JPH05252933A - Method for supplying oxygen and device for culturing organismic cell by the method - Google Patents

Abstract

PURPOSE:To prevent the clogging of a bulkhead by disposing a flow- straightening stirring blade at a place near to the lower opening of the oxygen- supplying zone bulkhead disposed in a culture tank and further disposing a sliding member-having eccentric member on a stirring shaft. CONSTITUTION:An oxygen-supplying zone bulkhead 2 is disposed in a culture tank 1, and a stirring blade equipped with a flow-straightening plate 6 is disposed at a place near to the lower opening of the bulkhead to produce an extrusion flow. An eccentric member 9 equipped with a sliding member 10 is disposed on the stirring shaft 8, and the change in the pressure is imparted to the oxygen-supplying zone bulkhead. A bubble layer 11 is broken and eliminated with a bubble-breaking layer 12.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for supplying oxygen to cells for in vitro growth of cells in the living body, and a culture apparatus therefor.

[0002]

2. Description of the Related Art Regarding the method of supplying oxygen to cells by a submerged aeration system and a culture apparatus, Chemical Engineering, January 16, 1987, pages 121-129 (Ch.
emicalEngineering, February, 16, 1987, p1
21-129), and Site Technology, 1990 Volume 3, pages 39-42 (Cytotechnology, 3, p39-
42, 1990) and JP-A-63-164879. In addition, when cells are kept in the culture tank, only the used medium is extracted and fresh medium is supplied, that is, cells that are dispersed in the culture medium by a screen having fine pores when performing so-called perfusion culture. For the method of separating liquid and liquid, see Development. Biol 1. Standard, 66, 169-175 pages, 1987 (De
velop. Biol. Standard, 66, p169-175, 19
87). Also, development. Biol. Standard, pages 66, 227-240, 19
1987 (Develop. Biol. Standard, 66, p227-2
40, 1987), vibrating the tube unit for oxygen supply to prevent attachment of cells and the like.

[0003]

Although the submerged aeration method is excellent in oxygen supply capacity, it may affect cell growth or substance production, or a large number of cells may aggregate to form a cell mass, or a micro It may not be applicable when culturing anchorage-dependent cells in a carrier. In the prior art, in order to be applicable to these cultures, a region surrounded by a screen where cells and microcarriers do not exist is provided in the culture tank, and liquid is aerated in the region. In these conventional techniques, the decrease in oxygen supply capacity due to the clogging of the screen that prevents the infiltration of cells and microcarriers is a major problem, and in order to prevent the clogging, rotation and vertical vibration are applied to the screen. However, the effect is not sufficient.

The present invention prevents clogging of partition walls for forming compartments in which cells and microcarriers do not exist,
It is an object of the present invention to provide a method and a culture device for efficiently supplying oxygen by submerged aeration.

[0005]

The present invention provides a method for supplying oxygen to cells in a culture medium by submerged aeration when culturing cells of a living body, wherein submerged aeration is provided in a culture tank. Oxygen supply zone having micropores that are substantially impermeable to aggregates of cultured cells and / or microcarriers and capable of changing position, performed in an oxygen supply zone separated by partition walls, said oxygen supply zone The upper end of the partition wall is opened above the culture liquid surface and the lower end is opened in the culture tank, and the oxygen supply zone partition wall is fixed to the culture tank by an elastic member so that its position can be changed. A means for discharging the solution inside the oxygen supply zone into the culture tank was provided in the vicinity of the lower end opening, a means for causing pressure fluctuation in the oxygen supply zone partition micropores, and a culture solution. And an oxygen supply method which is characterized in that a foam-breaking means above the oxygen supply zone bulkhead upper.

As a means for discharging the solution in the oxygen supply zone into the culture tank, a stirring blade having a straightening plate perpendicular to the rotary shaft is installed near the lower end opening of the partition wall of the oxygen supply zone for discharge. A method of supplying oxygen characterized by generating a flow.

Further, the present invention is characterized in that a rectifying partition wall is provided outside the oxygen supply zone partition wall provided in the culture tank, and a culture liquid circulation portion is provided between both partition walls.

Further, the oxygen supply zone partition wall provided in the culture tank has a mechanism for changing the pore diameter.

The present invention, in a device for culturing cells of a living body, uses a submerged aeration method in which an oxygen-containing gas is directly aerated in the liquid as a means for supplying oxygen to the cells in the culture liquid, and Aeration in the liquid is carried out in the oxygen supply zone separated by the oxygen supply zone partition having micropores that do not substantially permeate aggregates and / or microcarriers. Part is opened in the culture solution, and the oxygen supply zone partition wall is fixed to the culture tank by an elastic member so that its position can be changed, and oxygen is supplied near the opening of the lower end of the oxygen supply zone. A device for culturing cells of a living body, characterized by having a stirring blade as a means for discharging the solution in the zone into the culture tank and having a foam breaking means on the surface of the culture liquid. A.

Outside the oxygen supply zone partition provided in the culture tank, a rectifying partition having an upper end opening below the liquid surface and a lower end opening above the stirring blade is provided. It is an apparatus for culturing cells, which is characterized in that a circulation portion of a culture solution is provided therebetween.

[0012] Further, the apparatus for culturing cells is characterized in that the partition for distinguishing the oxygen supply zone provided in the culture tank has a mechanism for changing the pore diameter.

Further, an eccentric member eccentric from the shaft center is installed on a part of a shaft for driving a stirring blade provided in the culture tank, and slides following the rotation of the eccentric member. An apparatus for culturing cells characterized in that a sliding member is installed and the sliding member is connected to an oxygen supply zone partition so that an external force is applied to the partition by rotation of a stirring shaft to change its position. Is.

[0013]

The method of supplying oxygen to the cells in the culture solution by the submerged aeration method of the present invention is a method of forming agglomerates of cultured cells and / or micropores that are substantially impermeable to microcarriers, provided in the culture tank. The submerged aeration is performed in the oxygen supply zone which is divided by the oxygen supply zone partition wall which has a variable position. Oxygen supply zone partition wall The upper end is opened above the culture solution surface and the lower end is opened inside the culture tank, and the oxygen supply zone partition wall is fixed to the culture tank by an elastic member and its position can be changed. It is in. In the vicinity of the opening at the lower end of the partition wall, a stirring blade is installed as a means for discharging the solution inside the oxygen supply zone into the culture tank to generate a discharge flow. Further, by providing a rectifying partition wall outside the oxygen supply zone partition wall and providing a culture solution flow portion between both partition walls, the flow of the culture solution in the vicinity of the oxygen supply zone partition wall is efficiently generated. As a result, it is possible to effectively prevent clogging of the oxygen supply zone partition wall into the fine holes.
Furthermore, by the mechanism that vibrates the oxygen supply zone partition,
By causing a pressure fluctuation in the oxygen supply zone partition wall fine holes, it is possible to further effectively prevent clogging of the fine holes. Further, it has a mechanism for changing the pore diameter as a means for eliminating the substance in the fine pores that have been clogged. When a foam layer is formed by submerged aeration, foam breaking means is provided above the culture solution surface and above the upper end of the oxygen supply zone partition wall.

The biological cell culturing apparatus of the present invention comprises a culture tank,
A fresh medium storage tank as a medium supply system, a culture solution storage tank, an oxygen source, a carbon dioxide gas source, a nitrogen source, an air source as an oxygen supply system, and a sterilization filter for maintaining a sterile state of the culture system,
And a computer for monitoring and controlling the culture condition. The culture tank uses an in-liquid aeration method in which an oxygen-containing gas is directly aerated in the liquid as a means for supplying oxygen to the cells in the culture liquid, and an oxygen supply zone having fine pores that does not substantially permeate the cultured cells. Aeration is submerged in the oxygen supply zone separated by the partition. Oxygen supply zone partition wall The upper end is opened above the culture solution surface and the lower end is opened in the culture solution, and the oxygen supply zone partition wall is fixed to the culture tank by an elastic member and its position can be changed. .. A stirring blade is provided in the vicinity of the lower end opening of the oxygen supply zone as a means for discharging the solution inside the oxygen supply zone into the culture tank. Outside the oxygen supply zone partition, a rectifying partition with an upper end opening below the liquid surface and a lower end opening near the agitating blade in the upper part was provided, and a culture fluid flow section was provided between both partition walls. There is. An eccentric member that is eccentric from the shaft center is installed on a part of the shaft for driving the stirring blade, and a sliding member that slides following the rotation of the eccentric member is installed. By combining with the supply zone partition,
An external force is applied to the partition wall by the rotation of the stirring shaft to change its position. The partition for distinguishing the oxygen supply zone has a mechanism for changing the pore size. A foam breaking means is provided on the culture liquid surface.

[0015]

EXAMPLES Examples of the present invention will be described in detail below.

Example 1 FIG. 1 is a diagram showing an example of the method for supplying oxygen to cells in a culture medium according to the present invention. An oxygen supply zone partition wall 2 is provided in the culture tank 1. The oxygen supply zone partition wall 2 has micropores that are substantially impermeable to aggregates of cultured cells and / or microcarriers. The oxygen supply zone partition has an upper end opened above the culture solution surface and a lower end opened into the culture tank, and the oxygen supply zone partition is fixed to the culture tank by a support 4 using an elastic member 3 as a part thereof. , It has a structure that can change its position. Oxygen is supplied to the culture solution directly from the nozzle 14 through the gas supply pipe 20 through the pipe line 13 provided on the stirring shaft 8 in the oxygen supply zone separated by the oxygen supply zone partition wall. By doing. In the vicinity of the lower end opening of the partition wall, a stirring blade 5 is installed as a means for discharging the liquid inside the oxygen supply zone into the culture tank, and a discharge flow is generated. The stirring blade 5 is provided with a straightening plate 6,
The liquid inside the oxygen supply zone is efficiently discharged into the culture tank, and the invasion of cell aggregates and / or microcarriers into the oxygen supply zone is prevented. Further, a rectifying partition wall 7 is provided outside the oxygen supply zone partition wall, and a culture solution flow portion is provided between both partition walls to efficiently cause the flow of the culture solution near the oxygen supply zone partition wall. As a result, it is possible to effectively prevent clogging of the oxygen supply zone partition wall 2 into the fine holes. Further, an eccentric member 9 which is eccentric from the shaft center is installed on a part of the stirring shaft 8, and a sliding member 10 that slides following the rotation of the eccentric member 9 is installed. By combining with the oxygen supply zone partition wall 2, vibrating the oxygen supply zone partition wall 2 by rotating the stirring shaft, and applying pressure fluctuation to the oxygen supply zone partition wall micropores, the clogging into the micropores is further effective. Can be prevented. When the foam layer 11 is formed on the surface of the culture solution by submerged aeration, the foam breaking layer 12 is provided on the surface of the culture solution and above the upper end of the partition wall of the oxygen supply zone. For the foam breaking layer 12, a stainless wire mesh (about 10 mesh) whose surface is hydrophobized with polysiloxane is used. The foam formed on the liquid surface bursts and disappears when it comes into contact with the foam-breaking layer, and returns to the culture solution as droplets. The aerated gas is discharged from the culture tank through the exhaust pipe 15.

<Embodiment 2> FIG. 2 shows another embodiment of the present invention. The feature of this embodiment is that the drive shaft for stirring blades in the culture tank and the vibrating shaft for vibrating the oxygen supply zone partition wall 2 are separated from each other in the first embodiment. That is, the stirring blades for stirring in the tank and discharging the liquid in the oxygen supply zone were driven by the stirring shaft 8 provided at the bottom of the culture tank, and the vibration of the oxygen supply zone partition wall 2 was provided at the top of the culture tank. It is performed by the vibrating shaft 16.

According to this embodiment, the stirring strength of the culture solution,
The vibration of the partition walls of the oxygen supply zone can be adjusted separately, and the culture conditions can be set according to changes in the cell concentration during culture and the clogging of the micropores of the partition walls of the oxygen supply zone.

<Third Embodiment> FIG. 3 shows another embodiment of the present invention. The feature of this embodiment is that in the second embodiment, the oxygen supply zone partition wall 2 is vibrated by the stirring shaft 8 provided at the bottom of the culture tank. That is, an eccentric member 9 which is eccentric from the shaft center is installed on the stirring shaft 8, and a sliding member 10 which slides following the rotation of the eccentric member 9 is installed. The oxygen supply zone partition wall 2 is vibrated by being combined with the partition wall 2 and rotating the stirring shaft.

According to this embodiment, the distance between the bearing portion of the stirring shaft and the eccentric member for exciting the oxygen supply zone partition wall can be reduced, and as a result, the stirring shaft and The stress applied to the bearing is reduced, and a simpler structure can be obtained.

<Embodiment 4> FIG. 4 is a schematic view of a culture apparatus using the oxygen supply method according to the present invention.

The apparatus for culturing cells of a living body according to the present invention comprises a stainless steel culture tank 1, a stirring blade driving motor 31, a fresh medium storage tank 33 as a medium supply system, so as to enable heat sterilization of the inside. Culture solution storage tank 32, oxygen source 34, carbon dioxide gas source 35, nitrogen source 36, air source 37 as an oxygen supply system, sterilization filter 38 for maintaining the sterility of the culture system, and the culture condition are monitored. In addition, it is composed of a computer 50 for controlling.

The oxygen supply method of the present invention is used in the culture tank 1. That is, an oxygen supply zone partition wall 2 having micropores that do not substantially permeate aggregates of cultured cells and / or microcarriers is provided, and the partition wall upper end is above the culture liquid surface and the lower end portion is opened in the culture tank. There is. The oxygen is supplied by supplying the oxygen-containing gas from the gas supply pipe 20 into the oxygen supply zone, which is distinguished by the oxygen supply zone partition wall.
It is carried out by directly ventilating the culture solution through the conduit provided in. In the vicinity of the opening at the lower end of the partition wall, a stirring blade 5 is provided as a means for discharging the liquid inside the oxygen supply zone into the culture tank.
To generate a discharge flow. The stirring blade 5 is provided with a straightening plate 6 to efficiently discharge the liquid inside the oxygen supply zone into the culture tank and prevent the infiltration of cell aggregates and / or microcarriers into the oxygen supply zone. To do. The rectifying partition 7 is provided outside the oxygen supply zone partition.
Is provided and the flow portion of the culture solution is provided between both partition walls, so that the flow of the culture solution in the vicinity of the oxygen supply zone partition wall is efficiently generated. An eccentric member that is eccentric from the shaft center on a part of the stirring shaft 8, a sliding member that follows the rotation of the eccentric member, and a sliding member are connected to the oxygen supply zone partition wall 2 and the oxygen is generated by rotating the stirring shaft. By vibrating the supply zone partition walls 2 and applying pressure fluctuations to the oxygen supply zone partition wall fine holes, clogging of the fine holes is prevented. When the foam 11 is formed on the surface of the culture solution by aeration in the liquid, the foam breaking layer 12 is provided on the surface of the culture solution and above the upper end of the partition wall of the oxygen supply zone.
For the foam-breaking layer 12, a stainless wire mesh (about 10 mesh) whose surface was made hydrophobic with polysiloxane was used. The foam formed on the liquid surface bursts and disappears when it comes into contact with the foam-breaking layer, and returns to the culture solution as droplets. The aerated gas is discharged to the outside of the culture tank through the sterilization filter 38.

Living cells cultured in the present culture device are animal cells, microorganisms, plant cells and the like. Particularly, a culture solution in which particles of about 100 to 1000 μm are suspended in the culture solution is suitable.

First, the culture tank 1, the fresh medium storage tank 33, the culture solution storage tank 32, and the inside of the pipe are sterilized by high-pressure steam in advance. Then, a medium in which the nutrient components necessary for the growth of living cells for culture are dissolved is prepared, sterilized, and stored in a fresh medium storage tank. Then, a part thereof is transferred into the culture tank 1, and seed cells are aseptically inoculated into the culture tank 1 to start the culture. The culture is performed by warming the culture solution through warm water through a jacket (not shown) provided outside the culture tank. The culture temperature is controlled to the optimum temperature of the cells to be cultured.
The physicochemical properties of the culture solution change due to cell growth, causing depletion of nutrients and accumulation of waste components such as lactic acid. Therefore, the temperature sensor 39 and the pH sensor 4 are provided in the culture tank 1.
0, DO sensor 41, and DCO ₂ sensor 42 are installed, and temperature, pH, DO (dissolved oxygen concentration), and DCO ₂ (dissolved carbon dioxide concentration) are measured. These measurement signals are transmitted to the computer 50, and the culture status is determined.
In addition, if necessary, a part of the culture solution may be extracted to obtain turbidity of the solution, lactate concentration, glucose concentration, cell concentration, and shape,
Image information of cells such as size and brightness is input to a computer (not shown). The computer 50 infers and judges the culture state at that time based on these information and the past culture information accumulated therein, and commands to adjust the gas ventilation amount, the oxygen concentration in the ventilation gas, and the carbon dioxide concentration. Is given to each control valve. Further, in response to the depletion of nutrients in the culture solution and the accumulation of lactic acid, each pump is instructed to supply the fresh medium and withdraw the used culture solution to perfuse the medium. In addition,
When extracting the used culture solution, it is desirable to separate the solid matter floating in the culture solution.

With the main culturing device, oxygen can be supplied by submerged aeration with a required amount of aeration without concern about the occurrence of foam even when the culture contains solid matter such as cell aggregates and microcarriers. Therefore, high density culture of living cells becomes possible.

[0027]

EFFECTS OF THE INVENTION According to the present invention, oxygen can be supplied by submerged aeration even in a culture containing solid matters such as cell aggregates and microcarriers in the culture liquid, and the required amount can be obtained without fear of foam formation. Since it can be aerated with the aeration amount of, a high-density culture becomes possible.

[Brief description of drawings]

FIG. 1 is a cross-sectional view of one embodiment using the oxygen supply method of the present invention.

FIG. 2 is a sectional view of one embodiment using the oxygen supply method of the present invention.

FIG. 3 is a cross-sectional view of one embodiment using the oxygen supply method of the present invention.

FIG. 4 is a system diagram of one example of a culture device using the oxygen supply method of the present invention.

[Explanation of symbols]

1 ... Culture tank, 2 ... Oxygen supply zone partition wall, 5 ... Stirring blade, 6
... rectifying plate, 7 ... rectifying partition wall, 8 ... stirring shaft, 9 ... eccentric member,
10 ... Sliding member, 12 ... Foam breaking layer, 14 ... Nozzle.

Claims (8)

[Claims] 1. In culturing cells of a living body, in a method of supplying oxygen to cells in a culture solution by a submerged aeration method, submerged aeration is provided in a culture tank, and aggregates of cultured cells and / or Alternatively, it has micropores that are substantially impermeable to microcarriers, and is carried out in the oxygen supply zone distinguished by the partition walls of the oxygen supply zone whose position can be changed. The upper end of the partition wall of the oxygen supply zone is the culture medium. The lower end above the surface is opened in the culture tank, the oxygen supply zone partition wall is fixed to the culture tank by a member having elasticity and its position can be changed, and the lower end opening of the partition wall A means for discharging the solution inside the oxygen supply zone into the culture tank in the vicinity of the section, and a means for causing a pressure fluctuation in the micropores of the oxygen supply zone partition wall, Oxygen supply method, wherein a serial provided foam-breaking means above the partition wall upper end of the culture liquid surface a and the oxygen supply zone. 2. The means for discharging the solution inside the oxygen supply zone to the culture section in the culture tank according to claim 1, wherein the oxygen supply zone is orthogonal to the rotary shaft near the opening at the lower end of the partition wall of the oxygen supply zone. A method of supplying oxygen to generate a discharge flow by installing a stirring blade having a current plate. 3. The oxygen supply method according to claim 1, wherein a rectifying partition is provided outside the oxygen supply zone partition provided in the culture tank, and a portion for circulating the culture solution is provided between the partition walls. 4. The oxygen supply method according to claim 1, 2, or 3, wherein the oxygen supply zone partition wall provided in the culture tank has a mechanism for changing the pore diameter. 5. A device for culturing living cells, comprising a culture tank, a fresh medium storage tank as a medium supply system, a culture solution storage tank,
An oxygen source, a carbon dioxide gas source, a nitrogen source, an air source as an oxygen supply system, and a sterilization filter for maintaining the sterility of the culture system, and a computer for monitoring and controlling the culture condition, An in-liquid aeration method in which an oxygen-containing gas is directly aerated is used as a means for supplying oxygen to cells in the liquid, and it has fine pores that do not substantially permeate aggregates of cultured cells and / or microcarriers. Aeration in the liquid is carried out in the oxygen supply zone separated by the oxygen supply zone partition, the upper end of the oxygen supply zone partition is opened above the culture solution surface, and the lower end is opened in the culture solution, the oxygen supply zone The partition wall is fixed to the culture tank by an elastic member and its position can be changed, and the solution inside the oxygen supply zone is placed in the vicinity of the lower end opening of the oxygen supply zone. Having a stirring blade as a means for discharging the culture tank, a culture apparatus for biological cells, characterized by having a foam-breaking unit on the culture surface. 6. The method according to claim 5, wherein an upper end is opened below the liquid surface and a lower end is opened above the stirring blade outside the oxygen supply zone partition wall provided in the culture tank. An apparatus for culturing living cells, in which a rectifying partition is provided, and a portion where the culture solution flows is provided between both partitions. 7. The apparatus for culturing living cells according to claim 5, wherein the partition wall for distinguishing the oxygen supply zone provided in the culture tank has a mechanism for changing the pore diameter. 8. The eccentric member eccentric to the shaft center is installed on a part of a shaft for driving the stirring blade provided in the culture tank according to claim 5, 6 or 7. By installing a sliding member that slides following the rotation of the core member and connecting the sliding member with the oxygen supply zone partition wall, an external force is applied to the partition wall by the rotation of the stirring shaft and its position A device for culturing living cells that changes temperature.