JPH0585156B2 - - Google Patents

Description

Detailed Description of the Invention (Field of Industrial Application) The present invention relates to a cell culture device for growing animal cells (adherent cells) on a double plate using a special method to continuously produce physiologically active substances. It is something.

(Prior art) Conventionally, when culturing adherent cells, there is a large-scale culture device (A) in which a large number of plates are installed in parallel in a container in order to increase the adhesion area of cells. The mold is what can be called a liquid seal type, in which cells are attached to a plate and then the culture medium is circulated while controlling PH and DO (dissolved oxygen).The other type is a type in which something like a record plate is placed in parallel. Arranged in a cylinder,
First, the cell suspension is poured into the plate in a vertical position (the plate is horizontal), the cells are allowed to adhere to the plate, and then the plate is turned on its side and cultured in a rotational manner.

In addition to the above items, (B) Cell culture is carried out in the same way as roller culture using a spiral plastic film placed in a cylinder.Then, it is placed vertically and the culture solution is circulated by blowing in air, etc. There is a format.

Furthermore, (C) has a multi-box structure, and the difference from (A) is that there is a gas phase. The principle of this method is the same as that of Rubin, which is static culture, and there is no need to circulate the culture solution. This is called the multi-tray method.

The next type is (D), which has a plastic bag permeable to oxygen or carbon gas wrapped around it like a fire hose, making it easy to control DO and PH. The culture solution is in the form of a plastic bag that runs inside the hose. The hollow fiber type (E) is used in artificial kidneys, etc., to attach and proliferate cells on the surface of hollow fibers for dialysis, and nutrients are also supplied from the back side of the cell layer. I can do it.

In addition, (F) a glass bead-packed column format that allows cells to easily adhere and proliferate on the surface of filled glass beads and circulates a culture solution with adjusted PH and DO, and (G) a 20-40 rotation column instead of glass beads. There is a microcarrier method in which cells are cultured and grown on the surface of microbeads whose specific gravity is such that they float in a culture solution under gentle agitation of about 1/2 minute. However, microcarrier culture is very difficult for cells that are easily damaged by shear forces and have a low colony forming rate.

(Problems to be Solved by the Invention) As mentioned above, various devices and methods, each with their own characteristics, have been devised for the mass culture of adherent cells, but there are no practical mass production systems for cell products. However, many of them have not been used much yet and have ended up in the experimental stage.

Even now, the roller bottle method is still used for the mass cultivation of this type of cells and the mass production of interferon and vaccines, which is close to a manual industry level.

(Means for Solving the Problems) Therefore, the technical problem of the present invention is to provide a practical cell culture device capable of mass culturing adherent cells, and to solve this technical problem. The technical means of the present invention employs a multi-stage tray culture system.

That is, culture dishes with bottoms sloped downward toward the outer periphery and a raised edge with a chevron-shaped cross section on the outer periphery are provided in multiple stages at regular intervals in a culture tank, and the center part of each culture dish is A sintered metal filter is provided so that the culture medium is supplied from there,
Furthermore, an overflow medium outlet was provided at the bottom of the culture tank.

(Effect of the invention) According to this technical means, a medium is provided in each culture dish from a medium supply port and is supplied through a sintered metal filter, and the medium whose medium components have been consumed by cell proliferation is piled up. It overflows from around the culture dish and is collected. The collected medium can be reused by mixing it with waste liquid or fresh medium, and the bottom of the culture dish is sloped, so when the medium is supplied, the medium accumulated in the culture dish is pushed out from the center and forms a mountain. Although it overflows from the raised edge of the plate, since the area around the plate is mountain-shaped, the overflowing medium scatters around and does not mix into the plate below.

Therefore, the method of the present invention allows continuous culture, and the culture medium is radially exuded and circulated between the cells grown on the carrier through a sintered metal filter without stirring the cells. Therefore, compared to the microcarrier method, damage to cells due to shear force is extremely small, and cell growth can be performed under optimal conditions.

Since the liquid depth of the culture dish is small and the head space is large, gas movement is easy and, unlike the multi-tray system, all steps can be automated.
In addition, high-density culture is possible, and by using a steam sterilizable carrier, a series of production processes can be steam sterilized in-line, thereby minimizing the risk of contamination.

(Example) An example shown in the drawings will be described below. 1st
In the figure, 1 is a culture tank, 2 is a central cylinder erected in the center of the culture tank 1, and 3 is a culture dish attached to this at regular intervals.

The bottom surface 3a of the culture dish is inclined downward toward the outer periphery, and a raised edge 3b having a chevron-shaped cross section is formed on the outer periphery. A sintered metal filter 4 is provided in the opening 2d of the central cylinder 2 facing the culture dish 3.

2a is a culture medium inlet of the central barrel 2, and 2b is a culture medium outlet of the central barrel 2. Reference numeral 5 denotes a lid of the culture tank 1, which has a gas phase inlet 6 and a gas phase outlet 7. 8 is an overflow medium outlet opened at the bottom of the culture tank 1, and 9 is a viewing window of the culture tank 1.

A culture medium is supplied to the culture tank 1 as described above from the culture medium supply port 2a through the sintered metal filter 4 provided on the culture dish 3.

The medium whose medium components have been consumed by cell proliferation overflows around the mountain-shaped culture dish 3 from the ridge 3b, is discharged from the medium outlet 8, and is collected. The recovered medium is reused by mixing it with waste liquid or fresh medium.

The bottom surface 3a of the culture dish 3 is sloped downward, so when the culture medium is supplied, the culture medium accumulated in the culture dish 3 is pushed out from the center as described above, and the raised edge 3b
overflow from. Since the vertical edge 3b is mountain-shaped, overflowing culture medium scatters around and does not mix into the lower plate.

The pore size of the sintered metal filter 4 is 100 μm, and the size of animal cells is 10 to 100 μm, so that they can sufficiently pass through this sintered metal filter.
In addition, the sintered metal filter, culture dish, and apparatus are all made of SUS316, since the elution of metal ions may inhibit cell growth. Generally, SUS316 and SUS316L are used for animal cell devices.

Regarding the culture method using the above-mentioned device, the culture dish 3 is covered with a fine mesh filter that has cell adhesion properties.

Cover with porous ceramic fragments.

The S/V value can be increased by the following methods. Incidentally, S is the surface area and V is the volume of the medium.
In other words, although the S/V value is inferior to that of microcarriers, multi-tray, plastic bag,
It is much superior to spiral bags, glass beads, etc., and can be made to rival hollow eye bars.

For this purpose, a filter having the same size as the sloped portion of the culture dish 3 is stretched in the shape of a disk, and a filter made of the same material is assembled by folding it into pleats on top of it. 10 in FIG. 2 indicates this.

By doing this, the filter surface area becomes
It becomes ^7000cm2 . The surface area of the special filter is approximately 7 times the apparent surface area, and the surface area of a 10cm square is 10x
10×7= ^700cm2 .

Since one disk is 7000cm ² , this is equivalent to eight commonly used roller bottles (850cm ² ). In other words, this device has 5 stages, so 35,000
cm ² (41 roller bottles).

Also, by covering the surface with porous ceramic fragments, a considerable amount of surface area is obtained.

The advantages of this device compared to other devices will be described below.

As a culture device for animal cells, especially adhesive cells,
Various methods have been devised, and some have been put into practical use. Among them, minute beads (100~
Ton-class culturing devices for microcarriers that utilize cell adhesion (200 μm) have been put into practical use.

However, microcarrier culture is very difficult for cells that are easily damaged by shear forces and have a low colony formation rate. In order to reduce the influence of shear force, a system such as the static method in which the cells themselves are fixed to the adhesive surface and in which the medium can be exchanged efficiently is desirable.

In this sense, the multi-tray method in which glass plates are arranged in multiple stages and the hollow fiber method in which cells are attached to hollow fibers have been put into practical use. Although the multi-tray method is a compact and simple method, many steps such as cell inoculation, medium exchange, and product collection are performed manually, making it unsuitable for commercial production. In addition, the hollow eye bar method uses the adhesion surface area per medium volume,
In other words, it has a high S/V value and can be cultured at high density, but currently the hollow fiber itself is expensive, difficult to reuse, and cannot be steam sterilized, making it difficult to commercially produce. This seems to be a hindrance.

However, the main culture device compensates for the shortcomings of the above methods. That is, in this device, the cells adhere to and proliferate on the carriers on each disk, the medium circulates between them, and the medium is radiated out from the sintered metal filter, so the shearing force is extremely small.

Also, unlike the multi-tray method, all steps can be automated.

When circulating culture medium into a multi-stage system, regardless of trays or disks, if the culture medium is supplied to each stage in sequence, the fresh culture medium will consume more of its nutrients at the stage near the inlet, while the nutrients will be consumed at the stage near the outlet. Conversely, the concentration of growth-inhibiting substances such as lactic acid and ammonia produced by the cells themselves increases. As a result, although good growth is observed in the stages near the inlet, growth is inhibited in the stages near the exit.

In this device, the culture medium is evenly distributed to each stage via a sintered metal filter, and moves radially through the fine mesh of the filter, so that growth can be localized between stages and even within a single stage. is hard to occur.

Uniform culture medium supply to each stage using this device is
Although it is possible to adjust the length of the metal filter portion through which the culture medium seeps out, it is possible to improve the method as shown in Figure 5 in order to more evenly control the medium supply rate to each stage. I can do it. Although the S/V is inferior to microcarriers, by loading carriers to which cells can adhere, such as glass, plastic films, especially porous ceramic fragments with a three-dimensional structure, and fibrous films, onto the disk of this device. , multi-trays, plastic bags, spiral films, glass bead columns, etc., and can rival hollow fibers.

In addition, by using carriers that can be steam sterilized, a series of production processes can be steam sterilized in-line.
The risk of contamination is thereby extremely low.

Furthermore, since the liquid depth in the disk of this device is small and the head space is large, gas movement is facilitated, and the lack of oxygen in the culture medium that occurs in the packed column type can be prevented.

Furthermore, although it is difficult to reuse filters, ceramics can be regenerated with acid or alkali or steam sterilized, and can be obtained at a lower cost than hollow fibers and microcarriers. Hollow eye bars and microcarriers cannot be recycled with acid or alkali or steam sterilized, and are expensive.

3 and 4 show a modified embodiment, in which the culture medium is supplied from a culture medium supply pipe 11 arranged corresponding to each culture dish 3, and is supplied to each culture dish 3 via a sintered metal filter 4.

There is an overflow medium outlet 8 at the bottom of the culture tank 1.
There is also a hole 12 for use as a sensor for detecting the amount of medium in each culture dish 3 and for sampling.

Reference numerals 13 and 14 indicate a safety valve and a diaphragm pressure gauge attached to the lid. Also, 9 is a viewing window.

According to the system shown in FIG. 1, since the medium is supplied from below, the amount of medium supplied to each culture dish 3 is not uniform due to the head pressure, and therefore it is necessary to adjust the amount of culture medium in each culture dish 3. However, in the case of the one shown in FIG. 3, this is not necessary because the water is supplied to each culture dish 3 individually through the supply pipe 11.

The one in FIG. 5 is for adjusting the amount of medium supplied to the culture dish as described above, and the degree of opening of the sintered metal filter 4 is adjusted by the degree of shielding of the ring 15.
One end of a lever rod 16 is attached to the fixture 15a of the ring 15, and the other end is pinned 18a to the suspension rod 18 of the lid body 5. The middle of the lever 16 is fixed to a support 17 of the culture dish 3 at a fulcrum 17a.

Therefore, depending on the degree of engagement of the nut 19 of the suspension rod 18, the suspension rod 18 moves up and down, and thereby the lever rod 16
Since one end 18a moves up and down, the other end of the lever 16 moves up and down in the opposite direction, raising and lowering the ring 15 and adjusting the degree of opening of the sintered metal filter 4.

The lever rod 16 and the fixture 15a are connected as follows. That is, the inner hole 20 of the lever rod 16
A spring 21 is interposed in the spring 2.
A slidable pin 22, which is pressed by 1, is pinned to the fixture 15a. The ring 15 is therefore damped by the spring 21.

In the illustrated one, a ring 15 is positioned on a boss 3c of a culture dish 3 attached to a central shaft cylinder 2.

The sintered metal filter 4 within the central barrel 2 is ring-shaped and faces the slit portion 2d of the enlarged diameter portion 2c of the central barrel 2.

Therefore, the internal shape of the ring 15 is also shaped accordingly.

[Brief explanation of drawings]

Fig. 1 is a cross-sectional view of the culture device of the present invention, Fig. 2 is a slope view when a special filter is used in pleated form, and Fig. 3 and 4 are cross-sectional views of the inventive device according to other embodiments. Figure 5 is a cross-sectional view showing the medium supply amount adjustment device, Figures 6 and 7 are a cross-sectional view and front view of the sintered metal filter portion, and Figures 8 and 9 are cross-sectional views of the ring. and a front view, and FIGS. 10 and 11 are a sectional view and a front view of a state in which a ring is attached to a sintered metal filter portion. 1... Culture tank, 2... Central barrel, 3... Culture dish, 4... Sintered metal filter, 5... Lid body,
6, 7... Gas phase inlet/outlet, 8... Overflow medium outlet, 9... Viewing window.

Claims (1)

[Claims] 1 Culture dishes with bottoms sloped downward toward the outer periphery and a raised edge with a chevron-shaped cross section on the outer periphery are arranged in multiple stages in a culture tank at regular intervals, and a baking tray is placed in the center of each culture dish. 1. A cell culturing device, characterized in that it is provided with a crystalline metal filter so that a culture medium is supplied therefrom, and is further provided with an overflow medium outlet at the bottom of the culture tank.