JPS62118878A - Cell culture unit - Google Patents

Abstract

PURPOSE:Cultivation dishes of a specific shape is set in a culture tank in multiple stages with a certain interval and each dish is equipped with a sintered metal filter at its center to supply the culture medium therefrom whereby mass cultivation of adhesive cells is possible. CONSTITUTION:Cutivation dishes 3 which have the bottom 3a inclining toward the periphery and the cross section of reversed V-shape at the periphery, are set in multistages in the tank 1. Individual dishes 3 is equipped with a sintered metal filter 4 at the center to feed the culture medium therefrom. Further, an overflow outlet 8 is formed at the bottom of the tank. In other words, the medium is fed from inlet 2a, passed through filters into each dish. The medium whose nutrients have been consumed by proliferation of the cells is allowed to overflow from the periphery of the dishes for recovery. The recovered medi um can be utilized again, by mixing with waste or the make-up medium. Thus, mass cultivation of adhesive cells becomes possible.

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 culture dish using a special method and continuously producing physiologically active substances. It is.

(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. After attaching cells to the plate using what can be called a sealing method, p
One method is to circulate the culture medium while controlling H10 (dissolved oxygen).The other method is to arrange record boards in parallel in a cylinder and stand them vertically (the boards will be horizontal).
In this format, a cell suspension is poured into the plate, the cells are allowed to adhere to the plate, and then the cells are placed horizontally and cultured in a rotational manner.

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

Furthermore, (C) has a multi-box structure, which differs from (A) in that a gas phase is present. The principle of this method is the same as for lupine, which is static culture, and there is no need to circulate the culture solution. This is called a multi-train one-way system.

The next type (D) is a type in which a plastic bag permeable to oxygen or carbon dioxide gas is wrapped around it like a fire hose, making it easy to control DO and PN. The culture medium is in the form of a plastic bag that runs inside the hose. Hollow fiber type (E) is used in artificial kidneys, etc., to attach and grow 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 pea-packed column type that allows cells to adhere and grow on the surface of glass peas filled in a container and circulates a culture solution with adjusted pH and DO, and (G) a 20 to 40 rotation column instead of glass peas. There is a microcarrier method in which cells are cultured and grown on the surface of microbeads with a specific gravity that 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 have been devised for mass culturing adherent cells, but there are no practical cell products. It has not yet been used much as a mass-produced system, and many 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. The technical means of the invention is to adopt a multi-stage tray culture system.

That is, culture dishes whose bottom surfaces are inclined downward toward the outer periphery and which have a raised edge with a chevron-shaped cross section on the outer periphery are provided in multiple stages in a culture tank at regular intervals, and in the center of each culture dish, A sintered metal filter is provided from which the culture medium is supplied, and an overflow medium outlet is provided at the bottom of the culture tank.

(Effect of the invention) According to this technical means, the medium is provided in each culture dish from the medium supply port and is supplied through the sintered metal filter.
The medium whose medium components have been consumed by cell proliferation 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, forming a mountain-like shape. Although the culture medium overflows from the vertical edge of the plate, since the area around the plate is ridged, the overflowing culture medium scatters around the plate and does not contaminate the lower plate.

Therefore, the present invention allows continuous culture, and the culture medium is circulated 71 radially 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 culture dish has a small liquid depth and a large head space, gas movement is easy to perform, and unlike the multi-tray one-type system, all steps can be automated. In addition, high-density culture is possible,
The use of steam sterilizable carriers allows the production process to be steam sterilized in-line, thereby minimizing the risk of contamination.

(Example) The embodiments shown in the drawings will be described below.

In Fig. 1, (1) is a culture tank, (2) is a central shaft cylinder installed in the center of the culture tank (1), and (3) is a central cylinder installed at regular intervals on this cylinder. It is a culture dish.

The bottom surface (3a) of the culture dish faces downward toward the outer periphery (Ll
1, and a raised edge (3b) with a chevron-shaped cross section is formed on the outer periphery. A sintered metal filter (4) is located in the opening (2d) of the central barrel (2) facing the culture dish (3).

(2a) is the culture medium inlet of the central barrel (2), (2b)
is the medium outlet of the central barrel (2).

(5) is the lid of the culture tank (1), which has a gas phase inlet (6) and a gas phase outlet (7). (8) is the overflow medium outlet opened at the bottom of the culture tank (1), and (9) is the overflow medium outlet opened at the bottom of the culture tank (1).
).

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

The medium whose medium components have been consumed by the proliferation of cells overflows from the raised edge (3b) around the culture dish (3), which is shaped like a hill, and is discharged and collected from the medium outlet (8).

The recovered medium is reused by mixing it with waste liquid or fresh medium.

The bottom surface (3a) of the culture dish (3) is tilted 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 overflows from the erected edge (3b). do.

Since the standing edge (3b) is curved, the overflowing medium will scatter around and will 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 elution of metal ions may inhibit cell growth, so all sintered metal filters, culture dishes, and equipment must be 5US316.
Manufactured by Generally, animal cell devices are 5US316.5OS
316L is used.

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

■Laying porous ceramic fragments.

The S/V value can be increased by the following methods. Incidentally, S is the surface area, and ■ is the amount of medium. That is, S
Although the /V value is inferior to that of microcarriers, it greatly exceeds multi-trays, plastic bags, spiral bags, glass beads, etc., and is comparable to hollow fibers.

Regarding (2), a filter having the same size as the slope part of the culture dish (3) is stretched in the shape of a disk, and a filter made of the same material is assembled on top of it by folding it into pleats. Second
(10) in the figure shows this.

By doing this, the filter surface area is 7000
It becomes cJ. The surface area of the special filter is approximately 7 times the apparent surface area, and the surface area of 10cm square is 10XIOX
7 = 700cnl.

One disc has 7,000cni, which is equivalent to eight normally used roller bottles (850cni).In other words, this device has 5 stages, so it costs 35,000cni.
n ((41 bottles of o-rahotl) tonal.

As for (2), a considerable amount of surface area is obtained by laying porous ceramic fragments.

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

Various apparatuses for culturing animal cells, particularly adhesive cells, have been devised, and some have been put into practical use. Among these, a ton-class culturing device for microcarriers that utilizes the adhesion of cells to minute beads (100 to 200 μm) has 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 effects of shearing forces, it is desirable to use a system such as the standing method in which the cells themselves are fixed on the adhesive surface and in which the medium can be exchanged in a single manner.

In this sense, the multi-tray method using multiple tiers of glass plates and the hollow fiber method where 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 fiber method has a high adhesion surface area per medium volume, that is, a high S/V value, and allows for high-density culture, but currently the hollow fiber itself is expensive.
Since it is difficult to reuse and cannot be steam sterilized, it is thought to be a major obstacle to commercial production.

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 radially exuded from the sintered metal filter, so the shearing force is extremely small.

Also, unlike the multi-train system, all steps can be automated.

When supplying culture medium to a multi-stage system, regardless of tray or disk, if the culture medium is supplied to each stage sequentially, the fresh culture medium will consume more of its nutrients at the stage near the inlet, and the nutrients will be consumed at the stage near the outlet. In contrast, the concentration of growth-inhibiting substances such as lactic acid and ammonia produced by the cells themselves increases in the stages. As a result, although good growth was observed in the stages near the entrance,
At the stage near the exit, growth will be inhibited.

In this device, the culture medium is evenly distributed to each stage through a sintered metal filter, and the medium is passed through the fine holes of the filter.
Because it moves radially, localized proliferation is unlikely to occur in the groin or even in one stage.

Although it is possible to uniformly supply the medium to each stage using this device by adjusting the length of the part of the metal filter through which the medium seeps out, it is possible to control the medium supply speed to each stage more evenly. Therefore, it is possible to improve the method as shown in FIG.

Although 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. It greatly exceeds multi-trays, plastic bags, spiral films, glass beads columns, etc., and is comparable to hollow fibers.

Furthermore, the use of steam sterilizable carriers allows the production process to be steam sterilized in-line, thereby minimizing the risk of contamination.

Furthermore, since the liquid depth in the disk of this device is small and the head space is large, gas movement is easy (
This prevents the lack of oxygen in the culture medium that occurs in packed column formats.

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 fibers and microcarriers cannot be regenerated with acid or alkali or steam sterilized, and are expensive.

Figure 3.4 shows a modified embodiment in which the medium is supplied from a medium supply pipe (11) arranged corresponding to each culture dish (3), and is passed through a sintered metal filter (4) to each culture dish (3). ).

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

(13) (14) shows the safety valve and diaphragm pressure gauge attached to the lid. Also, (9) is a viewing window.

According to the system shown in Figure 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, according to the one in Figure 3, each culture dish (
3) It is not necessary because it is supplied to IrfJ 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). Ring (15
One end of a lever rod (16) is attached to the fixture (15a) of ), and the other end is pinned (18a) to the suspension rod (18) of the lid (5). The middle of the lever rod (16) is attached to the support (17) of the culture dish (3) as a fulcrum (17a).
) has been stopped.

Therefore, the wide suspension rod (18) moves up and down depending on the degree of engagement of the suspension rod (18) (naso) (19), and this causes the lever rod (1
6) One end (18a) is a lever rod (16) for lifting and lowering.
The other end moves up and down in the opposite direction, raising and lowering the ring (15) to adjust the opening degree of the sintered metal filter (4).

The lever rod (16) and the fixture (15a) are connected as follows. In other words, the inner hole (2) of the lever rod (16)
0) is interposed with a spring (21), and a slidable pin (22) pressed by the spring (21) is pinned to the fixture (15a). The ring (15) is therefore damped by the spring (21).

The one shown is a culture dish (3) attached to a solid barrel (2).
) A ring (15) is located on the boss (3c).

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

Therefore, the internal shape of the ring (15) also has a shape corresponding to this.

[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 the form of pleats. Fig. 3.4 is a cross-sectional view of the inventive device according to another embodiment. Plan view Figure 5 is a cross-sectional view showing the medium supply amount adjustment device Box 6.7 is a cross-sectional view and front view of a portion of the sintered metal filter Figure 8.9 is a cross-sectional view and front view of the ring Figure 11 is a cross-sectional view and a front view of the sintered metal filter with a ring attached to a portion thereof. (1)...Culture tank (2)...Solid barrel (3)...Culture dish (4)...Sintered metal filter (5)...Lid ( 6) (7) ・Gas phase inlet/outlet (8)... Overflow medium outlet (9)...
Viewing window Figure 1, Figure 2, Figure 4, Figure 6, Figure 8

Claims (1)

[Claims] 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 each culture dish has a sintered plate in the center. 1. A cell culture device comprising a metal filter from which a medium is supplied, and an overflow medium outlet provided at the bottom of the culture tank.