely affected by the force.
In addition, since the ventilation pipe 6 is connected to the side of the container 3, the upward force of the bubbles against the container 3 is biased.
Therefore, the flow of the culture solution becomes unbalanced, causing local stagnation.

[Means for Solving the Problems] The present invention was made primarily for the purpose of solving the problems of the conventional devices described above and providing a plant cell culture device that does not have an adverse effect on cells. in,
The container 11 is formed into a curved surface from the lower end to the upper end, and the support mechanism 19 of the stirring blade 21 is positioned above the culture medium surface 18 to suspend and support the stirring blade 21. It is characterized by being eccentrically connected to the axis of the two wings.

[Example 1] The present invention will be explained below by referring to an example shown in the drawings.
Note that the same components as those of the conventional device described above are denoted by the same reference numerals in the drawings, and the explanation thereof will be omitted.

In FIG. 1, a container 11, which is a culture bottle of the present invention, is formed of a curved shape having no flat surface from its lower end to its upper end. This curved surface is preferably a spherical surface or an elliptical surface.

Therefore, the container 11 is supported by the pedestal 12.
At the lower end of this container 11, stirring χ2, which will be described later,
A plurality of openings I3.13 are located eccentrically from the axis of I3.13.
A sleeve 14.14 is formed on the outer periphery of each opening 13.13 and protrudes toward the child.
A vent pipe 15.15 is connected to each sleeve 14.14, and a bubble forming part 16.16 is provided at the upper end of this vent pipe 15.15.
A rotating magnet 17 is arranged on the underside of the container 11 between each sleeve 14,14.

On the other hand, the take-out tube 8 above the culture medium surface (culture liquid surface) 18
A support mechanism I9 is formed on the outer periphery of the take-out tube 8. The support mechanism 19 supports the upper end of the support tube 2o rotatably disposed on the outer periphery of the take-out tube 8, and the lower end of the support tube 20. , a stirring blade 2 is placed opposite the rotating magnet 17.
1 is suspended.

Note that the support mechanism 19 may be separated from the extraction tube 8 and provided separately.

According to the configuration described above, each vent pipe 15.15 has a container l.
The fine bubbles rising inside the container 11 are affected by the centrifugal force generated by the rotation of the stirring blade 21, and rise along the inner circumferential surface of the curved surface of the container 11 while remaining in a fine bubble state, and rise to the surface of the culture solution. It disappears when it reaches 18.

Thereafter, the culture solution descends through the center of the container 11.

Therefore, a smooth circulation flow is formed symmetrically with respect to the center line of the container 11 over the entire area inside the container 11, and the culture solution can be stirred well.

As a result, stirring χ which was previously 200 to 600 revolutions/min
5.21 rotation speed to 50 to 100 rotations/min and 1/1 of the conventional speed.
It can be slowed down to 2 to 1/12 to prevent cell damage.

Further, since the support mechanism 19 of the stirring blade 21 is positioned above the culture medium surface 1B, there is no fear that sliding heat and shear force will adversely affect the culture solution.

Furthermore, since the opening of each vent pipe 15.15 is provided eccentrically from the axis of the stirring χ21, these bubbles are
Since large bubbles are not formed together on the lower surface of the cell 21, there is no risk of impairing the agitation efficiency of the cells or damaging the cells due to large bubbles bursting on the liquid surface.

It goes without saying that the apparatus according to the present invention can also be used for culturing animals, microorganisms, etc. without any problems.

[Effects] As described below, according to the present invention, the stirring efficiency of the culture solution is decreased to reduce the rotational speed of the stirring blade, and
Since the sliding portion does not have an adverse effect on the culture solution, plant cells can be cultured well.

[Brief explanation of the drawing]

FIG. 1 is a longitudinal sectional view showing an embodiment of the plant cell culturing apparatus according to the present invention, and FIG. 2 is a longitudinal sectional view showing a conventional plant cell culturing apparatus. l, 17... Rotating magnet 3.11... Container 4.1
9... Support mechanism 5.21... Stirring blade 6.15.
...Vent pipe patent applicant Noko Eida Vario Glass Co., Ltd. Figure 2 Diagram ■...Rotating magnet 3...Container 4...Support groove 5...Agitation blade 6, -1 Vent pipe

Claims (1)

[Claims] In a plant cell culture device, a rotating magnet is disposed below a container, a stirring blade equipped with a magnet is disposed at the bottom of the container so as to face the rotating magnet, and a ventilation pipe is connected to the container, The container is formed into a curved surface from the lower end to the upper end, the support mechanism for the stirring blade is positioned above the culture medium surface to suspend and support the stirring blade, and the ventilation pipe is connected to the lower end of the container so that the axis of the stirring blade is A plant cell culture device characterized in that the device is connected in an eccentric manner.