JPS63109772A - Culture of vegetable cell or the like - Google Patents

Abstract

PURPOSE:To enable the production of vegetable cell in high yield, to remarkably improve its production efficiency and to simplify the operation, by culturing vegetable cell by a particular method using a preculture tank and a main culture tank. CONSTITUTION:Vegetable cells, etc., are precultured in a preculture tank 11 and then cultured in a main culture tank 12 until the concentration of the cell reaches a prescribed level. The cultured liquid is diluted with a fresh medium and a part of the diluted cultured liquid is supplied to the preculture tank 11 via a circulation path 14. The cultured liquid is again cultured in the preculture tank 11 until the vegetable cell concentration reaches a prescribed level and taken out of the tank through the delivery port 16.

Description

DETAILED DESCRIPTION OF THE INVENTION (Field of Industrial Application) The present invention relates to a method for efficiently culturing a large amount of plant cells and tissues.

(Prior art) Useful substances such as medicines can be obtained from plant cells and tissues, and in order to permanently obtain such useful substances, it is necessary to industrially cultivate plant cells and tissues. is being attempted. Submerged culture of plant cells and the like in a culture tank is industrially preferred because it allows for mass culture. For this reason, traditionally.

Attempts have been made to culture in large quantities by increasing the size of the culture tank. However, as culture tanks become larger, plant cells become more susceptible to physicochemical damage due to water head pressure, gravity, etc., and control of culture conditions (control of carbon dioxide concentration, pit value, cell agitation speed, etc.) becomes necessary. becomes difficult. This resulted in the following drawbacks. For this reason, it has not been possible to improve culture efficiency by increasing the size of the culture tank.

Therefore, attempts have been made to improve the cell culture speed by using continuous culture, in which cells are continuously cultured under certain conditions. Theoretically, continuous culture has the highest production efficiency, but it is technically difficult to harvest plant cells continuously and aseptically, and the equipment for continuous culture is not available. .

It must be designed and manufactured under the most suitable conditions for continuous culture, and it is also extremely difficult to determine the operating conditions, making it difficult to commercially cultivate plant cells using continuous culture. The current situation is that it has not been standardized.

On the other hand, in contrast to continuous culture, batch culture, in which plant cells and the like are cultured in a fixed amount of medium, is also performed.

FIG. 4 shows an example of a conventional batch culture device. To explain batch culture based on this diagram, first, plant cells etc.
Next, the shake-cultured plant cells are transplanted into a pre-culture tank 52 containing an adjusted and bacterium cultured medium, and the plant cells are multiplied. On the other hand, in parallel with the preculture in the preculture tank 52, in preparation for the main culture, the main culture tank 53 adjusts the culture medium, removes bacteria, and sterilizes the transfer pipe 54 communicating from the preculture tank 52 to the main culture tank 53. conduct. When the main culture tank 53 has a large capacity, the culture medium membrane surface is separately sterilized using a continuous sterilizer, and the culture medium is continuously fed into the sterilized main culture tank.

Next, in order to prevent bacterial contamination, the main culture tank 53 is cooled to culture temperature while being ventilated and pressurized, and the precultured plant cells are transferred from the pre-culture tank 52 to the main culture tank 53 via a transfer pipe 54.
Main culture is performed in the main culture tank 53. Main culture tank 53
During the culture, the temperature and PTI in the main culture 53 are controlled to the set values, and in the case of aerobic culture, stirring is performed using the stirring blades 53a, and sterile air is supplied from the air supply pipe 55. Ventilate. and.

With this main culture, the culture is terminated when the plant cells reach a predetermined concentration. After culturing, the culture solution containing a predetermined concentration of plant cells is transferred to the product recovery process from the harvest port 53b of the main culture tank 53, and the main culture tank 53 is washed and sterilized for secondary culture. It will be done. The above steps start from preserved plant cells.

Plant cells are produced by mass culture.

In batch culture centered on the main culture tank 53, the preparation time of the culture tank (time required for cleaning, medium adjustment, sterilization, cooling, etc.), culture time (including inoculation time), and culture solution drain time are The sum is the time required for one batch culture.

Thus, in the double culture, several "seed" culture stages are required before the production stage in the main culture tank 53. Since the main culture is approximately 10 times larger than the preculture, the preculture process must be performed in multiple stages.
Furthermore, the capacity of the preculture tank 52 itself must be increased. Therefore, in double culture.

This is because equipment efficiency is poor and mass production is not possible.

It is not suitable for industrial use. In batch culture, there is also a repetitive batch culture that eliminates the need for preculture, which is a single culture stage, but such repetitive batch culture also has poor production efficiency.

The N yield itself does not increase.

(Problem to be solved by the invention) The present invention solves the above-mentioned conventional problems.

The purpose is to provide a culture method that can significantly improve the production efficiency of plant cells, etc. by using a preculture tank for preculture and a main culture tank for main culture.

(Means for Solving the Problems) The method for cultivating plant cells, etc. of the present invention uses a main culture tank for main culture and a pre-culture tank for pre-culture at the stage before the main culture tank. It is a method of culturing cells, tissues, etc., which includes the step of culturing plant cells, etc. to a predetermined concentration in a main culture tank,
A step of diluting the culture solution in which plant cells, etc. have been cultured to a predetermined concentration with a medium, and a step of culturing a part of the diluted culture solution in a pre-culture tank until the plant cells, etc. reach a predetermined concentration. and a step of taking out the culture solution in the preculture tank in which the plant cells have been cultured to a predetermined concentration, thereby achieving the above object.

(Example) The present invention will be described below with reference to Examples.

In the culture method of the present invention, for example, as shown in FIG. 1, plant cells and tissues are cultured in a preculture tank 11 that performs preculture and a main culture tank 12 that performs main culture. The lower part of the pre-culture tank 11 is the upper part of the main culture tank 12.

They communicate via a transfer pipe 13. In addition, the pre-culture tank 1
The lower part of 1 also communicates with the harvesting port 16. Main culture tank 12
The lower part thereof is in communication with the upper part of the pre-culture tank 11 via the circulation path 14. At the lower part of the pre-culture tank 11 and the lower part of the main culture tank 12, discharge ports of sterile air supply pipes 15 are provided, respectively. Stirring blades 11a and 12a are provided in the preculture tank 11 and the main culture tank 12, respectively.

To explain the method of the present invention using such a culturing device, first, plant cells are cultured in a flask 17 with shaking and propagated. next,! j1 Prepared and sterilized medium is transferred to preculture tank 11
The plant cells grown by shaking culture are transplanted into the medium in the preculture tank 11. Then, plant cells are precultured in the preculture tank 11.

During this pre-culture, the culture medium is adjusted and sterilized and the transfer tube 13 is sterilized in preparation for the main culture in the main culture tank 12. When the main culture tank 12 has a large capacity, culture medium sterilization is performed separately by a culture medium adjustment tank or a continuous culture medium sterilizer, and the sterilized culture medium is continuously charged into the main culture tank 12.

Next, in order to prevent bacterial contamination, the main culture tank 12 is cooled to culture temperature while being ventilated and pressurized with sterile air from the supply pipe 15 , and then the plant cells precultured in the pre-culture tank 11 are transferred from the transfer pipe 13 . After transplanting into the main culture tank 12, main culture is started.

During the main culture in the main culture tank 12, the temperature and pH are controlled to set values, and in the case of aerobic culture, sterile air is aerated into the main culture tank 12 from the supply pipe 15 while stirring blades 12a Stir at . When a predetermined concentration of plant cells is reached, the main culture is terminated.

At the time when the main culture in the main culture tank 12 is completed.

The culture medium is adjusted and sterilized in the preculture tank 11, and the medium is fed under pressure to the main culture tank 12 from the transfer pipe 13.

When a culture medium adjustment tank or a continuous culture medium sterilization device is provided, the required amount of fresh culture medium is fed into the main culture tank 12.

The medium fed into the main culture tank 12 is stirred and mixed with a culture solution containing a predetermined concentration of plant cells.

The culture solution is diluted. In the main culture tank 12, the culture solution is sufficiently stirred to uniformly disperse the plant cells. Next, the internal pressure of the main culture tank 12 is made equal to or higher than the sum of the internal pressure of the pre-culture tank 11 and the culture solution pressure using sterile air supplied from the supply pipe 15, and the diluted culture solution is sterilized. It is fed into the preculture tank 11 through the circulation path 14 . As a result, the preculture tank 11 and the main culture tank 12 are filled with culture solutions having the same concentration of plant cells, medium components, and the like.

The culture solution fed into the preculture tank 11 is cultured until the plant cells reach a predetermined concentration, and when the plant cells reach a predetermined concentration, the culture in the preculture tank 11 ends. The culture solution in the pre-culture tank 11 is harvested from the harvest port 16. Culture is carried out in the main culture tank 12 even while culture is being carried out in the pre-culture tank 11.

In the pre-culture tank 11 from which the culture solution has been harvested, the medium is subsequently adjusted and sterilized, and the medium is fed into the main culture tank 12 from the transfer pipe 14. In this case, as described above, if a culture medium adjustment tank or continuous culture medium sterilizer is provided, fresh culture medium is fed into the main culture tank 12 from the culture medium adjustment tank or continuous culture medium sterilizer. The culture solution in which the plant cells were cultured in the main culture tank 12 during the culture in the pre-culture tank 11 is.

It is diluted by the medium being delivered.

Thereafter, as described above, a portion of the diluted culture solution is fed to the preculture tank 11 and cultured, and when the plant cells reach a predetermined concentration, they are harvested from the harvest port 16.

FIG. 2 is a schematic diagram showing another embodiment of the apparatus used to carry out the method of the present invention. In this example.

The preculture, which is a stage before the main culture in the main culture tank 12,
The process is carried out in two preculture tanks 11 and 11'. In such an apparatus, a culture solution in which plant cells are cultured to a predetermined concentration in a main culture tank 12 is used.

After diluting with a medium, the plant cells are again cultured in each pre-culture tank 11 and 11'' until a predetermined concentration is reached, and the plant cells are harvested from the harvest ports 16 and 16' in each pre-culture tank 11 and 11°.

Unlike 'R1 organisms, plant cells etc. are grown outside the body (medium).
In order to avoid producing growth substances, as in the method of the present invention,
If given fresh medium, oxygen, and adequate space, they will grow efficiently.

The production amount by the method of the present invention and the production amount by conventional batch culture will be compared using a simple model. now.

In batch culture for 30 days, the concentration (WET) was 3% to 3.
Consider a plant cell growing at 0%. Usually, the induction period is considered to be about 3 to 5 days, assuming that the specific growth rate (μ) is constant and the growth is always logarithmic.

The specific growth rate (μ) is usually expressed by the formula (l).

Where, Δt: Time (days) χo: Initial concentration of callus

...(1).

becomes.

What is the annual harvest Y?

Y- (Callus concentration at harvest (%)) ・ [Main culture capacity (1)] ・ [Number of culture in 11 years] ・ ・ ・ ・ (
2) Therefore, if the capacity of the main culture tank is 1001,
What is the annual yield Y of conventional batch culture?

= 360 (kg/year)...
...(2)' (However, the cleaning time of the main culture tank is not included).

Consider the case where such plant cells are cultured by the method of the present invention. However, in the method of the present invention, plant cells grown to a concentration of 30% are diluted to 27% with a medium,
Thereafter, the seeds will be grown to 30% in a preculture tank and harvested. From the formula (1)', since the specific growth rate μ of the plant cell is 0.0767 (day"), the time Δt for the plant cell to grow from 27% to 30% is:
From equation (1). 1.5 (da) considering loss time during harvesting.
Assuming that the crop is harvested once in y), the annual harvest amount Y is calculated from equation (2) as follows. However, according to the method of the present invention, plant cells are grown from 3% to 30%, and then diluted to 27% and then grown to 30%. It will not be harvested for 30 days. Therefore.

+ao-690 (kg/year) becomes.

As described above, according to the method of the present invention, approximately twice as many plant cells can be harvested in one year as compared with conventional batch culture.

(Experiment example) Using the apparatus shown in Figure 1, a medium of 8 (H!) was placed in a pre-culture tank with a fixity of 1 oz, and a medium of 4001 was placed in a main culture tank of 16 oz.・Agitation culture was carried out.B5 medium was used as the medium,
Culture temperature: 25℃9 Aeration air volume: 0.1-0.4VVM
And so. Plant cells could not be harvested during the first month, but after one month, plant cells were harvested from the preculture tank every 7 days. In conventional batch culture, 80 kg (WET
), and 480 kg of cells can be obtained in 6 months; however, with the method of the present invention, 770 kg (
60% more plant cells than before were obtained. Figure 3 shows the results.

(Effects of the Invention) As described above, the method of the present invention can always maintain the concentration of plant cells, etc. in the main culture tank at a high concentration near the predetermined concentration at the time of harvest, and in the pre-culture tank with a small capacity. This is because it is harvested after culturing to a predetermined concentration.

Plant cells etc. can be harvested with high efficiency. Initially, if the plant cells seed-cultured in the pre-culture tank are transplanted to the main culture tank, subsequent seed culture is unnecessary and the scraping process is simplified. For conventional batch culture apparatuses, a circulation path may be provided using a sterile filter.

Yields can be increased using existing equipment.

4. U of P Figure 1 is a schematic diagram showing an example of the apparatus used to carry out the method of the present invention, Figure 2 is a schematic diagram showing another example thereof, and Figure 3 is a schematic diagram showing an example of the apparatus used to carry out the method of the present invention. The graph showing the amount, FIG. 4, is a schematic diagram showing an example of a conventional batch culture device.

11, 11'... Preculture tank, 12... Main culture tank,
13... Transfer pipe, 14... Circulation path, 15... Supply pipe, 16.16'... Harvesting port.

that's all

Claims (1)

[Scope of Claims] 1. A method for culturing plant cells, tissues, etc. using a main culture tank that performs main culture and a preculture tank that performs preculture before the main culture tank, A step of culturing plant cells etc. to a predetermined concentration in a tank, a step of diluting the culture solution in which the plant cells etc. have been cultured to a predetermined concentration with a medium, and a step of diluting a part of the diluted culture solution beforehand. A method for culturing plant cells, etc., comprising: culturing the plant cells, etc. in a culture tank until they reach a predetermined concentration; and removing the culture solution in the preculture tank in which the plant cells have been cultured to the predetermined concentration. .