JP3412364B2 - Cell culture device and cell culture method - Google Patents

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an apparatus and method for culturing cells. More specifically, a cell comprising a gas supply device having a hollow fiber converging body housed in a tubular body, a cell culture tank having a hollow fiber in a tubular culture tank, a medium exchange device, a pump, and a filter. The present invention relates to a culture device and a cell culture method using the device.

[0002]

2. Description of the Related Art Conventionally, for cell culture, a batch-type culture device having a simple structure and easy control of culture conditions has been used. Since this batch-type culture device performs culture using a fixed amount of medium, the culture gas and nutrients contained in the medium are limited. When culturing is performed using this device, the cell growth rate decreases to some extent and then stops. Therefore, in order to carry out continuous culture of cells, a cell culture device has been found which supplies a new medium into the culture tank and discharges an old medium containing waste products to the outside of the culture tank. That is, a medium containing nutrients and gas (hereinafter referred to as a circulation medium).
Is circulated by separating it from a medium containing cultured cells (hereinafter referred to as growth medium) by a membrane, supplying culture gas and nutrients to the growth medium through the membrane surface, and collecting waste gas and waste products from this medium. Is to do.

In this cell culture device, the efficiency of cell culture is affected by the surface area of the membrane that divides the culture tank. Therefore, it is convenient for the cell culture to have a large membrane surface area.
Therefore, the membrane is formed into a hollow fiber shape, and a plurality of hollow fibers have a structure in which they penetrate the culture tank.

In a cell culture device using a hollow fiber, for example, the cultured cells are outside the hollow fiber together with a small amount of growth medium,
Circulating medium passes inside the hollow fibers. This circulation medium supplies the growth medium with the gas and culture medium necessary for cell growth,
Since the waste gas and waste products are exchanged through the hollow fiber membrane, it is possible to continuously culture the cultured cells while they are separated from the circulation medium. The circulating medium must contain sufficient gas and culture medium necessary for cell growth. Therefore, conventionally, as a means for supplying gas to the circulating medium, for example, a method has been used in which oxygen gas, nitrogen gas, carbon dioxide gas, air, or a mixed gas thereof is blown into the circulating medium as bubbles using a nozzle or the like. Further, the supply of the culture solution to the circulating medium was carried out by removing the old medium during the circulation and adding a fresh medium to maintain a constant culture condition.

[0005]

However, in the method of blowing gas into the circulating medium with a nozzle, the efficiency of gas supply is low and sufficient gas required for culture cannot be introduced, and the circulating medium is always kept under constant culture conditions. It was difficult to maintain at. In addition, the gas blown into is likely to foam due to the influence of the polymeric substances contained in the circulating medium, and the medium may overflow from the tank supplying the gas, or the gas may remain in the circulating medium as fine bubbles. there were. This residual air bubble circulates in the medium and adheres to the inner wall of the hollow fiber when passing through the cell culture tank using the hollow fiber and stays there, which hinders the contact between the hollow fiber and the circulating medium, or when The circulation medium sometimes prevented the passage through the hollow fiber. As a result, the supply of the gas and the culture solution to the culture medium or the removal of the unnecessary gas and waste products from the culture medium is delayed, the culture efficiency is lowered and the growth rate of the cells is lowered, or in the worst case, the cells are killed. There was a problem such as.

Therefore, there has been a demand for an apparatus for efficiently supplying gas and culturing cells without generating bubbles in the circulating medium.

[0007]

Means for Solving the Problems As a result of intensive studies by the present inventors, a gas supply device having a hollow fiber converging body housed in a tubular body, and a cell culture tank having a hollow fiber in the tubular culture tank. Then, a cell culture device comprising a medium exchange device, a pump, and a filter was found, and the present invention was completed.

That is, the present invention provides a gas supply device having a hollow fiber converging body housed in a tubular body, a cell culture tank having a hollow fiber in a tubular culture tank, a medium exchange device, a pump, and a filter. And to form a circulation path,
An apparatus for culturing cells by circulating a circulating medium containing a culture solution and gas in this path is provided.

Hereinafter, each part of the cell culture device of the present invention will be described in detail.

(Gas Supply Device) The gas supply device used in the cell culture device of the present invention has a function of supplying a gas for culturing to the circulation medium flowing through the circulation path, and is a porous hollow. The culture gas is supplied to the circulation medium by a thread.

The gas supply device comprises a cylindrical body for receiving the circulating medium, and a hollow fiber converging body for supplying gas, which is housed inside the cylindrical body. Further, in the gas supply device, in addition to a cylinder for storing compressed gas such as oxygen gas, nitrogen gas, carbon dioxide gas, air or a mixed gas thereof, a pressure regulator for further adjusting the flow rate of the culture gas, a gas mixing device, etc. Is added for use. The tubular body has a tubular shape such as a cylinder or a prism, and can be manufactured using a material such as plastic, glass, and various metals (stainless steel). Among these materials, it is preferable to use a plastic cylindrical body that is easy to handle, has good workability, and is easy to confirm the inside.

The hollow fiber bundled body housed in the tubular body is held at both ends by hollow fiber support members, further penetrates through the lid of the tubular body and is guided to the outside of the tubular body, and the end is It is connected to the gas cylinder. This hollow fiber bundle is composed of porous hollow fibers. The pores of this porous hollow fiber can be used in the range of 0.01 μm to 1.0 μm, but in order to efficiently supply gas to the circulation medium without generating bubbles, 0.03 μm to 0. It is preferable to use hollow fibers having pores with a size of 4 μm. Further, the inner diameter of the hollow fiber to be used can be changed depending on the capacity of the gas supply device, the number of porous hollow fibers, and the like. Further, the length of the hollow fiber can be appropriately changed depending on various conditions of the cell culture device such as the gas supply device, similarly to the inner diameter.

Further, the hollow fiber is a porous membrane produced by using a material of a polymer compound such as polypropylene, cellulose diacetate, silicon and polyethylene.
It can be manufactured from a dense film. As these hollow fibers, various kinds of commercially available porous hollow fibers can be used. Since gas is blown into the hollow fiber from an external cylinder or the like to increase the pressure, the material and the thickness of the membrane can be selected and used. The thickness of the film is not limited, but is usually 20 μm to 1
It is preferable to use one having a diameter of 00 μm.

(Cell culture tank) The cell culture tank is connected to the gas supply device, and the circulation medium supplied with gas from the gas supply device is supplied. This cell culture tank is composed of a tubular culture tank, a plurality of hollow fibers accommodated in the tubular culture tank through which the circulating medium passes, a support member for the hollow fibers, and the like.

This culture tank is made of the same material and structure as the tubular body used in the gas supply device. The size of this tubular culture tank depends on the amount of cell culture, the type of cultured cells,
It can be changed according to other cell culture conditions.

The hollow fibers are various porous hollow fibers used for cell culture, and are made of a polymer compound material such as polypropylene, cellulose diacetate, or silicone rubber. The pores of this hollow fiber can be selected and used according to the type of cultured cells and the molecular weight of the culture product, but usually it is preferably 0.05 μm to 0.15 μm. The hollow fibers contained in the tubular culture tank can be used by bundling a plurality of hollow fibers, and the number, diameter, length, etc. of the hollow fibers at this time should be changed according to the capacity of the tubular culture tank. You can Furthermore, both ends of the hollow fiber are held by hollow fiber support members, and the growth medium is sealed inside the culture tank outside the hollow fiber.

The tubular culture tank contains culture cells and a growth medium, and is supplied with a culture solution and a culture gas from a circulation medium that passes through a hollow fiber to remove wastes and unnecessary gases, and thus the inside of the culture tank. Cells can be cultured. The circulation medium that has passed through the hollow fiber contains waste products and unnecessary gas and is discharged from the discharge port and collected in the medium exchange device. The growth medium and the circulation medium are serum-free medium containing saccharides such as amino acids, vitamins, inorganic salts and glucose as nutrients necessary for cell culture, and a serum-containing medium supplemented with serum such as fetal bovine serum. Can be used.

Further, it is preferable for continuous culture that the cell culture tank is provided with two or more outlets for taking out the cultured cells or the culture product from the growth medium after the culture. Further, this culture tank can be placed in a constant temperature bath or the like to keep the culture temperature in order to keep the cell culture conditions constant.

(Medium Exchange Device) The medium exchange device is a device which is connected to the cell culture tank and stores the circulating medium that has passed through the cell culture tank. This medium exchange device includes a reservoir having an inlet for circulating the circulating medium and an outlet for discharging the circulating medium, a medium discharging valve for discharging the medium containing waste products, a medium feeding valve for supplying fresh circulating medium, etc. It is constructed by attaching. Further, a stirring device for mixing the supplied fresh culture medium in the reservoir can be added.

Further, the reservoir of the medium exchange device has a double structure and it is preferable to circulate the circulating liquid adjusted to the culture temperature inside the double structure to keep the circulating medium at the culture temperature for efficient culture. .

The circulating medium that has passed through the medium exchanging device described above is sent to the gas supply device through a pump for feeding the circulating medium to form a circulating path. As a pump for feeding the circulating medium, for example, a magnet gear pump, a peristaltic pump or the like can be used. In addition, a filter for removing insoluble matters or air bubbles in the medium, monitoring of essential components in the circulating medium, and culture conditions such as the flow rate of the circulating medium, temperature, pH, dissolved oxygen amount, dissolved carbon dioxide amount, etc. are provided in this route. A control device for controlling can be added and used. The pump, filter and control device can be installed anywhere in the circulation path. Each device of these circulation paths can be connected by a tube made of, for example, plastic, silicon rubber, Teflon, or the like.

In the culture apparatus of the present invention, animal cells, microbial cells or plant cells can be cultured. For example, various cells such as adhesion-dependent cells, floating cells, normal cells / tumor cells, fibroblasts, epithelial cells. Can be propagated. Examples of animal cells include various cells such as vertebrates, invertebrates and protozoa. As microbial cells,
Bacteria, yeast, actinomycetes, etc. can be mentioned. Generally, oxygen-containing gas can be used for culturing animal cells, and carbon dioxide-containing gas can be used for culturing plant cells.

[0023]

EXAMPLES The present invention will now be described in more detail with reference to examples.

EXAMPLE In the cell culture device of the present invention, the gas supply device 1, the cell culture tank 2, the medium exchange device 3 and the pump 4 are connected so as to form a circulation path as shown in the configuration diagram of the device in FIG. It is configured. Further, in this route, a filter 5 for removing insoluble matters and air bubbles in the circulation medium and a control device 6 are provided.
Are connected.

The gas supply device 1 is composed of a transparent plastic tubular body, a polypropylene hollow fiber converging body and a hollow fiber supporting member housed inside the tubular body. In this gas supply device 1, a culture gas is supplied from a culture gas supply device (not shown) composed of an oxygen gas cylinder, a nitrogen gas cylinder and a carbon dioxide gas cylinder having a pressure adjusting device and a mixing device at a gas inlet 7 into a cylindrical body. It is introduced into the apparatus through a gas introduction port 7 attached to the end of the hollow fiber converging body provided at the top,
Circulation medium inlet 1 filled with hollow fiber and passing through the hollow fiber membrane
Gas is supplied to the circulating medium introduced from 6. On the other hand, the surplus gas is discharged from the gas discharge valve 8 attached to the end of the other hollow fiber bundle. This hollow fiber bundle is composed of porous polypropylene, and the gas for culturing is a mixture of oxygen gas, nitrogen gas and carbon dioxide gas of 0.2 to 0.5.
It is supplied from a gas introduction tube attached to the gas introduction port 7 at a pressure of Kg / cm ² .

The circulating medium supplied with the gas is then sent to the cell culture tank 2 via the control device 6 for controlling the culture conditions. Culture information obtained from various sensors connected to each part of the cell culture device is input to the control device 6 to control culture conditions.

The cell culture tank 2 is a cell culture tank having a hollow fiber inside a cylindrical culture tank into which the circulating medium supplied with the culture gas by the gas supply device 1 is introduced. This culture tank is composed of an inlet 9 for introducing the circulating medium, a hollow fiber, and an outlet 10 for discharging the circulating medium. Further, valves 11 and 12 for introducing a culture medium, cultured cells and the like into the culture tank and for taking out the culture from the culture medium can be added. This cell culture tank is approximately 25 cm in length and 5 c in diameter.
The cylindrical culture tank contains 0.2 liters of a growth medium, and a hybridoma producing a monoclonal antibody can be cultivated.

The hollow fiber of the cell culture tank is made of polypropylene and is made up of about 1000 bundles of porous hollow fibers having an inner diameter of 200 μm and a length of 15 cm. Both ends of this hollow fiber are put together in one place to form an inlet 9 and an outlet 10.

The circulating medium used for the culture is sent to the medium exchange device 3 through the medium introduction port 13. The medium exchange device
It has a medium supply valve 14 for supplying fresh circulation medium and a medium discharge valve 15 for discharging circulation medium. It is preferable to add a stirring device to the medium exchange device 3 for further mixing the medium. Further, in order to control the temperature of the circulating medium, the medium exchange device can be held in a constant temperature bath.
Further, as the pump 4 used in the cell culture device, a magnet gear pump can be used. Further, the circulation medium path is constructed by adding a filter 5 having pores of 40 μm.

[0030]

In the cell culture device of the present invention, the gas for culturing can be efficiently supplied to the circulating medium as compared with the conventional gas supply method using a nozzle. Using this device, cells can be efficiently cultured for a long period of time.

[Brief description of drawings]

FIG. 1 is a configuration diagram of a cell culture device of the present invention.

[Explanation of symbols]

1 gas supply device 2 cell culture tank 3 Medium exchange device 4 pumps 5 filters 6 Control device 7 gas inlet 8 gas discharge valve 9, 16 Circulation medium inlet 10, 17 Circulation medium outlet 11, 12 valves 13 Circulation medium inlet 14 Medium supply valve 15 Medium discharge valve

Claims (4)

(57) [Claims] 1. A gas supply device having a hollow fiber converging body housed in a tubular body, a cell culture tank having a hollow fiber in a tubular culture tank, a medium exchange device, a pump, and a filter. A cell culture device comprising: 2. The cell culture device according to claim 1, wherein the gas is a mixed gas of oxygen gas, nitrogen gas and carbon dioxide gas. 3. A gas supply device having a hollow fiber converging body housed in a tubular body, a cell culture tank having a hollow fiber in the tubular culture tank, a medium exchange device, a pump, and a filter. A cell culture method is characterized in that the cells are bound to form a circulation path, a circulation medium containing a culture solution and gas is circulated in the circulation path, and the cells are cultured in a cell culture tank. 4. The cell culture method according to claim 3, wherein the gas is a mixed gas of oxygen gas, nitrogen gas and carbon dioxide gas.