JPH0775549A - Living cell culture device - Google Patents

Abstract

(57) [Summary] [Structure] A screen 7 is installed in the culture tank 1, a submerged aeration pipe 2 and a circulation pipe 3 in which an aeration sparger 14 is installed form a closed circulation system, and a shaker 4 is provided. A stirring shaft 6 to which a vibrating shaft 5 and a stirring blade 8 are attached is provided. An oxygen-containing gas is aerated from the aeration sparger 14 to agitate the culture solution in the culture tank by air lift. Activate the shaker 4,
The vibrating shaft 5 and the stirring shaft 6 are vibrated to stir the culture solution in the vicinity of the screen 7 and the stirring blade 8 to perform finer and uniform stirring. [Effect] Even for a culture solution containing solid particles such as microcarriers, efficient oxygen supply and air lift agitation by submerged aeration can be performed, and the agitation by vibration can be used to finely and uniformly agitate the culture solution for long-term living cells. High density culture is possible.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a culture device for living cells, and more particularly to a culture device using a culture carrier such as a microcarrier.

[0002]

2. Description of the Related Art The stirring method used for microcarrier culture is described in Microcarrier Cell Culture.
“The Principles and Mens” (Microcarrie
r Cell Culture-the principles & methods-) (P3
5 to P37, technical data collection for microcarrier culture method, published by Pharmacia Fine Chemical Co., Ltd.). For other agitation methods and airlift agitation in cell culture, refer to "World of bioreactors".
(P167 to P200, 1992, published by Hario Research Institute Shibata Co., Ltd.).

[0003]

The air lift method is a method of flowing / stirring a culture solution with a driving force of gas-permeable bubbles, and an internal circulation air lift method in which a gas is directly aerated in the culture tank and outside the culture tank. It is roughly classified into an external circulation type in which gas is aerated and liquid is circulated. The air lift method is
It is an excellent stirring method that requires less power and can be applied to large equipment. However, in the air lift method, the flow state of the culture solution in the culture tank can only be roughly controlled, and there is a case where a stagnant flow or a partial flow occurs and the mixed state of the culture solution becomes uneven. This phenomenon becomes a problem especially in the case of a culture medium containing solid particles much larger than cells such as microcarriers (diameter 100 to 1000 μm).

An object of the present invention is to provide a cell culture device for a living body, which utilizes the advantages of the air-lift method, uniformly mixes culture solutions, and carries out high-density culture using a culture carrier such as a microcarrier. It is in.

[0005]

Means used for achieving the above object will be described in detail with reference to FIG. 1 as an example. In the apparatus for culturing living cells according to the present invention, first, the culture tank 1
A screen 7 having a pore size that allows microcarriers to pass but liquid components to pass therethrough is installed therein. The screen 7 prevents the microcarriers from leaking into the submerged aeration pipe 2 and enables air lift stirring of the culture liquid in the culture tank 1. As shown in FIG. 1, it is more desirable that two screens 7 are installed in the culture tank 1 one above the other, and the microcarriers are completely confined in the space between the two screens 7. By injecting gas from the sparger 14 installed in the lower part of the submerged aeration pipe 2 to reduce the density of the liquid in the pipe, the liquid rises in the pipe, and then the circulation pipe 3 descends. A circulation flow is generated which flows from the lower part of the culture tank 1 toward the upper part and returns to the submerged aeration pipe 2. In this way, the circulating flow of the liquid generated by aeration, that is, the flow of the liquid generated by the air lift action by aeration mixes / stirs the culture liquid in the culture tank 1. Japanese Patent Application No. Hei 5 (1999) -9698 discloses a device for culturing living cells capable of achieving high-density culturing of living cells by performing such air-lift stirring.
-60581 has already been filed. by the way,
It was found that when the concentration of microcarriers was increased to increase the culture density, the microcarriers were unevenly dispersed in the culture solution. Then, we found a method of attaching a stirrer that vibrates up and down as a method of solving this problem by further experiments. The stirrer includes a stirring shaft 6 and a stirring blade 8 attached to the stirring shaft, and is connected to the vibration shaft 5 of the screen 7. Further, the vibrating shaft 5 is connected to the vibrator 4 installed outside the culture tank. When the shaker 4 is operated, the mixed flow shown in FIG. 2 is generated in the vicinity of the screen 7 and the stirring blade 8, and the microcarriers are uniformly suspended and stirred. It is more desirable to install a stirring blade so that a mixed flow occurs in a portion where the liquid flow due to the air lift action is weak. According to the culture device equipped with the stirrer of the present invention, it is possible to achieve more uniform mixing / stirring of the culture solution containing solid particles such as microcarriers and reduction of the gas aeration amount in the submerged aeration pipe. , A milder agitation can be performed.

[0006]

The apparatus for culturing living cells of the present invention comprises a culture tank 1 equipped with a screen 7 that allows liquid components in a culture medium to pass but not microcarriers, and at least one gas aeration sparger at the lower end. Liquid aeration pipe 2
And a closed circulation system pipe connected so that the culture liquid in the culture tank 1 is agitated by the air lift action of aeration in the submerged aeration pipe 2, and further comprises a screen 7 and / or a stirring shaft 6. It is characterized by having means for uniformly stirring the culture solution by vibrating.

The screen 7 installed in the culture tank prevents the microcarriers from leaking into the submerged aeration pipe which causes the liquid to flow due to the air-lifting action of gas aeration. When the microcarriers leak into the submerged aeration pipe, the microcarriers are entrained in gas bubbles due to aeration and taken into the foam layer formed in the gas phase portion on the upper surface of the culture solution, so that the culture cannot be continued. Therefore, it is necessary to confine the microcarriers in the culture tank to enable air lift stirring. In the culture device of the present invention,
The setting place of the screen 7 is not particularly limited as long as it is a position where it is immersed in the liquid in the culture tank. However, since the space in which the microcarriers are confined becomes a culture zone in which living cells actually survive / proliferate, it is desirable to install the screen 7 at a position where the culture zone can be as large as possible. Further, it is more desirable to install the two screens 7 on the upper part of the culture solution and the bottom part of the culture tank so as to confine the microcarriers in the two screens 7. Further, in the present invention, since the portion other than the culture zone in the culture tank, that is, the outside of the screen 7, the medium containing no solid content such as microcarriers can be extracted without using other separation means,
Perfusion culture can be easily performed.

In order to enhance the filtration efficiency of the culture solution, the screen 7 is preferably installed so as to face the circulation channel of the culture solution and be oriented perpendicular to the circulation direction. Also,
The pore diameter of the member forming the screen 7 is selected in accordance with the size of the microcarrier used, so that the microcarrier does not leak from the culture zone and mix into the oxygen-dissolved solution. Although the member of the screen 7 is not particularly limited, it is a material that can withstand these sterilization treatments because it undergoes autoclave sterilization and steam sterilization at the start of culture, and it is not corrosive and exhibits cytotoxicity. The absence is a prerequisite.

The submerged aeration pipe 2 allows the liquid to flow by the air-lifting action of the gas aeration, and at the same time, can efficiently dissolve oxygen by the aeration of the gas. Therefore, the gas aeration in the submerged aeration pipe of the culture device of the present invention serves as an efficient oxygen supply means to the cells of the living body in the culture tank, in addition to the flow / agitation of the liquid by the air lift action. . The air lift function and oxygen supply by gas ventilation are
It can be controlled by the gas flow rate and the composition of the gas. Usually, air, oxygen, nitrogen, and carbon dioxide gas are appropriately mixed and used as an aeration gas. Further, a detailed description will be given with reference to FIG. 1 showing an embodiment of the present invention. An aeration gas containing oxygen is aerated from a gas aeration sparger installed at the bottom of the submerged aeration pipe 2. The liquid is caused to flow by the air-lifting action of aeration, and rises in the pipe 2 and then descends the connecting pipe 3 to reach the outside of the screen 7 installed under the culture tank 1. The liquid passes through the screen 7 and rises in the culture tank 1, passes through the screen 7, flows into the connecting pipe 3, descends, and returns to the pipe 2. Oxygen is enriched in the liquid in the pipe for submerged aeration by submerged aeration by aeration of oxygen-containing gas. The oxygen-enriched liquid supplies oxygen and nutrients to the cells in the culture tank in the process of rising in the culture tank, and receives waste products instead. A place to ventilate in the submerged aeration pipe is preferably provided with a gas aeration sparger under the inside of the submerged aeration pipe so that a stronger circulation force can be obtained with a small amount of aeration.

Next, the agitation of the culture solution by the vibration of the screen and the vibration of the agitator, which is a feature of the present invention, will be described in detail with reference to FIG. A vibration shaft 5 and a stirring shaft 6 are connected to the screen 7. A stirring blade 8 is attached to the stirring shaft 6. The vibration shaft 5 is connected to the vibrator 4.
When the vibration exciter 4 is operated, the vibrating shaft 5, the screen 7, and the stirring shaft 5 integrally vibrate vertically. Due to the vibration, the culture solution near the screen 7 and the solution near the stirring blade 8 are stirred. The stirring blade 8 is preferably installed in a region where the liquid flow due to the air lift effect of ventilation is weak. The shape of the stirring blade is not particularly limited, but it is preferably a shape in which solid matter such as microcarriers does not deposit on the stirring blade. For example, as shown in FIG. 3, (a) a disc with a circular hole, and (b) a conical disc are available. The members of the stirring blade, stirring shaft, and vibration shaft are not particularly limited, but since they are autoclave sterilized or steam sterilized at the start of culture, they are materials that can withstand these sterilization treatments, and
It is essential that it is not corrosive and does not show cytotoxicity. The stirring state of the culture solution changes depending on the magnitude of vibration, that is, the vertical swing range and the number of vibrations. ADVANTAGE OF THE INVENTION According to this invention, the stirring state in a culture tank can be optimized by the flow of the liquid by an air lift action, and the vibration of a stirring shaft and a screen.

The shaker is a member that can be easily installed in and / or removed from the culture tank, does not affect the aseptic condition in the culture apparatus by its vibration operation, and can withstand the sterilization operation of the culture apparatus. It is essential that it is configured.
Specifically, 1) a method of attaching an expandable and contractible bellows-shaped member to the vibration shaft 5 and expanding and contracting the expandable and contractible member by changing the air pressure to vertically vibrate the screen, 2) install a solenoid, and attract the magnetic material. A method in which the screen is vertically vibrated by the repulsive force, and 3) a eccentric member is installed on a separately provided rotating shaft, and the eccentric member and the vibration shaft 5 are connected by interposing a sliding member therebetween, There is a method of applying vertical vibration to the screen by rotating the core member. The vibration by the shaker 4 has the effect of suppressing clogging of the screen 7 at the same time as stirring the culture solution. Due to the vibration, the solid content in the culture solution does not adhere to the screen 7 and clogging is suppressed, which enables long-term culture.

[0012]

The present invention will be described in detail below with reference to examples.

(Embodiment 1) FIG. 1 shows an embodiment of the culture apparatus of the present invention.

The main culturing apparatus has a stainless steel internal volume of 8
Liter, culture tank of 6 liters of culture volume 1, submerged aeration pipe 2, connection pipe 3, vibrator 4, vibrating shaft 5, stirring shaft 6, stirring blade 8, although not shown, fresh culture medium storage tank, culture medium Storage tank,
The gas supply system is composed of an air source, an oxygen source, a carbon dioxide gas source, a nitrogen source, and a control unit for monitoring and controlling the culture condition.

In the culture tank 1, a suspension of a culture solution and a microcarrier is stored, and a screen 7 composed of a stainless steel mesh with a pore diameter of 75 μm is installed so that the microcarrier does not enter the submerged aeration pipe. Has been done. Culture tank 1
A defoaming layer 13 for breaking bubbles generated by aeration is provided in the gas phase portion above the culture liquid surface. Furthermore, in the culture tank 1, a level sensor 11 and a temperature sensor 1
2, pH sensor 9 and DO sensor 10 are installed.

Living cells that can be cultured by the present culture apparatus include animal cells, microorganisms, plant cells and the like. In particular, it is suitable for culturing using a culture medium in which living cells are suspended in the culture medium in the form of particles of 100 μm or more and a culture carrier such as a microcarrier.

First, the culture tank 1, the fresh medium storage tank, the medium storage tank, and the pipes connecting them are sterilized by high-pressure steam in advance.

When the cells of the living organism to be cultivated are adherent cells, a cell-adhering microcarrier for adhering cells is prepared in advance by a conventional method, and then the screen 7 in the culture tank is used. Sterilize by injecting into the culture zone partitioned by. Separately, a cell adhesion microcarrier preparation tank may be installed to prepare, sterilize and equilibrate the cell adhesion microcarrier, and then aseptically supply to the culture tank 1.

Next, a medium in which the nutrients necessary for the growth of the living biological cells to be cultured are dissolved is prepared, sterilized, and stored in a fresh medium storage tank. A required amount of the stored fresh medium is transferred to the culture tank 1. The amount of liquid in the culture tank is adjusted by the information from the level sensor 11. Next, warm water is passed through a water jacket provided outside the culture tank 1 to adjust the temperature in the culture tank to a temperature suitable for the growth of cells of the living body to be cultured. The temperature condition in the culture tank is sent to the control unit by the temperature sensor 12, and the control unit keeps the temperature in the culture tank constant based on the information.

The cell growth causes changes in pH and dissolved oxygen concentration (DO) in the culture solution, depletion of nutrients, and accumulation of waste products. Therefore, the temperature sensor 1 is installed in the culture tank 1.
2, pH sensor 9, DO sensor 10 is installed, temperature,
Measure pH and DO. These pieces of measurement information are transmitted to the control unit to determine the culture status.

Supply of oxygen to living cells and airlift stirring of the culture solution are performed by the following methods. That is, the control unit determines from the information from the DO sensor 10 that oxygen in the culture solution has been consumed by the cells of the living body and the dissolved oxygen concentration has decreased. Based on this determination, the control unit vents the oxygen-containing gas from the vent sparger 14 at an appropriate ventilation rate. Oxygen is dissolved in the medium by aeration, and the medium in which oxygen is dissolved flows and circulates due to the airlift effect of aeration, contacts the culture solution in the culture zone through the screen 7, and oxygen is diffused and supplied to the cells. . The amount of oxygen supplied to the culture solution is adjusted by adjusting and adjusting the oxygen concentration in the aeration gas, by increasing or decreasing the aeration amount, and the culture tank 1.
This is carried out in combination with the three methods of changing the internal pressure of the solution to increase the amount of oxygen dissolved in the culture solution. The defoaming layer 13 breaks the foam layer generated on the liquid surface of the culture tank and on the gas phase inside the submerged aeration pipe by aeration. The defoaming layer 13 is composed of a stainless wire mesh whose surface is hydrophobized with polysiloxy acid, and when the foam comes into contact with the surface, the foam breaks. Further, the vibrator 4 is operated at the same time as the air lift agitation to vibrate the screen 7 and the agitation shaft 7 to adjust the gas aeration amount.

FIG. 4 is a diagram showing an example of an actual perfusion culture result of this example and the conventional method. Asahi Kasei Microcarriers (manufactured by Asahi Kasei Corporation) was used at a concentration of 6 g / liter,
The medium was 5% newborn calf serum (purchased from Dainippon Pharmaceutical) (V
/ V) ERDF medium (manufactured by Kyokuto Pharmaceutical Co., Ltd.) was used to culture CHO-K1 cells. The vertical axis of the graph represents the cell concentration and the horizontal axis represents the number of culture days. The cell concentration is determined by sampling the culture medium at a suitable time, measuring the number of cells attached to the microcarriers as the number of viable cells by the conventional nuclear release method, and converting it to the number of cells per 1 ml of the culture medium. And The culture result according to this example is shown as A in the graph, and the culture result according to the conventional method is shown as B. The conventional method is the case where the stirring shaft is not attached in this embodiment. A
In culturing, the agitation shaft and screen were shaken with a width of 5-10.
It was vibrated once every 5 seconds in mm. In both A and B, cells proliferate smoothly, and the perfusion rate is adjusted to 1 to 1 according to the proliferation of cells.
It was increased to 0 and a long-term culture of 30 days was achieved.
Comparing the reaching cell concentrations, the reaching cell concentration in A was about twice the reaching cell concentration in B and was about 4 × 10 ⁷ cells / ml. It can be said that this is a manifestation of the effect that the mixing state of the culture solution was optimized by the stirring shaft of the present invention.

[0023]

EFFECTS OF THE INVENTION According to the present invention, even in a culture medium containing solid particles such as microcarriers, efficient oxygen supply and air lift agitation are carried out by submerged aeration, and further, the culture medium is finely and uniformly mixed by agitation by vibration. Can be stirred,
A long-term high-density culture of living cells can be realized.

[Brief description of drawings]

FIG. 1 is an explanatory view showing an embodiment of a culture device of the present invention.

FIG. 2 is an explanatory view showing a mixed state of the liquid in the culture tank of the culture device of the present invention.

FIG. 3 is an explanatory view showing two examples of stirring blades of the stirrer of the present invention.

FIG. 4 is a characteristic diagram showing an example of a culture result in the culture device of the present invention.

[Explanation of symbols]

1 ... Culture tank, 2 ... Submerged aeration pipe, 3 ... Circulation pipe, 4 ...
Exciter, 5 ... Vibration axis, 6 ... Stirring axis, 7 ... Screen, 8
... stirring blade, 9 ... pH sensor, 10 ... DO sensor, 11 ...
Level sensor, 12 ... Temperature sensor, 13 ... Defoaming layer, 14
… Ventilated spargers.

Claims (4)

[Claims] 1. A culture device equipped with a screen having pores for preventing the passage of solid particles and for allowing the culture solution to pass, equipped with means for vibrating the screen and means for stirring the culture solution near the screen. A biological cell culture device characterized by the above. 2. The biological cell culture apparatus according to claim 1, wherein the means for stirring the culture solution is a stirring shaft connected to a vibration shaft for vibrating the screen, and a stirring blade attached to the stirring shaft. . 3. The biological cell culture device according to claim 1, wherein a plurality of stirring blades are attached to the stirring shaft to stir the culture solution. 4. A culture tank equipped with a screen having pores for preventing the passage of solid particles and allowing passage of the culture solution, a submerged aeration pipe for supplying oxygen to the culture solution, and the submerged aeration. The culture tank and the submerged aeration pipe have a means for aeration to generate air lift in the submerged pipe, and the culture tank and the submerged aeration pipe are directed upward to the culture liquid in the subculture tank by an airlift action inside the submerged aeration pipe. A living cell culture device forming a closed circulation system interconnected to generate a flow, comprising a vibrating shaft for vibrating the screen, and at least one stirring blade connected to the vibrating shaft. A living body cell culture device comprising a stirring shaft and means for vibrating the screen.