JPS633592B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for culturing cell tissues in which the density of cell tissues can be controlled within a predetermined range, thereby allowing efficient proliferation of cell tissues. Regarding culture tanks.

"Culture volume" as used herein refers to the volume in which a given amount of cell tissue is stirred in a culture solution and cultured while moving, and the volume of a section divided by providing a partition in the culture solution. can be.
In addition, the "culture volume range" is the volume range in which a given cell tissue is cultured while maintaining the density of the cell tissue necessary for its proliferation. It can be the volume of a partitioned area, but since the density of cell tissue required for cell tissue to proliferate has a certain range of values, the culture required to maintain it The volume range will also have a corresponding range of numerical values.

[Background of the invention and description of prior art]

The invention of a method for producing a specific substance by culturing microorganisms and thereby cultivating the specific substance in the cells or culture of the microorganism is a method for producing various antibiotics, amino acids, vitamins, etc. It has been developed for some time and is becoming widely known. For this purpose, a method for culturing large amounts of microorganisms was developed. To cultivate a large amount of microorganisms, air or oxygen is blown into the culture solution, and at the same time, the culture solution is vigorously stirred to bring the microorganisms into contact with air or oxygen and to reduce nutrients between the microorganisms and the culture solution. It is widely known to maintain good contact with the source.

Recently, methods have been used to proliferate animal cell tissues by inoculating animal cell tissues into a medium and culturing them. As a result, methods of propagating the cell tissues of plants have also become popular. Especially in the case of plant tissue,
Attempts have been made to produce substances contained in plant cell tissues, such as alkaloids, steroids, terpenoids, quinones, or pigment glycosides, by propagating plant cell tissues in culture. However, since both animal and plant cell tissues are multicellular, if they are cultured with vigorous agitation, similar to the culture of microorganisms, the cell tissues will often collide and be damaged. There is a tendency. For this purpose, in culturing animal or plant tissue,
The culture solution was stirred by blowing air without mechanical stirring using a stirring blade or stirring blade. For example, when producing anthraquinones by culturing the cell tissue of Morinda citrifolia, Wagner established that a ventilation tube (air supply tube) was opened near the bottom of a round-bottomed culture tank. Air was supplied from the trachea to the bottom of the culture solution, and the rise of the supplied air bubbles (air lift) stirred the culture solution and supplied oxygen to the culture solution. Hashimoto et al. also used a similar device to produce shikonin.
They also produced serpentin using a similar device.

The present inventors intended to produce reserpine by culturing cell tissues of plants belonging to the genus Rauolphia, and installed a ventilation pipe in a round-bottomed culture tank so as to open near the bottom of the culture tank. , tried culturing the cell tissue of the plant, but found that the proliferation of the cell tissue of the plant was undesirable, both when the density of the cell tissue in the culture solution was too high and when the density was too low. I found out.
It has also been found that in order for plant cell tissues to grow smoothly by aeration and agitation culture in a culture solution, it is necessary to culture them at an appropriate cell density. In other words, when there is a certain amount of plant cell tissue and we try to culture it at an appropriate cell density, there is a volume of culture solution necessary to culture that certain amount of cell tissue. The volume of the culture solution required to culture a certain amount of cell tissue can be secured by providing an appropriate partition in the culture solution in the culture tank. The present invention was based on this finding.

[Object of the invention and summary of the invention]

An object of the present invention is to provide a culture tank in which the culture volume within the culture tank can be arbitrarily divided and cell tissues of a given plant can be cultured in the compartments.

The present invention provides an air lift agitation type culture tank having a ventilation pipe and an exhaust port that open near the bottom of the culture tank. , a culture tank characterized by being able to arbitrarily increase the culture volume of cell tissue. In the culture tank of the present invention, the guide plate is one of the partitions that can partition the culture solution in the culture tank and allow cell tissue to be cultured within the partition,
This guide plate can be slid and moved along the mounting axis (optionally also a vent tube), thereby increasing the volume of the compartment in which the tissue is to be cultured (cultivation volume). It is something that can multiply. Furthermore, the movement and fixation of the guide plate in the culture tank of the present invention can be carried out by the lifting and lowering of the guide plate and the fixing device attached to the outside of the culture tank, thereby ensuring smooth growth of the cell tissue to be cultured. can be made to do so.

In addition, when the guide plate is shaped like an inverted one, and culture is carried out by air lift agitation,
It is also possible to form an air layer inside the inverted one-type guide plate to supply air (oxygen) to the deep part of the culture solution.

[Specific description of the invention]

An example of the culture tank of the present invention will be explained with reference to the drawings.

FIG. 1 is a side view showing a cross section of an example of the culture tank of the present invention, FIGS. 2 and 3 are side views showing a cross section of another example of the culture tank of the present invention with a part cut away, FIG. 4 is a plan view of an example of the culture tank of the present invention shown in FIGS. 1 and 3, and FIG. 5 is a plan view of an example of the culture tank of the invention shown in FIG. .

In FIG. 1, 1 is the culture tank, 2 is the hemispherical bottom of the culture tank 1, 3 is the lid of the culture tank 1, and 4 is the
A ventilation pipe for supplying air to the culture tank 1, one end of which opens near the bottom 2 of the culture tank 1,
The other end is typically a sterilizer (not shown)
is connected to an air supply device (not shown, for example, a compressed air reservoir, an air compressor, or a blower) through which air is supplied near the bottom 2 of the culture tank 1, and the air is The culture solution is stirred by rising in the culture solution (agitation by air lift). 5 is a raw material supply port, 6 is an exhaust pipe, 7 is a filter attached to the pipe system of the exhaust pipe 6, and 8 is a ventilation pipe 4.
This is a valve in the pipe system. Further, 9 is a guide plate attached to the ventilation pipe 4 inside the culture tank 1 so as to be able to slide up and down, and 10 is a
An attachment portion of the guide plate 9 to the ventilation pipe 4,
It can be moved by sliding up and down. Furthermore, 11 is a lifting device for moving the guide plate 9 up and down and fixing it in a predetermined position; 12 is a connecting rod for moving the guide plate 9 up and down by the lifting device 11 and fixing it in a predetermined position; 13 is a shaft sealing portion of the ventilation pipe 4 and the connecting rod 12.

In Fig. 2, 1 is the culture tank, 2 is the conical pyramid-shaped bottom of the culture tank 1, 3 is the lid of the culture tank 1, and 4 is the lid of the culture tank 1.
is a ventilation pipe that supplies air to the culture tank 1,
Similar to the culture tank of FIG. 1, one end thereof opens near the bottom of the culture tank 1, and the other end is connected to an air supply system (not shown), usually through a sterilizer (not shown).
For example, by connecting to a compressed air reservoir, an air compressor, or a blower, air is supplied from this air supply device to near the bottom of the culture tank 1, and the air rises in the culture solution. The culture solution is stirred. Reference numeral 5 designates a raw material supply port, 6 an exhaust pipe, 7 a filter in the exhaust pipe 6 pipe system, and 8 a valve in the ventilation pipe 4 pipe system. Also 9
1 is a guide plate that is attached to the ventilation pipe 4 inside the culture tank 1 so that it can be moved up and down by sliding the attachment part 10 thereof, and 14 is a screw that fixes the guide plate 9 in an appropriate position.

In Fig. 3, 1 is the culture tank, 2 is the round-shaped fiber-shaped bottom of the culture tank 1, 3 is the lid of the culture tank 1, and 4 is the lid of the culture tank 1.
is a ventilation pipe that supplies air to the culture tank 1,
Air is supplied from an air supply device (not shown) in the same way as in the culture tank shown in FIGS. 1 and 2. 5 is a raw material supply port, 6 is an exhaust pipe, 7 is a filter in the pipe system of the exhaust pipe 6, and 8 is a valve in the pipe system of the exhaust pipe 4. Further, 9 is a ventilation pipe 4 inside the culture tank 1.
1, an inverted one-shaped guide plate 11 attached so as to be able to move up and down by sliding the attachment part 10 moves the guide plate 9 up and down, as in the culture tank shown in FIG. A lifting device 12 similarly moves the guide plate 9 up and down by the lifting device 11, and a connecting rod 13 fixes the guide plate 9 in a predetermined position. This is the shaft seal part. 15 more
is an outlet for taking out the culture product of the culture tank 1.

In Fig. 6, 1 is the culture tank, 2 is the conical pyramid-shaped bottom of the culture tank 1, 3 is the lid of the culture tank 1, and 4 is the lid of the culture tank 1.
is a ventilation pipe that supplies air to the culture tank 1,
Air is supplied from an air supply device (not shown) in the same way as in the culture tank shown in FIGS. 1 and 2. 5 is a raw material supply port, 6 is an exhaust pipe, 7 is a filter for the exhaust pipe 6, and 8 is a valve for the ventilation pipe 4. Further, 9 is a ventilation pipe 4 inside the culture tank 1.
, three inverted one-shaped guide plates are attached so as to be able to move up and down by sliding the attachment part 10, and 14 is an inverted one-shaped guide plate 9.
It is a screw that fixes it in an appropriate position.

In the culture tank of the present invention, the horizontal cross-sectional shape of the culture tank 1 may be either circular or quadrangular as shown in FIGS. 4 and 5, but may also be other polygonal shapes. good. The shape of the bottom of the culture tank 1 is shown in Figures 1, 2 and 3.
As shown in the figure, the shape may be either hemispherical or pyramidal, but any shape may be used as long as the culture solution can be stirred by introducing air. Furthermore, the culture product can be taken out by opening the lid 3 of the culture tank 1;
A culture product take-out port 15 is provided at the bottom of the culture tank 1, and the culture product can be taken out from the take-out port 15 without opening the lid 3 of the culture tank 1. Furthermore, the shape of the guide plate 9 may be a flat plate shape as shown in FIGS. 1 and 2, or may be an inverted one-shaped shape as shown in FIG.
The upper surface may be sloped towards the periphery and shaped to allow material falling onto the upper surface to fall through the peripheral gap into the culture volume below. Guide plate 9
When the shape of the culture tank is made into an inverted one-shape as shown in FIG. 3, an air layer 16 as shown in FIGS. However, since air is supplied from this air layer 16 to the culture volume at the bottom 2 of the culture tank 1, even if the cell tissue has a high oxygen demand, it can be cultured in a liquid medium without destroying the cells. This gives rise to advantages that can be achieved. Furthermore, in order to move and fix the guide plate 9 in place, it is possible not only to fasten it to the ventilation pipe 4 with screws 14, but also to attach the connecting rod 12 to the guide plate 9, as shown in FIG. It is also possible to install a lifting device 11 on the lid 3 of the culture tank 1 for lifting and fixing the guide plate 9 using the connecting rod 12. in this case,
Preferably, the shaft sealing portion 13 of the ventilation pipe 4 and the connecting rod 12 is attached.

When culturing cells using the culture tank of the present invention, the lid 3 of the culture tank 1 is opened, and culture solution and seed cells that have been sterilized in advance are charged into the culture tank 1. From the amount of seed cells to be used and the culture volume range experimentally determined in advance, the culture volume of the culture tank required for the smooth growth of the seed cells is calculated, and the guide plate 9 is set so that this culture volume can be obtained. After calculating the position of and fixing the guide plate 9 at that position in the ventilation pipe 4, the lid 3 of the culture tank 1 is closed, and air is blown into the culture tank 1 from the ventilation pipe 4, thereby blowing into the culture solution. Start culturing by supplying air and stirring the culture solution. When the guide plate 9 can be moved and fixed by the lifting device 11 on the lid 3 of the culture tank 1, as in the case of the culture tank 1 shown in FIGS. 1 and 3, the lid 3 of the culture tank 1 Charge the culture solution and seed cells into the culture tank 1 from the raw material supply port 5 without opening the tank, and if necessary, shake the culture tank 1 little by little to transfer all the loaded seed cells under the guide plate 9. Falling into
After moving the guide plate 9 to a predetermined position and fixing it, air can be introduced into the culture tank 1 from the ventilation pipe 4 to start culturing in the same manner. One to two days after starting the culture, the cells will proliferate and the amount of cells will exceed the culture volume range, increasing the culture volume required for the smooth growth of the cells. The position of the guide plate 9 is calculated so that the position of the guide plate 9 is obtained, and the guide plate 9 is pulled up and fixed to that position. Then, air is introduced into the culture tank 1 from the ventilation pipe 4 to start culturing. In the case of the culture tank shown in Fig. 2, the guide plate 9 is moved and fixed by opening the lid 3 of the culture tank 1, and then the lid 3 of the culture tank 1 is closed to restart the culture. In the case of a device such as the culture tank shown in FIG. 3, in which the guide plate 9 can be moved and fixed outside the culture tank 1, the guide plate 9 can be moved and fixed by opening the lid 3 of the culture tank 1. The culture can be restarted immediately.
After another 1 to 2 days, when the cells have proliferated, the above-mentioned lifting and fixing of the guide plate 9 and resumption of culture by introducing air into the culture tank 1 are repeated, and then the guide plate 9 is pulled up and fixed as the cells proliferate. The culture is started by fixing and introducing air into the culture tank 1, and the culture volume increases due to cell proliferation.
Repeat until the volume limit is reached to obtain proliferated cells. In cases where there is no outlet 15 for taking out the culture product, such as in the culture tank shown in FIGS. 1 and 2, the lid 3 of the culture tank 1 is opened to take out the grown cells, but as in the case of the culture tank shown in FIG. If there is a culture product outlet 15, this outlet 1
Take out the cells grown from 5 together with the culture medium,
Obtain the proliferated cells.

The culture tank of the present invention and the culture device using the same can not only be used for culturing plant cell tissues, but also suitably used for culturing other multicellular cell tissues, such as animal cell tissues. It can also be used for culturing microorganisms.

〔Effect of the invention〕

Since the culture tank of the present invention can arbitrarily change its culture volume, during the culture of cell tissues, the culture volume can be adjusted to a range necessary for the appropriate growth and proliferation of cell tissues (culture volume range). This allows for efficient cell tissue proliferation.

In addition, the culture tank of the present invention is equipped with an inverted one-shaped guide plate, so when culturing is carried out while performing air lift agitation, an air layer is created in the deep part of the culture tank, and oxygen is transferred from this air layer to the culture tank. Can be supplied deep. In addition, the top surface of this guide plate is sloped toward the periphery, so even if light cell tissue is lifted onto the guide plate outside the culture volume by air lift, the culture volume below the guide plate is It can be dropped into parts.

[Brief explanation of the drawing]

FIG. 1 is a side view showing a cross section of an example of the culture tank of the present invention, FIGS. 2 and 3 are side views showing a cross section of another example with a part cut away, and FIG. 1
FIG. 5 is a plan view of an example of the culture tank of the present invention shown in FIGS.
FIG. 6 is a side view showing a partially cut away cross section of another example of the present invention. Drawing code, 1: Culture tank, 2: Bottom of culture tank 1,
3: Lid of culture tank 1, 4: Ventilation pipe, 5: Raw material supply port, 6: Exhaust pipe, 7: Filter of pipe system of exhaust pipe 6, 8: Valve of pipe system of vent pipe 4, 9: Guide plate, 10: attachment part of guide plate 9, 11: elevating device for guide plate 9, 12: connecting rod between guide plate 9 and elevating device 11, 13: shaft sealing portion, 14: screw for fixing guide plate 9, 15 : Culture product outlet, 16: Air layer.

Claims (1)

[Scope of Claims] 1. In a culture tank that has a ventilation pipe and an exhaust port that open near the bottom of the culture tank and that stirs the culture solution by air introduced from the ventilation pipe, 1. A culture tank, characterized in that a culture tank is equipped with a guide plate that can be moved along with the guide plate, and by moving the guide plate, the culture volume in which cell tissue is cultured can be arbitrarily increased or decreased. 2. The culture tank according to claim 1, wherein the mounting shaft of the guide plate is a ventilation pipe. 3. The device according to claim 1 or 2, characterized in that the guide plate lifting device is mounted on the lid of the culture tank, whereby the guide plate is moved outside the culture tank. Culture tank. 4 In a culture tank that has a ventilation pipe and an exhaust port that open near the bottom of the culture tank and that stirs the culture solution by air introduced from the ventilation pipe, an inverted one that can move along its installation axis. A shaped guide plate is attached, and by moving the guide plate, the culture volume in which cell tissue is cultured can be arbitrarily increased or decreased, and an air layer can be formed deep in the culture medium during culture. Culture tank. 5. The culture tank according to claim 4, wherein the mounting shaft of the inverted one-shaped guide plate is a ventilation pipe. 6. Claim 4, characterized in that the lifting device for the inverted one-shaped guide plate is mounted on the lid of the culture tank, whereby the movement of the inverted one-shaped guide plate is performed outside the culture tank. The culture tank according to item 1 or item 5. 7. The culture tank according to any one of claims 4 to 6, characterized in that two or more inverted one-shaped guide plates are attached to the mounting shaft thereof.